HK1228499B - Use of compounds comprising two or more hydrophobic domains and a hydrophilic domain comprising peg moieties for stabilization of a cell - Google Patents

Description

Use of compounds comprising two or more hydrophobic domains and a hydrophilic domain containing a PEG moiety for stabilizing cells

The present invention relates to the use of compounds comprising two or more hydrophobic domains and a hydrophilic domain comprising a polyethylene glycol (PEG) moiety for stabilizing cells and methods related thereto.

Octavio T. et al. (Biotechnology and Bioengineering 1990 36:911-920) describe the effects of the shear protectant Pluronic F-68 (Poloxamer 188), a nonionic surfactant, on hybridomas grown under hydrodynamic stress. The paper discloses that the shear sensitivity of mammalian cells may be a problem that has prevented the development of large-scale animal cell cultures. Octavio et al. investigated the relationship between plasma membrane fluidity, shear sensitivity, and the effects of shear protectants added to the culture medium. They demonstrated that plasma membrane fluidity could be reduced by adding cholesterol to the culture medium, and they showed that cell survival of cells subjected to selected shear rates was higher when cholesterol was added to the culture medium compared to controls. Pluronic F-68 has been shown to have the same effect.

Tomeczekowski J. et al. 1993; Enzyme and microbial technology 15:849-853 describe cholesterol as a suitable, physiological agent to protect cells from shear stress by reducing plasma membrane fluidity.

Laura A. et al. (Enzyme and Microbial Technology 2000 26:324-331) describe Pluronic F-68 as a shear protectant for animal cells from hydrodynamic stress and investigate its mechanism of action. Laura et al. review various other publications showing that Pluronic F-68 exhibits two protective mechanisms: a physical and a biological/cellular mechanism. Pluronic F-68 reduces the level of frequency experienced by cells, e.g., it stabilizes the foam layer and reduces the rising velocity of the bubbles, thereby reducing shear forces. At the cellular level, Pluronic F-68 reduces plasma membrane fluidity.

Similar disclosures are found in Thomas C. et al. (Advances in Bioprocess Engineering 1998: 137-171); Ramirez O. et al. (Biotechnological and Bioengineering 1990; 36: 911-920); Michaels J. et al. (Biotechnological and Bioengineering 1991; 38: 169-180) and Sowana D. et al. (Biochemical Engineering Journal 2002; 12: 165-173).

However, cholesterol is a hydrophobic molecule and therefore it must be dissolved in solvents such as DMSO or alcohol, which show cytotoxicity at concentrations above 1%, resulting in limited cholesterol concentrations that can be used to stabilize cells.

Furthermore, it has been shown that monovalent molecules such as cholesterol or Pluronic F-68 have lower shear protection properties compared to divalent molecules. Therefore, the concentration of monovalent protective agents (as disclosed) needs to be higher than that of divalent molecules.

Ultimately, monovalent molecules may be internalized into the cell interior and thereby alter cell physiology.

Therefore, there is a need for new uses and methods that employ compounds and compositions that can bind to cells without affecting viability and/or stabilize cells. For example, such uses and methods should stabilize cells exposed to stress, such as shear stress.

The uses and methods of the present invention solve this problem and overcome the disadvantages of the prior art.The uses and methods of the present invention are particularly effective in stabilizing cells, in particular against shear stress.

In one embodiment, the invention relates to the use of a compound comprising, preferably consisting of, two or more hydrophobic domains linked to one hydrophilic domain, wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain, and wherein the two or more hydrophobic domains each comprise a linear lipid, a steroid or a hydrophobic vitamin, and wherein the hydrophilic domain comprises a polyethylene glycol (PEG) moiety, for stabilizing cells.

A polyethylene glycol (PEG) moiety is understood as a linear or branched moiety, preferably a linear moiety, comprising at least one -O- _CH2 - _CH2- moiety, preferably 1-50, more preferably 4-30 -O- _CH2 - _CH2- moieties.

The basic principle of stabilization is assumed to be that the terminal hydrophobic portion of the compound is anchored into the lipid bilayer of the cell membrane. This anchoring of the hydrophobic molecule reduces the fluidity of the plasma membrane and thus stabilizes the cell.

A stabilizing, in particular a shear-protecting effect, was demonstrated in particular for cholesterol, myristic acid and stearic acid as hydrophobic moieties in the compounds that can be used in the process according to the invention (see Example 5).

According to the present invention, "stable cells" are understood to mean cells with a higher viability compared to control cells treated under defined conditions without the methods or compounds of the present invention, preferably shear stress conditions such as centrifugation, e.g., centrifugation at 500 g for 20 minutes. Stabilization is typically determined for a cell population of, for example, 2, 10, 100, or more cells, and the average viability of each cell population is compared. A higher average viability of the treated cell population compared to the average viability of the control population indicates a stabilizing effect. Viability can be determined by measuring cell morphology, cell viability, and/or cell recovery. Methods for measuring cell morphology, cell viability, and cell recovery are known in the art. Specifically, the methods described in Example 5 can be used. To determine cell morphology, visual inspection by microscopy can be performed. To determine cell viability, a cell viability test using the WST-1 Proliferation Kit (RAS) can be performed. Specifically, a viability that is at least 5%, more preferably 10%, and even more preferably 20% higher than that of control cells or a control cell population indicates stabilization and, therefore, stabilization of the cells or cell population.

In a preferred embodiment of the invention, the stabilization is stabilization during exposure of the cells to shear forces.

In a further preferred embodiment, the stabilization is stabilization of the cells during exposure to shear forces due to centrifugation, large-scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead-based cell separation.

In a further preferred embodiment, the stabilization is stabilization of the cells upon exposure to centrifugation, large-scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead-based cell separation procedures.

"Shear stress" or "shear force" is understood to be the component of stress that is coplanar with the cross-section of a substance. Shear stress arises from the component of the force vector that is parallel to the cross-section. Such stress is applied to cells during centrifugation, large-scale cell culture, flow cytometry, fluorescence-activated cell sorting, and/or bead-based cell separation.

Centrifugation is a method used in industrial and laboratory settings that involves using centrifugal force to sediment a mixture with a centrifuge. The cells can be centrifuged, for example, at 100 g, 200 g, 500 g, 1000 g or higher for 5 minutes or longer, such as 1 hour or 5 hours.

Flow cytometry is a laser-based biophysical technique used for cell counting, cell sorting, biomarker detection, and protein engineering by suspending cells in a fluid stream and passing them through an electronic detection device. It allows simultaneous multi-parameter analysis of the physical and chemical characteristics of up to thousands of particles per second.

Fluorescence activated cell sorting (FACS) is a specialized type of flow cytometry that provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell.

In bead-based cell separation methods, beads, such as magnetic beads, are used, which are typically coated with a member of a bioaffinity binding pair, such as an antibody. Using these beads, cells of interest carrying a marker recognized by the binding pair member can be bound and subsequently separated, for example, using magnetic properties. The separation step applies shear forces to cells bound to the beads.

Large-scale cell culture is understood to be the cultivation of cells in volumes exceeding 10 ml, 50 ml, 100 ml or 1 l of liquid medium or more, in particular as a batch culture involving agitation. Such agitation also means shear stress on the cells to be cultured.

The compounds used in the present invention comprise, or preferably consist of, two or more hydrophobic domains and one hydrophilic domain.

Preferably, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hydrophobic domains are covalently bound to the hydrophilic domain.

With regard to the stabilizing effect, it has been found to be advantageous that the compounds used according to the invention preferably contain 2 or 3 or more, more preferably 2 or 3, hydrophobic domains.

With particular advantage, in a specific embodiment at least one lipid hydrophobic domain comprises a steroid, even more preferably cholesterol.

In a preferred embodiment of the present invention, two or three hydrophobic moieties of the hydrophobic domain are covalently bound to the hydrophilic domain.

For the general understanding herein, a "hydrophobic portion" is comprised in and forms the main part of a "hydrophobic domain", thus determining its hydrophobic character.

The hydrophobic moieties of a compound comprising two or more hydrophobic moieties may be the same or may be different. For example, a compound comprising two hydrophobic domains may comprise two myristic acid moieties, or one myristic acid moiety and one cholesteryl moiety.

Cholesterol moieties are particularly preferred hydrophobic moieties for compounds used in the present invention.

In a preferred embodiment of the use, the compound comprises, preferably consists of, two or more hydrophobic domains and one hydrophilic domain,

wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain, and

wherein the two or more hydrophobic domains each comprise a linear lipid, a steroid, or a hydrophobic vitamin, and

wherein the hydrophilic domain comprises a compound of formula (I):

X1-[A1 -(L1) _n ] _k1 -Z - [A2 -(L1) _n ] _k2 - X2 (I),

in

Z is a linear polyethylene glycol (PEG) moiety containing 1-100, preferably 1-50, more preferably 4-30 -O-CH ₂ -CH ₂ - moieties, wherein the polyethylene glycol moiety optionally comprises one or more spacer moieties SP linking two -O-CH ₂ -CH ₂ - moieties,

and wherein the linear PEG moiety optionally comprises a linker moiety L3 at one or both termini,

Each L1 is a linker moiety selected independently of each other,

Each n is 0 or 1, chosen independently of each other,

A1 and A2 are bifunctional or trifunctional moieties selected independently of each other, provided that at least one of A1 or A2 is trifunctional,

k1 and k2 are integers from 0 to 10, selected independently of each other, provided that at least one of k1 and k2 is not 0,

X1 and X2 are independently selected from hydrogen or a protecting group,

L3 is independently selected from a straight alkyl or alkenyl chain having 1-10 C atoms, which is optionally (i) interrupted by 1-3 N, O or S atoms, and/or (ii) substituted by 1-4 hydroxyl, carbonyl, amino or mercapto groups,

and

wherein the two or more hydrophobic domains are covalently bound to the hydrophilic domain via the trifunctional domain,

or a salt thereof.

In a preferred embodiment, k1 + k2 ≥ 2.

Lipids are small hydrophobic molecules selected from the group consisting of fats, waxes, sterols, soluble fats, hydrophobic vitamins such as vitamins A, D, E and K, fatty acid monoglycerides, diglycerides, triglycerides and phospholipids.

The hydrophobic domains each comprise, and preferably consist of, a linear lipid, a steroid or a hydrophobic vitamin.

A linear lipid, steroid, or hydrophobic vitamin can be directly bound to the trifunctional moiety or bound to the trifunctional moiety via a linker L2. An example of a compound in which a linear lipid is directly bound to the trifunctional moiety is the compound myristic acid-myristic acid-(spacer C18)7-Fluos-biotin-TEG. An example of a compound in which a steroid is bound to the trifunctional moiety via a linker L2 is the compound cholesteryl-TEG-cholesteryl-TEG-(spacer C18)7-Fluos-biotin-TEG. In this latter example, TEG (tetraethylene glycol) is the linker L2.

In a preferred embodiment, the hydrophobic domains are each composed of a straight-chain lipid, a steroid or a hydrophobic vitamin. In this case, it is obvious that the hydrophobic domain is hydrophobic, more preferably lipophilic, because the straight-chain lipid, steroid or hydrophobic vitamin is hydrophobic, more preferably lipophilic.

A hydrophobic moiety is understood to be a moiety which repels a mass of water. Preferably, the moiety is lipophilic; ie it tends to dissolve in other non-polar, lipophilic substances such as fats or fatty acids.

In another preferred embodiment, the hydrophobic domains each comprise a linear lipid, a steroid or a hydrophobic vitamin and one or more other moieties. In this embodiment, the hydrophobic moiety as a whole is hydrophobic, more preferably lipophilic.

In an even more preferred embodiment, the hydrophobic domain of the invention comprising a linear lipid, steroid or hydrophobic vitamin is capable of inserting into a cell membrane. This can be determined by methods known in the art.

In a preferred embodiment, the two or three or more hydrophobic moieties of the compounds used in the present invention are different hydrophobic domains, or in the case of three or more hydrophobic moieties, two or more are different or all are different from each other.

In a preferred embodiment of the present invention, the first hydrophobic domain comprises a saturated fatty acid, in particular myristic acid, stearic acid or behenic acid, in particular myristic acid, preferably consisting thereof; and/or the second hydrophobic domain comprises cholesterol, preferably consisting thereof. In the case of a third hydrophobic domain, this domain preferably comprises cholesterol or a saturated fatty acid, in particular myristic acid, stearic acid or behenic acid, in particular myristic acid, preferably consisting thereof, and/or this domain is identical to the first or second hydrophobic domain. In the case of more than four hydrophobic domains, the same preferred embodiments apply with respect to the third hydrophobic domain.

In particular, compounds comprising at least one cholesterol moiety, especially 1, 2 or 3 cholesterol moieties are especially preferred.

For stabilization, the compounds used in the present invention contain 2 or 3 or more, more preferably 2 or 3, hydrophobic domains.

This conformation shows a higher binding affinity to cells than monovalent molecules (i.e., molecules of the present invention comprising a hydrophobic portion). Therefore, lower concentrations of the compounds used in the present invention are required to achieve shear protection compared to monovalent molecules.

The hydrophilic part of the molecule inhibits internalization of the compounds used in the invention and the shear protection is induced by the incorporation of the hydrophobic part into the outer plasma membrane. Experiments with labeled compounds used in the invention have demonstrated that the compounds are just incorporated into the outer plasma membrane without affecting the interior of the cell.

In addition, the compounds used in the present invention surprisingly exhibit favorable binding or immobilization on cells, as shown in detail in the Examples. Further compounds that exhibit immobilization include a linking group. The compounds may further include a labeling moiety. Such compounds may also be used for targeting and/or detecting cells.

Regarding further applications for cell labeling and fixation, compounds having a hydrophobic moiety and further comprising a linker and/or a labeling moiety were found in the Examples to exhibit targeting and tight retention of all cell types (see Example 2 for details). Specifically, cholesterol, myristic acid, stearic acid, and behenic acid moieties were found to be particularly useful for this purpose. The compound 5′-cholesterylTEG-cholesterylTEG-PEG2000-Fluos-3 (internal reference: BMO 29.891133) represents a preferred compound for use in the present invention, having exemplary advantages and enabling quantitative cell targeting.

Furthermore, it was found that compounds containing two or three hydrophobic moieties further comprising a linker group were experimentally demonstrated to be useful for quantitative cell fixation.

According to the present invention, a "cholesterol-bilinker molecule" is understood to be a compound used according to the invention that contains two hydrophobic moieties, both of which are cholesterol. Thus, a "myristic acid-trilinker molecule" is understood to be a compound used according to the invention that contains three hydrophobic moieties, all of which are myristic acid.

The use of compounds comprising two hydrophobic moieties, both of which are cholesterol, is especially preferred.

Furthermore, the use of compounds comprising two hydrophobic moieties, both of which are myristic acid, is especially preferred.

Such compounds are effective in stable cells according to Example 5.

According to the invention, an "asymmetric double linker molecule" is understood to be a compound used according to the invention which comprises two hydrophobic moieties, wherein the two hydrophobic moieties are different from one another.

The compounds used in the present invention are described in the examples for the most part in this modular, schematic format.

According to the present invention, the compound "cholesteryl-TEG-cholesteryl-TEG-(Spacer C18)7-Fluos-Biotin-TEG" (as shown in Figure 6 A) is understood to be a compound in which two cholesterol moieties as hydrophobic parts are bound to a trifunctional part via TEG (tetraethylene glycol).

According to Figure 6, which shows a modular description of the compounds used in the present invention together with the chemical formula, "(Spacer C18)" is understood to mean a PEG moiety of 18 atoms in length followed by a phosphate moiety as a spacer moiety. Thus, -(Spacer C18)7- is understood to mean a moiety consisting of 7 "(Spacer C18)" moieties.

According to the present invention, "Fluos" is understood to be a fluorescein moiety directly bound to the trifunctional moiety A2.

According to the present invention, "biotin-TEG" is understood to be a biotin moiety bound to the trifunctional moiety A2 via the linker TEG.

In the case of the compounds used in the present invention disclosed in this schematic manner, the trifunctional moiety A1 is typically glycerol, a hydrophobic portion of the TEG binding (see Figure 6A). In addition, embodiments are also disclosed in which serinol or 6-[(2-hydroxyethyl)amino]-1-hexanol are substituted for glycerol as the trifunctional moiety. Other alternatives to such trifunctional moieties are available to those skilled in the art.

The trifunctional moiety A1 for the compound of FIG6A is serinol, wherein the hydrophobic portion is directly bound to the trifunctional moiety A1.

In an even more schematic manner, "cholesteryl-TEG-cholesteryl-TEG-(spacer C18)7-Fluos-biotin-TEG" can be described as the structure "5'-XXYYYYYYYFZ-3'," where Y = PEG + spacer moiety, X is a hydrophobic portion coupled to a hydrophilic portion via a trifunctional linker, F is the fluorescent tag fluorescein, and Z is a linker (biotin). 5' and 3' indicate the direction of synthesis similar to that of nucleotides by automated synthesis as shown in the Examples.

Similarly, -PEG2000- is understood to be a PEG2000 moiety; ie, a polyethylene glycol (PEG) chain composed of 45 C ₂ H ₆ O ₂ subunits.

In the compounds used in the present invention described in the experimental part, L1 is present (n=1) and is a phosphate, if not explicitly stated otherwise.

In the examples, "spacer" in the context of the specific disclosed compounds used herein is understood to be a PEG moiety including a phosphate moiety. The length of the PEG moiety is determined, for example, by C9 or C12, which indicates that the PEG moiety has a length of 9 or 12 atoms, respectively.

"dT" is to be understood as thymidine, as illustrated in FIG6B . This moiety dT can be used to determine the concentration of a compound by absorption and is a bifunctional moiety according to the present invention.

In addition, compounds used in the present invention that contain two or three hydrophobic molecules covalently bound to hydrophilic domains exhibit tight binding to cells, potentially exploiting cooperative binding effects. The binding of such molecules to cells is 100-1000 times stronger than that obtained using compounds containing only one hydrophobic molecule.

Furthermore, in one embodiment preferably two, three or more, in particular two or three, hydrophobic moieties are spatially separated by the use of a linker moiety L1.

In such preferred embodiments, n=1, and thus L1 is present.

The hydrophilic domain of the compounds used in the present invention comprises a PEG moiety and is therefore flexible.

The compounds used in the present invention have a terminal hydrophobic portion followed by a long, flexible hydrophilic domain.

The hydrophilic domain allows for flexible folding around target cells required for safe embedding of cells, thereby creating a cell-friendly, hydrogel-like environment that is important for maintaining cell morphology and function effectively, thereby stabilizing the cells.

Different linear PEG moieties can be used that differ in length and/or contain spacer moieties, such as phosphates between PEG moieties, to achieve a flexible hydrophilic domain. For example, a polyethylene glycol (PEG) chain (PEG2000) composed of 45 C ₂ H ₆ O ₂ subunits as described above (see Example 6B) or a PEG moiety with a phosphate spacer, such as -(SpacerC18)7-, can be used.

Suitable protecting groups are known in the art. Suitable protecting groups for use in phosphoramidite chemistry are, for example, (4,4'-dimethoxytrityl (DMT) and fluorenylmethoxycarbonyl (Fmoc). A particularly preferred protecting group is DMT (4,4'-dimethoxytrityl).

Various salts of the compounds used in the present invention can be used, such as Na ⁺ and/or TEA ⁺ salts of the compounds used in the present invention, as shown in FIG1 .

Other salts are possible and known to the skilled person. Preferably, salts are used which do not affect or do not substantially affect cell viability or function.

In a preferred embodiment of the present invention, the moiety Z has the following structure:

-(L3) _n2 - [O-CH ₂ -CH ₂ ] _y - (SP) _n1 ] _m -[O-CH ₂ -CH ₂ ] _y1 -(L3) _n2 - ,

in

SP is a spacer moiety,

Each spacer moiety SP is selected independently of each other,

Each n1 is 0 or 1, selected independently for each m part,

Each n2 is 0 or 1, chosen independently of each other,

m is an integer of 1-100, preferably 1-50, more preferably 4-30,

y is an integer of 1-100, preferably 1-50, more preferably 4-30,

y1 is an integer of 0-30, preferably 0-10, more preferably 0-4,

The condition is y*m +y1 ≤ 100

and wherein L3 is as defined above.

In a further preferred embodiment of the present invention, n1 is identical for the m moieties -[O-CH ₂ -CH ₂ ] _y -(SP) _n1 ]-.

As can be seen from the examples, n1 is generally either always 0 in the compounds used according to the invention or always 1 in the compounds used according to the invention.

An exemplary compound where n1=1 is Cholesteryl-TEG-SpacerC12-Cholesteryl-TEG-(SpacerC18)7-Fluos-Biotin-TEG.

An exemplary compound where n1=0 is cholesteryl--TEG-cholesteryl-TEG-PEG2000-Fluos-biotin-TEG.

In a further preferred embodiment of the present invention, y1 is 0.

An exemplary compound where y1 = 0 is Cholesteryl-TEG-SpacerC12-Cholesteryl-TEG-(SpacerC18)7-Fluos-Biotin-TEG.

In a further embodiment of the present invention, y1 is 1.

An exemplary compound where y1=1 is 5'-cholesterylTEG-cholesterylTEG-(Spacer C18)7-Spacer C3-dT-biotinTEG-3'.

In a further preferred embodiment of the present invention, y is 3, 4, 5 or 6, and n1 is 1. Even more preferably, m is 3, 4, 5, 6, 7, 8, 9 or 10.

In a further preferred embodiment of the present invention the spacer moieties SP are independently selected from the group consisting of phosphates and bifunctional moieties.

Preferably all spacer moieties SP are identical. Even more preferably all moieties SP are phosphates.

The difunctional moiety according to the present invention is understood to be a moiety comprising two functional groups prior to the synthesis of the compound used in the present invention. Such difunctional moieties are therefore suitable for the synthesis of straight-chain compounds. Suitable difunctional groups are preferably selected from phosphate groups, carbamate groups, amide groups, moieties comprising nucleobases, even more preferably dT, and straight-chain alkyl groups having 1-10 C atoms (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms), and the alkyl chain contains a functional group at the terminal C atom, especially independently selected from amine, carbonyl, hydroxyl, sulfhydryl, and carbonic acid groups. Examples of suitable straight-chain alkyl groups with terminal functional groups are diaminoalkyl moieties such as H ₂ N-(CH ₂ ) ₅ -NH ₂ or hydroxy-carbonyl moieties such as -C(O)-(CH 2 ) ₄ -O-.

According to the present invention, a trifunctional moiety is understood to be a moiety comprising three functional groups prior to the synthesis of the compounds used in the present invention. Such trifunctional moieties are therefore suitable for the synthesis of branched compounds. Suitable trifunctional moieties are preferably selected from trifunctional moieties having 1 to 10 C atoms (particularly 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms) and comprising at least one -OH, -SH and/or at least one -NH2 group, more preferably selected from amino acids such as lysine or serine, serinol, -O-CH2-CH(( _CH2 ) ₄ - _NH2 )-CH2-, glycerol and 1,3-diaminoglycerol moieties.

In further preferred embodiments of the present invention, X1 and/or X2, preferably X1 or X2, is replaced by a hydrophobic binding domain. In such embodiments, k1 + k2 can be 1. An exemplary compound in which X1 is replaced by a hydrophobic domain is biotin-PEG-Lys-(C18)2, as shown in the Examples.

In a further preferred embodiment of the invention, n2 are both 0. In such embodiments, the central linear PEG moiety is directly bound to the moieties X1-[A1-(L1)n]k1 and [A2-(L1)n]k2-X2.

In a further preferred embodiment of the present invention, one or both n2 = 1, and L3 is an alkyl group having 1-10 carbon atoms, which optionally contains an amide group, a carbonyl group, a carbamate group, and/or an NH group. In a further preferred embodiment of the present invention, L3 is an alkyl group having 1-10 carbon atoms, which optionally contains an amide group, a carbonyl group, a carbamate group, and/or an NH group. For example, one L3 can be -NH- _CH2 - _CH2NHCO - _CH2 - _CH2- , as in the compound biotin-PEG2000-Lys-(C18) ₂ of the present invention.

In a further preferred embodiment of the present invention, the linear lipid is

(a) saturated or unsaturated fatty acids, and/or

(b) Fatty acids having 8 to 26 C atoms, preferably 12 to 22 C atoms, more preferably 14 to 18 C atoms.

Fatty acids are carboxylic acids with long aliphatic tails (chains) that are either saturated or unsaturated. Most naturally occurring fatty acids have chains with an even number of carbon atoms (4-28).

Examples of saturated fatty acids are caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, lignoceric acid and cerotic acid.

Examples of suitable unsaturated fatty acids are:

In an even more preferred embodiment, the linear lipid is selected from oleic acid, myristic acid, stearic acid and behenic acid, more preferably from myristic acid and oleic acid.

The hydrophobic vitamin is a small molecule selected from vitamins A, D, E, and K. In a more preferred embodiment, the hydrophilic vitamin is α-tocopherol. An exemplary compound for use in the present invention comprising α-tocopherol is 5′-α-tocopherol TEG-PEG2000-Fluos-3′.

In a further preferred embodiment, a steroid may be used as the hydrophobic moiety.

Steroids are a class of organic compounds containing a characteristic arrangement of four cycloalkane rings linked together. The core of a steroid is composed of seventeen carbon atoms bonded together in the form of four fused rings: three cyclohexane rings (designated Rings A, B, and C) and one cyclopentane ring (Ring D). Steroids differ by the functional groups attached to this four-ring core and by the oxidation state of the rings. Sterols are a specific form of steroid, possessing a hydroxyl group at position 3 and a backbone derived from cholestane.

In a further preferred embodiment of the present invention,

(a) the steroid is a sterol, or

(b) steroids selected from cholesterol; steroid hormones, preferably gonadal steroids, more preferably androgens, such as anabolic steroids, androstenedione, dehydroepiandrosterone, dihydrotestosterone, or testosterone, estrogens, such as estradiol, estriol, or estrone; progestogens, such as progesterone or progestine, corticosteroids, in particular glucocorticoids or mineralocorticoids; ecdysteroids, such as ecdysterone; phytosterols; brassinosteroids; hopanoids; and ergosterol,

More preferably the steroid is cholesterol, or

(c) The hydrophobic vitamin is α-tocopherol.

In a further preferred embodiment of the present invention, two, three or four, preferably two or three, hydrophobic domains are covalently bound to the hydrophilic domain.

In a further preferred embodiment of the present invention, the two or more hydrophobic domains covalently bound to the hydrophilic binding moiety are different or identical.

In a further preferred embodiment of the present invention, the hydrophobic domain consists of a linear lipid, a steroid or a hydrophobic vitamin.

In a further preferred embodiment of the present invention, the hydrophobic domain comprises (preferably consists of) a linear lipid, steroid or hydrophobic vitamin covalently bound to the trifunctional moiety A1 via a linker moiety L2.

Such bifunctional and trifunctional moieties are successfully employed in the compounds used according to the invention for binding the hydrophobic moiety directly or via a linker L2.

Linker L2 is independently any linker moiety suitable for covalently binding a hydrophobic moiety to a hydrophilic moiety and having a length of 50, 30 or 20 atoms or less between the hydrophobic moiety and A1 or A2, respectively.

In a preferred embodiment, the linker L2 comprises, preferably consists of, a phosphate group, a moiety -[O- _CH2 - _CH2 ] _y2- (SP) _n ] _m1- , wherein SP and n are as defined above, preferably n=0, y2 is an integer from 1 to 30, preferably 3 to 10, and m1 is an integer from 1 to 10, preferably 1 to 3, a glycerol moiety, a carbamate group, an amide group, a linear alkyl group having 1 to 10 C atoms, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms, and the alkyl chain comprises a functional group at the terminal C atom, in particular independently selected from amine, carbonyl, hydroxyl, thiol, carbonate group, the alkyl group being optionally substituted by 1, 2, 3, 4 or 5 moieties R1, wherein R1 is independently a C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl, hydroxyl, thiol, amino or carbonyl moiety. Examples of suitable linear alkyl groups with terminal functional groups are diaminoalkyl moieties such as H ₂ N—(CH ₂ ) ₅ —NH ₂ or hydroxy-carbonyl moieties such as —C(O)—(CH 2 ) 4 —O—. Preferably, the linear alkyl group is unsubstituted. Even more preferably, the linear lipid, steroid or hydrophobic vitamin is bound to the trifunctional moiety A1 via a linker moiety —(O—CH 2 —CH 2 ) j —, wherein j is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably j is 3, in particular tetraethylene glycol (TEG), a phosphate moiety or a moiety comprising TEG, glycerol and a phosphate moiety, or a moiety comprising or consisting of —TEG-glyceryl-phosphate-O—(CH ₂ ) ₄ —C(O)—.

In a more preferred embodiment, the compounds used in the present invention comprise a linear lipid, steroid or hydrophobic vitamin covalently bound to a trifunctional moiety A1 via a linker moiety L2, preferably wherein L2 is selected from a phosphate, amide, carbamate, ester group or a moiety

- [O-CH ₂ -CH ₂ ]y ₂ - (SP) _n ] _m1 -,

in

SP and n are as defined above, preferably n=0,

y2 is an integer of 1-30, preferably 3-10, and

m1 is an integer of 1-10, preferably 1-3,

More preferably, wherein the linear lipid, steroid or hydrophobic vitamin is bound to the trifunctional moiety A1 via a linker moiety tetraethylene glycol (TEG) or phosphate.

In a further preferred embodiment of the present invention, k1 is 1, 2, 3, 4 or 5, preferably 1, 2 or 3.

In particularly preferred embodiments of the present invention, the hydrophobic domain is covalently bound to the hydrophilic domain only via the trifunctional moiety A1 or via the domain X1-[A1-(L1) _n ] _k1 described above. Further preferred embodiments of the compounds used according to the present invention also apply to these embodiments. In such compounds, the hydrophobic domain is located exclusively at one terminal portion of the molecule, while other groups, such as a linker or labeling moiety (if present), are located at the other terminal portion, spatially separated therefrom.

In a further preferred embodiment of the present invention, k1 is 1, 2, 3, 4, 5 or 6, preferably 1, 2 or 3.

In case the compounds used in the present invention comprise a dT moiety as the bifunctional moiety A2, k2 is preferably 3, 4, 5 or 6.

In another preferred embodiment of the invention, k1 is 0 and X1 is replaced by a hydrophobic domain, preferably comprising a steroid, more preferably cholesterol. In a particularly preferred embodiment, Z is the moiety -(L3) _n2 -TEG(L3) _n2- , wherein n2 is independently 0 or 1. In an even more preferred embodiment of the invention, k2 is 1, 2, 3, 4, 5, or 6, preferably 3, 4, 5, or 6. One or more (particularly one) further hydrophobic moieties are combined with the moiety -[A2-(L1)n]k2-X2, wherein the further hydrophobic moiety comprises a steroid, more preferably cholesterol. Even more preferably, L2 is a linker moiety tetraethylene glycol (TEG), a phosphate, or a moiety comprising TEG, glycerol, and a phosphate moiety, or a moiety comprising or consisting of -TEG-glyceryl-phosphate-O-(CH2)4-C(O)-. An exemplary compound used in the present invention is Chol-TEG-Chol-TEG-Doubler-Biotin-dT shown in FIG12.

In case the compounds used in the present invention comprise a dT moiety as the bifunctional moiety A2, k2 is preferably 3, 4, 5 or 6.

In a further preferred embodiment of the present invention, the compound used according to the present invention further comprises a labeling moiety and/or a linking group.

In a still even further preferred embodiment of the present invention, the compound further comprises a labeling moiety.

Such compounds may furthermore be used for cell labelling purposes.An exemplary compound for use in the present invention is 5'-cholesterylTEG-cholesterylTEG-PEG2000-Fluos-3'.

In one such preferred embodiment, the compound does not further comprise a linking group.

In another even further preferred embodiment of the present invention, the compound further comprises a linking group. An exemplary compound is 5'-cholesterylTEG-cholesterylTEG-PEG2000-biotinTEG-3.

In a preferred embodiment, the compound does not further comprise a labeling moiety.

In another more preferred embodiment of the present invention, the compound further comprises a labeling moiety and a linking group.

Such compounds are particularly suitable for applications where, in addition to stabilization, both cell immobilization and detection are desired, such as for localization of fixed cells and/or for cell quantification. An example of such a compound is 5'-(cholesteryl-TEG)2-PEG2000-Fluos-biotin-TEG-3', which was successfully used to immobilize cells to streptavidin-coated plates and detect these cells.

Suitable labeling moieties are moieties that are suitable for in vitro detection and are known to the skilled person. Detection can be direct (as in the case of luminescence, particularly fluorescence) or indirect (as in the case of enzymes or their substrates). Therefore, labeling moieties that are suitable for indirect or indirect detection can be used.

As used herein, "label" or "labeling moiety" refers to any substance capable of generating a signal for direct or indirect detection. Thus, the labeling moiety can be detected directly or indirectly. For direct detection, labeling moieties suitable for use in the present invention can be selected from any known group of detectable labels, such as chromogens, chemiluminescent groups (e.g., acridinium esters or dioxetanes), electrochemiluminescent compounds, dyes or fluorescent dyes (e.g., fluorescein, coumarins, rhodamines, oxazines, resorufins, cyanines and derivatives thereof), luminescent metal complexes, such as ruthenium or europium complexes, and radioisotopes.

In an indirect detection system, the first partner of the bioaffine binding pair is the labeled portion of the compound used in the present invention; that is, the portion to which the first partner covalently binds to the compound used in the present invention. Examples of suitable binding pairs are haptens or antigens/antibodies, biotin or biotin analogs such as aminobiotin, iminobiotin or desthiobiotin/avidin or streptavidin, sugars/lectins, nucleic acids or nucleic acid analogs/complementary nucleic acids, and receptors/ligands, such as steroid hormone receptors/steroid hormones. Preferred first binding pair members include haptens, antigens, and hormones. Also preferred are haptens such as labels, digoxin, and biotin and their analogs. The second partner of such binding pairs, such as antibodies, streptavidin, etc., is typically labeled to allow direct detection, for example, by a labeling moiety as described above.

Therefore, in a preferred embodiment, the labeling moiety is a labeling moiety for direct labeling or for indirect labeling.

In a preferred embodiment, the labeling moiety is selected from (a) a direct labeling moiety selected from the group consisting of a chromogen, a chemiluminescent group (e.g. an acridinium ester or a dioxetane), an electrochemiluminescent compound, a dye or a fluorescent dye (e.g. fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and its derivatives), a luminescent metal complex such as a ruthenium or europium complex and a radioactive isotope; (b) or one of the partners of an indirect detection system, preferably wherein the labeling moiety is one of the members of a binding pair selected from the group consisting of: (i) hapten or antigen/antibody, (ii) biotin or a biotin analogue such as aminobiotin, iminobiotin or desthiobiotin/avidin or streptavidin, (iii) sugar/lectin, (iv) nucleic acid or nucleic acid analogue/complementary nucleic acid, and (v) receptor or receptor fragment/ligand, e.g. a steroid hormone receptor/steroid hormone.

Preferred first binding pair members that are labeling moieties suitable for indirect detection include haptens, antigens, and hormones. Also preferred are haptens such as digoxigenin and biotin and their analogs. The second partner of such a binding pair, such as an antibody, streptavidin, etc., is typically labeled to allow direct detection, for example, by a direct labeling moiety as described above; however, it is also possible to utilize antibodies in the compounds used in the present invention and to use labeled antigens or haptens for detection.

In the above description of binding pair members, the term antibody is understood to cover both antibodies and antigen-binding fragments thereof.

In a preferred embodiment, the labeling moiety is a labeling moiety for direct labeling, and even more preferably, the labeling moiety is a fluorescent moiety or a dye.

Suitable fluorescent moieties (or dyes) are known in the art and include fluorescein, Cy3, Cy5, Cy5.5, Cy2, Cy3.5, Cy3b, Cy7, Alexa Fluor dyes, xanthene derivatives such as rhodamine, Oregon green, eosin, or Texas red, cyanine derivatives such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine, naphthalene derivatives such as dansyl and sodium fluorosilicate derivatives, coumarin derivatives, oxadiazole derivatives such as pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole, pyrene derivatives such as cascade blue, oxazine derivatives such as Nile Red, Nile Blue, cresyl violet, oxazine 170, acridine derivatives such as proflavine, acridine orange, acridine yellow, arylmethine derivatives such as auramine, crystal violet, malachite green, tetrapyrrole derivatives such as porphine, phthalocyanine, and bilirubin.

In the examples, fluorescein is used as a representative label. This allows for sensitive detection of the label, allowing for localization and/or quantification of the label. Fluorescent labels are particularly preferred labeling moieties of the present invention.

Suitable radioactive isotopes or radioisotopes for labeling and methods of labeling the compounds used in the present invention with such radioactive labels are known to the skilled person. For example, one of the following isotopes may be used: ¹⁴ C, ³ H, ³² P, ³³ P, ¹²³ I, ¹²⁵ I and ¹³¹ I.

In the case where an antibody or antigen-binding fragment is used as a member of the indirect system antibody/antigen or hapten, the antibody or antigen-binding fragment specific for the epitope or hapten can be part of the compound used in the present invention, or the epitope or hapten can be part of the compound used in the present invention. Thus, each other member can be directly labeled, for example, with a fluorescent label for subsequent detection. Suitable antibodies or antigen-binding fragments are described in more detail below.

In a preferred embodiment of the present invention, the linker and/or label binding moiety is [A2-(L1) _n ] _k2 -X2.

In a particularly preferred embodiment of the present invention, the hydrophobic domain is covalently bound to the hydrophilic domain only via the trifunctional moiety A1 (or via the domain X1-[A1-(L1) _n ] _k1 as described above), and the linker and/or labeling moiety is bound to the moiety [A2-(L1) _n ] _k2 -X2. This ensures that the hydrophobic domain that inserts into the cell membrane and the moiety for immobilization and/or labeling (if present) are spatially separated.

Such compounds, in addition to being stable, are also suitable for immobilization in the presence of a linking group.

Such compounds, in addition to being stable, are suitable for labeling and detection in the presence of a labeling moiety.

In a further particularly preferred embodiment of the present invention, the hydrophobic domain is covalently bound to the hydrophilic domain only via the trifunctional moiety A1 (or via the domain X1-[A1-(L1) _n ] _k1 as described above), and the linker and labeling moiety are bound to the moiety [A2-(L1) _n ] _k2 -X2.

Such compounds, in addition to stabilization, further allow both immobilization and labeling, detection and quantification.

In a still further particularly preferred embodiment of the present invention, the hydrophobic domain is covalently bound to the hydrophilic domain only via the trifunctional moiety A1 (or via the domain X1-[A1-(L1) _n ] _k1 as described above) and the linker group, instead of the labeling moiety, is bound to the moiety [A2-(L1) _n ] _k2 -X2.

Such compounds can be used if only fixation or only labeling, detection and/or quantification of the bound cells is intended in addition to stabilization.

A linking group is a moiety suitable for reversibly or irreversibly and/or covalently or non-covalently fixing a compound to a support, particularly a solid support. In a preferred embodiment, the linking group is an antibody or antigen-binding antibody fragment, a receptor or its binding site, a ligand for a receptor, an enzyme or its binding site, a substrate for an enzyme, a tag binding site, a tag, or a functional chemical group.

The functional chemical group may be, for example, a thiol group, which can be bound to the gold-coated substrate surface by forming a covalent, irreversible -S-S- bond.

The binding of biotin to streptavidin or the antibody or antigen-binding antibody fragment is non-covalent and reversible. Such linkers utilizing non-covalent binding to a solid support are preferred when—in addition to stabilization—the intention is to separate the cells again for further use, for example for administration in an animal model.

In preferred embodiments, the linking group can be, for example, a biotin moiety that allows non-covalent binding to a streptavidin-coated surface, or a thiol group that can bind to a gold-coated substrate surface as a solid support.

In an even more preferred embodiment of the present invention, the compound for use according to the present invention comprises a labeling moiety and/or a linking group, wherein said labeling moiety is a fluorescent label and/or said linking group is biotin.

In an even more preferred embodiment, the compound used in the present invention comprises a labeling moiety and a linking group, wherein the labeling moiety is a fluorescent label and the linking group is biotin.

In an even more preferred embodiment, the compounds used in the present invention comprise a linker group which is biotin.

The term "solid support" refers to a material in a solid phase that interacts with a reagent in a liquid phase through a heterogeneous reaction. The use of solid supports is well known in the fields of chemistry, biochemistry, pharmacy, and molecular biology. Many types of solid supports have been developed based on the technical problems to be solved. Any of these can be used in the context of the present invention. For example, the solid support can include the following components: silica, cellulose acetate, cellulose nitrate, nylon, polyester, polyethersulfone, polyolefin, or polyvinylidene fluoride, or a combination thereof. Further suitable solid supports include, but are not limited to, controlled pore glass, glass plates or slides, polystyrene, and activated dextran. In other aspects, synthetic organic polymers such as polyacrylamide, polymethacrylate, and polystyrene are also illustrative support surfaces. In addition, polysaccharides such as cellulose and dextran are further illustrative examples of support surfaces. Other support surfaces such as fibers are also feasible.

The solid support can be contained in a container, wherein the container is a tube, such as a centrifuge tube or spin tube, a syringe, a cartridge, a chamber, a multi-well plate, or a test tube, or a combination thereof. The solid support can be pretreated or functionalized to allow cell fixation. For example, the well plate can be pretreated with streptavidin, as shown in the examples. In one embodiment, the solid support can be fibrous or microparticles, usually allowing suitable contact. The size of the solid support suitable for use can be changed. The cell can only be combined with one solid support (such as a container or multi-well plate) or can be combined with many solid supports (such as beads). The shape of the solid support suitable for use can be, for example, a sheet, a pre-cut disk, a cylinder, a single fiber, or a solid support composed of microparticles. In a preferred embodiment, the solid support is flat or substantially flat with a cavity. In one embodiment, the solid support can be fibrous or microparticles. The size of the solid support can be changed and can be selected according to the method to be carried out or application.

In some embodiments, the solid phase is a test strip, a chip, particularly a microarray or nanoarray chip, a microtiter plate, or microparticles.

In a more preferred embodiment, where present, the labeling moiety and/or linking group is covalently attached via the trifunctional moiety A2, as described above.

In another embodiment, one or more moieties A2 are bifunctional or trifunctional labeling moieties or linking groups, more preferably moiety A2 is a moiety comprising a nucleobase, and even more preferably moiety A2 is dT (thymidine). Such compounds comprising dT are used for concentration determination of the compound.

In a further preferred embodiment of the present invention, the linker L1 is independently selected from phosphate, amide, carbamate and ester groups.

In a further preferred embodiment of the present invention, the moieties A1 and A2 are independently selected from difunctional groups selected from the group consisting of phosphate groups, carbamate groups, amide groups, moieties comprising nucleobases (even more preferably dT), and straight-chain alkyl groups having 1-10 C atoms (and the alkyl chain comprising a functional group at the terminal C atom, in particular independently selected from amine, carbonyl, hydroxyl, thiol, carbonate groups), and trifunctional moieties having 1-10 C atoms and comprising at least one -OH, -SH and/or at least one -NH ₂ group, preferably selected from lysine, serine, serinol, -O-CH ₂ -CH((CH ₂ ) ₄ -NH ₂ )-CH ₂ -, glycerol, and 1,3 diaminoglycerol moieties.

In a further more preferred embodiment of the present invention, the linker L2 is independently selected from phosphate, amide, carbamate, ester group and part

- [O-CH ₂ -CH ₂ ] _y2 - (SP) _n ] _m1 -,

in

SP and n are as defined above, preferably n=0,

y2 is an integer of 1-30, preferably 3-10, and

m1 is an integer of 1-10, preferably 1-3.

It is shown that a PEG-based linker, namely a TEG linker, can be used in exemplary compounds used in the present invention. The exemplary compound is 5'-cholesterylTEG-cholesterylTEG-(Spacer C18)7-Fluos-BiotinTEG-3'.

The compounds used in the present invention and their intermediates can be prepared by methods known to those skilled in the art. An exemplary synthesis of the compounds used in the present invention is shown in Figure 6C. Additionally, intermediates used in the synthesis of the compounds used in the present invention are shown in Figure 12. Furthermore, the general concept of compound synthesis is briefly described in Example 1. Compounds can be prepared on a solid phase similar to the phosphoramidite-based synthesis of nucleotides. Compounds can be synthesized by synthesis on a solid support, such as CPG, as described in the Examples. Specifically, compounds can be synthesized by a subsequent coupling step under conditions known to those skilled in the art and cleavage from the solid support (in this example, CPG (controlled pore glass)). Other solid supports, such as macroporous polystyrene, can also be used for synthesis. Synthesis can be performed with or without the protecting groups. Specifically, compounds can be synthesized with or without DMT (DMT on), leaving the DMT molecule at the terminus of the molecule, known as the 3' end, or by cleaving the DMT group. The compounds can optionally be further purified, for example, by dialysis.

The synthesis of biotin-PEG-Lys-(C18)2 is described in detail in Figure 6C).

Other compounds used in the present invention can be prepared in a similar manner according to methods known in the art.

In still further embodiments, the present invention relates to compositions comprising at least one compound as described above that binds to at least one cell, preferably a living cell. Such compositions are provided for fixed cells. Depending on the further presence of a labeling moiety and/or a linking group, the compositions can further be used for detection and/or fixation of cells, respectively.

In a preferred embodiment, such compositions further comprise a solid support to which at least one compound used in the present invention is bound via a linker. In such embodiments, at least one fixed cell is immobilized on a solid support via a compound used in the present invention. Where the compound further comprises a labeling moiety, localization, detection, and quantification of cells is possible.

In another preferred embodiment, the composition comprising at least one compound used in the present invention that binds to at least one cell comprises an aqueous buffer solution, wherein the at least one cell to which the at least one compound used in the present invention binds is suspended. Such a composition is suitable for adequate stabilization of the cell, for example, during FACS or centrifugation.

The compounds are suitable for binding to any cell comprising a lipid bilayer. Preferably, the cell is a eukaryotic cell, more preferably an animal cell, even more preferably a vertebrate cell, most preferably a human cell.

In a further preferred embodiment, the cell is a leukocyte, a rare cell, a tumor cell or a mutant cell, more preferably a vertebrate or human leukocyte, a rare cell, a tumor cell or a mutant cell.

In a further embodiment, the invention relates to the use of a composition comprising one or more compounds as described above for stabilizing cells.

Therefore, in a further embodiment, the present invention relates to the use of a composition comprising at least three different compounds which can be used according to the invention for stabilizing cells, wherein the different compounds differ at least in their hydrophobic domain.

By using multiple compounds for use in the present invention, at least two of which differ in their hydrophobic domains, compositions can be obtained that effectively bind to and stabilize all cell types.

In an even more preferred embodiment, the composition thus comprises at least four, five, six, seven, eight, nine or ten different compounds for use according to the invention. In an even more preferred embodiment, two, three, four, five, six, seven, eight, nine, ten or all of the compounds of such a composition differ at least in their hydrophobic domain.

Suitable preferred hydrophobic domains are those defined above.For example, a composition comprising 5'-cholesterylTEG-cholesterylTEG-PEG2000-Fluos-3' may be used.

In a more preferred embodiment, one or all hydrophobic domains of at least one compound comprise, preferably consist of, a saturated fatty acid, in particular myristic acid, stearic acid or behenic acid, in particular myristic acid; and/or one or all hydrophobic domains of at least one compound comprise, preferably consist of, a steroid, in particular cholesterol, or a hydrophobic vitamin, in particular α-tocopherol.

In a preferred embodiment, the present invention relates to the use of an aqueous solution comprising one or more compounds for stabilizing cells.

The aqueous solution is preferably buffered. For example, the solution of the present invention may be phosphate buffered saline (PBS), Tirs, and/or Hepes buffer.

The pH of the solution of the present invention is preferably about 5.5-8.5, more preferably 6.5-7.5.

In a further embodiment, the present invention relates to a method of stabilizing cells, the method comprising:

a) providing a compound as defined above; and

b) contacting the cell with the composition under conditions that allow the compound to interact with the cell membrane, thereby stabilizing the cell, and

c) Optionally, applying shear forces to the cells.

In a preferred embodiment of the invention, the stabilization is stabilization during exposure of the cells to shear forces.

In a further preferred embodiment, the stabilization is stabilization of the cells during exposure to shear forces due to centrifugation, large-scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead-based cell separation.

In a further preferred embodiment, the stabilization is stabilization of the cells upon exposure to centrifugation, large-scale cell culture, flow cytometry, fluorescence activated cell sorting and/or bead-based cell separation procedures.

With respect to the compounds useful in such methods, the same embodiments as described above with respect to the uses apply.Providing compounds to be used in the methods of the invention is described above.

Compositions comprising two or more compounds as described above may also be used in the methods of the invention. Such compounds and their use according to the invention are described above.

The compound can be contacted with the cells in step b) as an aqueous solution containing one or more compounds used in the present invention. Such solutions are suitable for pipetting or other methods of addition to the cells. The aqueous solution is preferably buffered. For example, the solution of the present invention can be phosphate buffered saline (PBS), Tis, and/or Hepes buffer, or a solution containing culture medium. The pH of the solution is preferably about 5.5-8.5, more preferably 6.5-7.5.

Since stabilizing cells is done with living cells or potentially living cells, the cells are typically present in an aqueous solution (which is preferably buffered and/or contains nutrients), for example, the cells are suspended in PBS or culture medium. The compounds used in the present invention can be added to the cells, for example, in the form of a solution, for example as an aqueous solution, by methods known in the art, such as pipetting.

The pH of the cell suspension is preferably about 5.5-8.5, more preferably 6.5-7.5.

Gentle mixing may be performed to maintain cell viability.

The compound is contacted with the cells in step b) of the method of the present invention. Typically, more than one cell will be present and contacted with the compound of the present invention. Thus, the compound is preferably added to a composition comprising a plurality of cells, e.g., 2 or more, 10 or more, 50 or more, 100 or more, or 1000 or more cells. Preferably, such a cell population is a suspension cell. Thus, a solution containing a compound as disclosed herein can be added to a suspension of cells to be stabilized.

The cell population can be cells of the same or different cell types. For example, a white blood cell population comprising different cell types can be used, as in the examples (see Example 5).

Typically, contacting occurs at a temperature of about 1°C to 45°C, preferably 10°C to 30°C, and more preferably 22°C to 38°C.

Furthermore, contact typically occurs at a pressure of approximately 900-1100 mbar to maintain cell viability.

Furthermore, the cells are preferably incubated with the compound for a sufficient time to allow binding to the cells. Typically, the cells are preferably incubated with the compound for 1 minute to 3 days, preferably 5 minutes to 24 hours, even more preferably 10 minutes to 8 hours.

Furthermore, the aqueous solution is typically selected so as not to affect the integrity and/or viability of the cells. Thus, the solution preferably does not contain cytotoxic compounds.

Such conditions allow interaction of the compound with the cell membrane, thereby stabilizing the cell.

The stabilization occurs already at the time of interaction, without further application of shear stress.Thus, in one embodiment, no shear stress is applied to the cells after step b) of the method of the invention.

However, in a preferred embodiment, after step b) of the method of the invention, shear forces or shear stress are applied to the cells.

In a preferred embodiment of the cells, shear forces are applied to the cells by centrifugation, large-scale cell culture, flow cytometry, fluorescence-activated cell sorting, and/or bead-based cell separation, as described above. Thus, in a preferred embodiment, after step b) of the method of the present invention, the cells are centrifuged, cultured on a large scale, subjected to flow cytometry, subjected to fluorescence-activated cell sorting, and/or separated using beads.

In a still further preferred embodiment of the present invention, the cells are suspended cells and/or the cells are animal or human cells, in particular vertebrate cells, especially mammalian cells. Such cells are particularly sensitive to shear forces and are therefore difficult to handle and manipulate without affecting viability. Even more preferably, the cells are suspended animal or human cells, in particular suspended vertebrate cells, especially suspended mammalian cells.

In an even more preferred embodiment, the cell or cell population is a leukocyte or a plurality of leukocytes, respectively, which are even more preferably human leukocytes.The method of the present invention is effective in stabilizing such cells (Example 5).

The method of the present invention is performed during centrifugation for cell stabilization.

For example, such a centrifugation step is performed to separate cells from surrounding liquids, such as culture medium. The cells must be centrifuged and thus exposed to shear stress. Very sensitive and fragile cell populations may be damaged by such a process. The methods and uses of the present invention improve the handling of such cell populations. In a preferred embodiment, the cells are centrifuged after step b) of the method of the present invention. Preferably, they are centrifuged, for example, at 100g, 200g, 500g, 1000g or higher for 5 minutes or more, for example 1 hour or 5 hours.

The method of the invention may also be used in biotechnology, for example for cell stabilization in large-scale animal cell cultures: It has been revealed that shear sensitivity of mammalian cells may be a relevant problem complicating the development of large-scale animal cell cultures. The method and use of the invention reduces these problems.

Therefore, the present invention also relates to the method according to the invention for stabilizing cells in large-scale animal cell culture. In a preferred embodiment, after step b) of the method according to the invention, the cells are cultured on a large scale, for example by batch culture and/or in a volume of liquid culture medium exceeding 10 ml, 50 ml, 100 ml or 1 l.

The method of the present invention can also be used for cell stabilization in flow cytometry and/or fluorescence activated cell sorting:

Flow cytometry is a very common method for isolating specific cell populations. In this method, cells are exposed to high shear stress depending on the flow rate. The method of the present invention reduces this shear stress.

Therefore, the present invention also relates to the method of the invention for stabilizing cells during flow cytometry and/or fluorescence activated cell sorting or wherein the cells are exposed to flow cytometry and/or fluorescence activated cell sorting after step b) of the method of the invention.

The methods of the present invention can also be used for cell stabilization in bead-based cell separation methods:

Cell populations with different phenotypes can be separated by specific antibodies coupled to magnetic beads. In this method, cells are exposed to high shear stress depending on the bead size. The method of the present invention reduces this shear stress.

Thus, the method of the invention can also be used for cell stabilization in bead-based cell separation methods.In a preferred embodiment, after step b) of the method of the invention the cells are coupled to beads, especially magnetic beads and separated, especially magnetically.

In a still further embodiment, the present invention relates to the use of a kit comprising at least one compound or composition as described above for stabilizing cells.

The kit may further comprise two or more compounds for use in the present invention stored separately (e.g., in containers or syringes). They may be stored in a dry form, e.g., lyophilized or dry, or as a solution, or in a frozen form, e.g., as a frozen solution.

In case the compound further comprises a labeling moiety and/or a linking group, the stable cells to which the compound is bound can furthermore be used for the detection and/or characterization of rare cells, preferably for a rare cell characterization.

In such applications, nucleated cells separated from leukocytes can be immobilized on a defined surface using a compound used in the present invention on an array, preferably a microarray or nanoarray. Rare cells within this population of nucleated cells, such as white blood cells (WBCs), such as circulating tumor cells, endothelial cells, or epithelial cells, can be quantitatively bound to the surface and identified using antibodies or specific binding molecules directed against antigens or biochemical properties specific to the rare cell population. This allows for accurate localization and relocalization for further characterization steps, if necessary.

Compounds further comprising a linking group can be further used to immobilize suspended cells, preferably for screening, or even more preferably for screening with antibodies or antigen-binding antibody fragments or other forms of binding molecules. Screening of cultured cell lines with antibodies or their antigen-binding fragments is a common application in antibody development. One application includes antibodies binding to specific receptor molecules on the cell surface. Using a secondary antibody (sandwich effect), the characteristics of the primary antibody can be studied. Such experiments are difficult to perform using suspended cells. The developed compounds for cell fixation allow careful fixation of suspended cells without losing any physiological cell properties and can therefore be used to perform such screening assays. In addition, using the compounds used in the present invention, suspended cells can be fixed for functional cell assays. In vitro or in vivo assays for studying cell function are important: functional cell assays are commonly used in pharmaceutical, pesticide, and biotechnology research and development to study small molecule compounds or biologics or to identify the class of small molecules using high-throughput screening. Some functional assays are based on surface-dependent assays and are therefore generally performed using adherent cells.

In a preferred embodiment, the uses and methods of the present invention are in vitro uses and methods.

Compounds further comprising a linker group can be further used to bind living cells to a solid surface, which can then be detached from the surface and implanted into mouse models. These types of functional assays are particularly important, for example, for studying the tumor-inducing potential of circulating abnormal cells.

The compounds can further be used in a lab on a chip: To study the morphology or function of a small number of cells, such as 2-50 cells or a single cell, a surface can be selectively or systematically spotted with a compound used in the present invention that further comprises a linker. This spotting allows for the targeted immobilization of a small number of cells or a single cell on such a spot. This allows for direct molecular analysis on the surface (chip). The chip can be an array, in particular a microarray or nanoarray.

The compound further comprising a linking group used in the present invention can be combined with a solid substrate. Such a solid substrate can be a particle such as a nanoparticle, particularly a magnetic nanoparticle, a column, or a flat substrate, an array or a well plate, particularly an oligo-well plate or a multi-well plate.

Disclosed is a method of labeling a cell, the method comprising:

a) providing a compound for use in the present invention, wherein the compound comprises a labeling moiety; and

b) contacting the cell with the compound under conditions that allow the compound to interact with the cell membrane, thereby fixing the label on the cell; and

c) optionally detecting the label.

As shown in the Examples, a compound used in the present invention (wherein the compound comprises a labeling moiety) is contacted with cells. Since labeling is preferably performed using living cells or potentially living cells, the cells are typically present in an aqueous solution (which is preferably buffered and/or contains nutrients), for example, the cells are suspended in PBS. The labeled compound used in the present invention can be added to the cells in the form of a solution, for example, as an aqueous solution, by methods known in the art, such as pipetting.

Typically, contacting occurs at a temperature of about 1°C to 45°C, preferably 10°C to 30°C, more preferably 22°C to 38°C.

Furthermore, contact occurs at a pressure of approximately 900-1100 mbar to maintain cell viability.

Furthermore, the cells are preferably incubated with the compound for a sufficient time to allow binding. Typically, the cells are preferably incubated with the compound for 1 minute to 3 days, preferably 5 minutes to 24 hours, even more preferably 10 minutes to 8 hours.

Furthermore, the aqueous solution is generally selected so as not to affect the integrity and/or viability of the cells.

Such conditions allow the compound to interact with the cell membrane, thereby immobilizing the labeled moiety on the cell.

The labeling moiety and thus the cells can be detected as described above, depending on the labeling moiety chosen. In the case of direct labeling, detection can occur directly, for example by detecting the fluorescence of fluorescein or the absorption of dT, as shown in the examples.

In the case of an indirect detection system, the second member of the binding pair can be detected. For example, biotin-labeled compounds used in the present invention can be used. For detection, streptavidin can be used, which is then labeled with a directly detectable label. Therefore, depending on the further steps, biotin can represent a linking group or a labeling moiety of the present invention.

For cell-independent labeling, certain compounds of the invention described above may be used.

A method for labeling cells is disclosed, the method comprising

a) providing a composition comprising at least three different compounds for use according to the invention, wherein the different compounds differ at least in their hydrophobic domain and wherein the different compounds comprise a labeling moiety,

b) contacting the cell with the composition under conditions that allow the compound to interact with the cell membrane, thereby labeling the cell; and

c) optionally detecting the label.

Thus, the composition is preferably a solution, more preferably an aqueous solution comprising the compound used in the present invention.

A method for immobilizing a linker on a cell surface is disclosed, the method comprising

a) providing a compound for use in the present invention, wherein the compound comprises a linking group; and

b) contacting the cell with the compound under conditions that allow the compound to interact with the cell membrane, thereby immobilizing the linker group.

With respect to antibodies and antigen-binding antibody fragments, the skilled artisan is aware of such molecules: naturally occurring antibodies are globular plasma proteins (~150 kDa (http://en.wikipedia.org/wiki/Dalton_unit)), also known as immunoglobulins, which have a basic structure. They are glycoproteins because they have sugar chains attached to amino acid residues. The basic functional unit of each antibody is the immunoglobulin (Ig) monomer (comprising only one Ig unit); secreted antibodies can also be dimerized with two Ig units, such as IgA, tetramerized with four Ig units, such as bony fish IgM, or pentameric with five Ig units, such as mammalian IgM. In the present invention, examples of suitable forms include forms of naturally occurring antibodies, including the antibody isotypes known as IgA, IgD, IgE, IgG, and IgM.

In addition to naturally occurring antibodies, artificial antibody forms including antibody fragments have also been developed, some of which are described below.

While the general structure of all antibodies is very similar, the unique properties of a given antibody are determined by its variable (V) regions, as detailed above. More specifically, the variable loops (three each on the light (VL) chain and three on the heavy (VH) chain) are responsible for antigen binding and, therefore, for its antigenic specificity. These loops are called complementarity-determining regions (CDRs). Because CDRs from both the VH and VL domains form the antigen-binding site, it is the combination of the heavy and light chains, not either alone, that determines the ultimate antigenic specificity.

Thus, the term "antibody" as used herein means any polypeptide that has structural similarity to naturally occurring antibodies and is capable of specifically binding to each target, wherein the binding specificity is determined by the CDRs. Thus, "antibody" is intended to refer to immunoglobulin-derived structures that bind to each target, including but not limited to full-length or intact antibodies, antigen-binding fragments (fragments derived from (physically or conceptually) antibody structures), derivatives of any of the former, chimeric molecules, fusions of any of the former with another polypeptide, or any optional structure/composition that selectively binds to each target. An antibody or a functionally active portion thereof may be any polypeptide comprising at least one antigen-binding fragment. An antigen-binding fragment consists of at least a heavy chain variable domain and a light chain variable domain, arranged in such a way that both domains are capable of binding to a specific antigen simultaneously.

A "full-length" or "complete" antibody is a protein comprising two heavy (H) and two light (L) chains interconnected by disulfide bonds, comprising: (1) on the heavy chain, a variable region and a heavy chain constant region comprising three domains, CH1, CH2, and CH3; and (2) on the light chain, a light chain variable region and a light chain constant region comprising one domain, CL.

"Antigen-binding antibody fragments" or "antigen-binding fragments thereof" also include at least one antigen-binding fragment as defined above and exhibit substantially the same function and binding specificity as the complete antibody from which the functionally active portion (or fragment) is derived. Limited proteolytic digestion with papain cleaves the original Ig into three fragments. Two identical amino-terminal fragments (each containing a complete L chain and approximately half an H chain) are the antigen-binding fragments (Fab). The third fragment (of similar size but containing the carboxyl-terminal halves of both heavy chains with their interchain disulfide bonds) is the crystallizable fragment (Fc). The Fc contains carbohydrates, complement binding sites, and FcR binding sites. Limited pepsin digestion produces a single F(ab')2 fragment containing both the Fab segment and the hinge region, including the H-H interchain disulfide bond. The F(ab')2 is divalent for antigen binding. The disulfide bonds of the F(ab')2 can be cleaved to yield the Fab'. Furthermore, the variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment (scFv).

The variable domain (Fv) is the smallest fragment with a complete antigen-binding domain consisting of one VL and one VH. Such fragments, containing only the binding domain, can be produced enzymatically or by expressing the relevant gene fragments (e.g., in bacteria and eukaryotic cells). Different methods can be used, such as a single Fv fragment or a 'Fab' fragment containing one of the upper arms of the "Y," which includes the Fv plus the first constant domain. These fragments are often stabilized by introducing a polypeptide link between the two chains, resulting in a single-chain Fv (scFv). Alternatively, disulfide-linked Fv (dsFv) fragments can be used. The binding domain of the fragment can be combined with any constant domain to produce a full-length antibody or can be fused to other proteins and polypeptides.

Recombinant antibody fragments are single-chain Fv (scFv) fragments. Dissociation of scFv results in monomeric scFv, which can complex into dimers (diabodies), trimers (tribodies), or larger aggregates such as TandAbs and Flexibodies.

Antibodies with two binding domains can be generated by combining two scFvs with a simple polypeptide linker (scFv) or by dimerization of two monomers (diabodies). The simplest design is a diabody with two functional antigen-binding domains that can be identical, similar (bivalent diabodies), or specific for different antigens (bispecific diabodies).

In addition, antibody forms comprising four variable domains of heavy chain and four variable domains of light chain have been developed. Examples of these include tetravalent bispecific antibodies (TandAbs and Flexibodies, Affimed Therapeutics AG, Heidelberg.Germany). Flexibodies are combinations of scFv and double antibody polymer motifs, resulting in multivalent molecules with a high degree of flexibility for connecting two molecules very far from each other on the cell surface. If there are more than two functional antigen-binding domains and if they have specificity for different antigens, then the antibody is multispecific.

In general, specific immunoglobulin types representing antibodies or antigen-binding fragments thereof include, but are not limited to, the following antibodies: Fab (a monovalent fragment having a variable light chain (VL), a variable heavy chain (VH), a constant light chain (CL), and a constant heavy chain 1 (CH1) domain), F(ab')2 (a bivalent fragment comprising two Fab fragments linked by a disulfide bond or, optionally, at a hinge region), Fv (VL and VH domains), scFv (single-chain Fv in which VL and VH are linked by a linker, e.g., a peptide linker), bispecific antibody molecules (antibody molecules having specificity as described herein and linked to a second functional moiety having a different binding specificity than the antibody, the second functional moiety including, but not limited to, another peptide or protein such as an antibody or a receptor ligand), bispecific single-chain Fv dimers, diabodies, triabodies, tetrabodies, minibodies (scFv linked to CH3).

The antibody can be a monoclonal antibody, a chimeric antibody, or a humanized antibody.

Tags are peptide motifs used for identification in biotechnology. A well-known tag is the His tag (6x histidine), which can bind to Ni ²⁺ columns.

In case nucleic acids or nucleic acid analogs/complementary nucleic acids are used as binding pairs, any nucleic acid sequence and its complement can be used.

Lectins are carbohydrate-binding proteins that are highly specific for sugar moieties. As a suitable lectin, concanavalin A can be used, which binds to α-D-mannosyl and α-D-glucosyl residues, branched α-mannosidic structures (high α-mannose type, or hybrid type and biantennary complex type N-glycans.

As receptor/ligand binding pairs, for example, steroid hormone receptor/steroid hormone can be used. For example, estrogen can be used as a steroid and its receptor as a corresponding binding partner.

Attached photos

Figure 1: Plate used in the experiment of Example 6: Streptavidin-treated MTP (Microcoat), 12 wells, NUNC, MC ID: 604 176, Batch No.: 1665 C2

Figure 2: Shows the experimental design for Example 6. 4x assay. Row A: 200µl of PBS was added, 1 nmol of compound was added, mixed, incubated for approximately 30 minutes, washed twice with PBS, 800µl of PBS was added, and 300,000 untreated WBCs were added. Row B: 800µl of PBS was added, and 300,000 untreated WBCs were added. Row C: 10x10^6 WBCs in 1ml were incubated with 10 nmol of the compound of the invention for 10 minutes. 800µl of PBS was added per well, and 300,000 treated WBCs were added. After 30 minutes, the first MTP plate was washed twice with PBS, covered with Höchst, and incubated for 15 minutes. Measurements were performed using a Cellavista (Operator s9s5). The second plate was measured after 90 minutes. The third plate was measured after 150 minutes.

Figure 3: shows the results of Example 6 after 30, 90 or 120 minutes of incubation.

Figure 4: Shown as a graph are the results of Example 6 after 30, 90 or 120 minutes of incubation.

Figure 5: Shows the plates of Example 6 after 30, 90 or 150 minutes of incubation.

Figure 6: A: Chemical structures of exemplary compounds used in the present invention and synthetic by-products. C: Synthesis of biotin-PEG-Lys-(C18)2 and synthetic by-products.

Figure 7: Shows cells stained with A) a compound containing a cholesteryl group with internal reference number 29.891180, B) a compound containing myristic acid with internal reference number 29.891194. C) Staining of MDA-MB468 cells. D) and E) Cells stained with various compounds used in the present invention at different exposure times as indicated. Representative images according to Example 3.

Figure 8: A) and B) show the results of an xCelligence experiment using Jurkat cells according to Example 3. B): 1: PBS + biotin linker; 2: PBS + 10% FCS + biotin linker; 3: PBS + 1% FCS + biotin linker; 4: PBS without (w/o) biotin linker; 5: PBS + 10% FCS without (w/o) biotin linker; 6: PBS + 1% FCS without (w/o) biotin linker; 7: PBS + biotin linker without (w/o) SA; 8: PBS without biotin linker and SA.

Figure 9 shows the results of an xCelligence experiment using WBC cells according to Example 3. B): 1: PBS + biotin linker; 2: PBS + 10% FCS + biotin linker; 3: PBS + 1% FCS + biotin linker; 4: PBS without (w/o) biotin linker; 5: PBS + 10% FCS without (w/o) biotin linker; 6: PBS + 1% FCS without (w/o) biotin linker.

Figure 10: Shows staining of fixed cells according to Example 3. Left column: DA-MB468 - Antibody: K5/8. Middle column: MDA-MB468 - Antibody: EpCAM Miltenyi FITC. Right column: MDA-MB468 - Antibody: EGFR.

Figure 11: Shows staining of fixed cells according to Example 3. Left column: MDA-MB468 - Antibody: EpCAM Biolegend. Middle column: MDA-MB468 - Antibody: EpCAM Miltenys APC. Right column: WBCs - Antibody: CD45 Biolegend.

Figure 12: shows the structures of further compounds and reference compounds and intermediates used in the present invention.

Figure 13: Shows WBC recovery after centrifugation and fixation using different molecules. Molecular probes HH1749*, HH1750*, and HH1755* (*Biotin-PEG-Lysin-(C18)2) demonstrate different performance regarding recovery after centrifugation: higher molecule concentrations result in higher cell recovery after centrifugation. Centrifugation profile: 10 min, 300 x g.

Figure 14: Shows WBC recovery after centrifugation and cell fixation using different molecules. Molecular probes HH1749*, HH1750*, and HH1755* exhibit different performance with respect to cell fixation efficiency at different concentrations. Higher compound concentrations result in higher cell fixation efficiency.

Figure 15: Shows WBC recovery after centrifugation using different compounds at different time points. Molecules A and B (A: Cholesteryl-TEG-Cholesteryl-TEG-(Spacer C18)7-Fluos-Biotin-TEG; B: Biotin-PEG-Lysin-(C18)2) demonstrate different performance regarding recovery after centrifugation. Each left column: no compound of the invention; each second column from the left: 0.35 nmol of molecule A; each third column from the left: 100 nmol of molecule B; each right column: 0.5 nmol of molecule B. Higher molecule concentrations result in higher cell recovery after centrifugation. Molecule B can immobilize cells within 3.5 hours. Centrifugation profile: 10 min, 300 x g.

Figure 16: Shows WBC recovery after centrifugation by different experimenters. The left, center, and right bars for each assay represent different experimenters 1, 2, and 3. Higher molecule concentrations resulted in higher cell recovery after centrifugation. Furthermore, cell stability was independent of the experimenter. Centrifugation profile: 10 min, 300 x g. Molecule: Cholesteryl-TEG-Cholesteryl-TEG-(Spacer C18)7-Fluos-Biotin-TEG.

Figure 17: Shows WBC recovery after centrifugation at various time points and centrifugation settings. The following molecules were tested: 1234: 5'-(Cholesteryl-TEG)2-Spacer C18-dT-Biotin-TEG-3'; 1248: 3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS; 1254: 3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS; 1255: 3'-(Myristic Acid)2-PEG2000-dT-Biotin-TEG-5' INVERS. All molecules were able to immobilize cells within 2 hours. WBCs in PBS were damaged during centrifugation at 300 x g for 20 min. Molecule 1234 showed the best performance, followed by compounds 1255 and 1254. Centrifugation profile: 20 min, 300 x g. Each left column: Incubate with molecule for 10 min. Each middle column: Incubate with molecule for 1 h. Each right column: Incubate with molecule for 2 h.

Figure 18: Shows WBC recovery after centrifugation at various time points and centrifugation settings. The following molecules were tested: 1255: 3'-(Myristic Acid)2-PEG2000-dT-Biotin-TEG-5' INVERS; 1234: 5'-(Cholesteryl-TEG)2-Spacer C18-dT-Biotin-TEG-3'; 1248: 3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS; and 1254: 3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS. All molecules enabled cell fixation within 2 hours. WBCs in PBS were damaged during centrifugation at 500 x g for 20 min. Molecule 1234 showed the best performance, followed by molecules 1255 and 1254. Centrifugation profile: 20 min, 500 x g. Each left column: Incubate with the molecule for 10 min. Each middle column: Incubate with the molecule for 1 h. Each right column: Incubate with the molecule for 3 h.

Figure 19: Shows WBC recovery after centrifugation at various time points and centrifugation settings. The following molecules were tested: 1255: 3'-(Myristic Acid)2-PEG2000-dT-Biotin-TEG-5' INVERS; 1234: 5'-(Cholesteryl-TEG)2-Spacer C18-dT-Biotin-TEG-3'; 1248: 3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS; 1254: 3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS. All molecules enabled cell fixation within 2 hours. WBCs in PBS were damaged during centrifugation at 1000 x g for 20 minutes. Centrifugation profile: 20 minutes, 1000 x g. Left column: 10-minute incubation with the molecule. Each middle column: 1 h min incubation with molecules. Each right column: 2 h incubation with molecules.

Figure 20: Shows Jurkat cell recovery after centrifugation at different time points. Columns from the left: 1: 10 min incubation with molecule. 2: 1 hour incubation with molecule. 3: 3.5 hour incubation with molecule. 4: 5.5 hour incubation with molecule. The following molecules were tested: 1255: 3'-(Myristic Acid)2-PEG2000-dT-Biotin-TEG-5' INVERS. 1234: 5'-(Cholesteryl-TEG)2-Spacer C18-dT-Biotin-TEG-3'; 1248: 3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS; 1254: 3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS. Jurkat cells cultured in PBS and treated with various molecules are stable for 5.5 hours during centrifugation. Centrifugation profile: 20 min, 500 x g.

Figure 21: Demonstrates that trifunctional linker moieties do not affect cell viability. Cell viability assays were performed using the WST-1 Proliferation Assay Kit (RAS) using various compounds of the present invention, which differ in their trifunctional linker moieties. The different linkers did not appear to affect cell viability over the 4-hour linker incubation period. A) Viability assay after 2 hours and B) 4 hours.

Figure 22: Demonstrates that trifunctional linker moieties do not affect cell viability. Test compounds used in this invention, namely A) compound 1244, which has a cholesterol moiety, and B) compound 1274, which has a stearic acid moiety, were found to have no effect on cell morphology during a 4.5-hour linker incubation period. Left panel: 1-hour incubation. Middle panel: 2.5-hour incubation. Right panel: 4.5-hour incubation. Top panel: Brightfield. Bottom panel: DAPI.

Figure 23: Shows cell morphology at different time points after incubation without a linker. Without the addition of a compound used in the present invention, cells diffused away during the 4.5-hour incubation period. Cell morphology was unaffected in the cells on the left during the incubation period. Left panel: 1-hour incubation. Middle panel: 2.5-hour incubation. Right panel: 4.5-hour incubation. Top panel: Bright field. Bottom panel: DAPI.

Figure 24: Shows the recovery rate of MDA-MB468 cells after centrifugation at different time points. Columns from the left: 1: 10 min incubation with molecule. 2: 1 h incubation with molecule. 3: 3 h incubation with molecule. 4: 5 h incubation with molecule. The following compounds used in the present invention were tested: 1234: 5'-(Cholesteryl-TEG)2-Spacer C18-dT-Biotin-TEG-3'; 1255: 3'-(Myristic Acid)2-PEG2000-dT-Biotin-TEG-5' INVERS. MDA-MB468 cells were stable during centrifugation in PBS and with various molecules used in the present invention for 5 h. Centrifugation profile: 20 min, 500 x g.

Example 5: Stabilization of cells using compounds useful in the methods of the invention

Compounds useful in the methods of the invention were assayed for their effects on stabilizing cells and on immobilization.

A) WBC recovery after centrifugation and fixation with different molecules

As shown in Figure 14, molecular probes HH1749*, HH1750*, and HH1755* (*Biotin-PEG-Lysin-(C18)2) exhibited different performance regarding post-centrifugation recovery: higher molecular concentrations resulted in higher cell recovery rates after centrifugation. Centrifugation profile: 10 min, 300 x g. As can be seen in Figure 15, molecular probes HH1749*, HH1750*, and HH1755* exhibited different performance regarding cell fixation rates at different concentrations. Higher linker concentrations resulted in higher cell fixation rates.

B) WBC recovery after centrifugation with different compounds at different time points

As can be seen in Figure 16, molecules A and B (A: Cholesteryl-TEG-Cholesteryl-TEG-(Spacer C18)7-Fluos-Biotin-TEG; B: Biotin-PEG-Lysin-(C18)2) exhibit different performance with respect to recovery after centrifugation. The higher the molecule concentration, the higher the cell recovery after centrifugation. Molecule B was able to immobilize cells within 3.5 hours. Centrifugation profile: 10 min, 300 x g. A: Cholesteryl-TEG-Cholesteryl-TEG-(Spacer C18)7-Fluos-Biotin-TEG. B: Biotin-PEG-Lysin-(C18)2

C) WBC recovery after centrifugation - different experiments

As shown in Figure 17, higher molecule concentrations resulted in higher cell recovery after centrifugation. Furthermore, cell stability was independent of the experimenter. Centrifugation profile: 10 min, 300 x g. Molecule: Cholesteryl-TEG-Cholesteryl-TEG-(Spacer C18)7-Fluos-Biotin-TEG.

D) WBC recovery after centrifugation - different time points and centrifugation settings

The results of the first experiment are shown in Figure 18. The following molecules were tested:

• 1234:5'-(cholesteryl-TEG)2-Spacer C18-dT-biotin-TEG-3'

• 1248:3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS 1254:3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS

• 1255:3'-(Myristic acid)2-PEG2000-dT-biotin-TEG-5' INVERS

• All molecules were able to be fixed to cells within 2 hours.

• WBCs in PBS are damaged during centrifugation at 300 x g for 20 min

• Molecule 1234 showed the best performance followed by compounds 1255 and 1254.

Centrifugation profile: 20 min, 300 x g.

The results of a second experiment in this context are shown in Figure 19. The following molecules were tested:

1255:3'-(Myristic Acid)2-PEG2000-dT-Biotin-TEG-5' INVERS 1234:5'-(Cholesteryl-TEG)2-Spacer C18-dT-Biotin-TEG-3'

1248:3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS

1254:3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS

Here are the results:

• All molecules were able to be fixed to cells within 2 hours.

• WBCs in PBS are damaged during centrifugation at 500 x g for 20 min

• Molecule 1234 showed the best performance, followed by compounds 1255 and 1254

Centrifugation profile: 20 min, 500 x g. The results of the third experiment in this context are shown in Figure 20. The following molecules were tested:

• 1255:3'-(Myristic acid)2-PEG2000-dT-biotin-TEG-5' INVERS

• 1234:5'-(cholesteryl-TEG)2-Spacer C18-dT-biotin-TEG-3'

• 1248:3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS

• 1254:3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS

Here are the results:

• All molecules were able to be fixed to cells within 2 hours.

• WBCs in PBS are damaged during centrifugation at 1000 x g for 20 min

Centrifugation profile: 20 min, 500 x g.

E) Jurkat recovery after centrifugation at different time points

The results of this experiment are shown in Figure 21. The following molecules were tested:

• 1255:3'-(Myristic acid)2-PEG2000-dT-biotin-TEG-5' INVERS

• 1234:5'-(cholesteryl-TEG)2-Spacer C18-dT-biotin-TEG-3'

• 1248:3'-(Cholesteryl-TEG)2-PEG2000-Fluos-Biotin-TEG-5' INVERS

• 1254:3'-(Cholesteryl-TEG)2-Spacer C18-Fluos-Biotin-TEG-5' INVERS

Here are the results:

• Jurkat cells were stable in PBS and treated with different molecules for 5.5 hours during centrifugation.

Centrifugation profile: 20 min, 500 x g.

F) The trifunctional linker does not affect cell viability

The results of the first experiment in this context are shown in Figures 22A and B.

A cell viability assay using the WST-1 proliferation kit (RAS) was performed with different molecules useful in the method of the invention, differing in the trifunctional linker moiety.

The different linkers did not affect cell viability during the 4-hour linker incubation time, as can be seen in FIG22 .

The results of a second experiment in this context are shown in Figures 23A and B. It was found that the tested molecules useful in the methods of the invention (nos. 1244 and 1274) did not affect the cell morphology during the linker incubation time of 4.5 hours.

G) Morphology of cells not incubated with molecules useful in the methods of the invention - different time points

The results of this experiment are shown in Figure 24. The following were found:

• Without the addition of molecules useful in the method of the invention, the cells diffused away during the 4.5 hour incubation time.

• Cell morphology was not affected in the cells on the left during the incubation time.

H) Recovery of MDA-MB468 after centrifugation at different time points

The results of this experiment are shown in Figure 25. The following compounds were tested for use in the methods of the invention:

• 1234:5'-(cholesteryl-TEG)2-Spacer C18-dT-biotin-TEG-3'

• 1255:3'-(Myristic acid)2-PEG2000-dT-biotin-TEG-5' INVERS

The following were found:

• MDA-MB468 cells were stable in PBS during centrifugation and in the presence of different molecules useful in the methods of the present invention over a period of 5 hours.

Centrifugation profile: 20 min, 500 x g.

Example 6: SA plates (streptavidin plates) incubated with compounds useful in the methods of the present invention compared to Comparison of WBC (leukocytes) incubated with compounds useful in the methods of the present invention

Using the starting material 5'-(cholesteryl-TEG)2-PEG2000-Fluos-biotin_TEG-3' INVERS

(14530 pmol/µl) (internal reference number: 29.891250) and streptavidin-treated MTP (Microcoat), 12-well plates.

Red blood cell lysis was performed as follows:

-EDTA-whole blood 59.423 6.400 WBC/µl (Ambulanz Roche)

Lysis buffer: 100mM NH4Cl + 5mM Hepes + 0.5mM KHCO3 + 0.1mM EDTA-K

1 x 8 ml of whole blood was placed in a 50 ml Falcon tube with lysis buffer and incubated at room temperature for 10 minutes.

Centrifuge at 250 g for 15 minutes and resuspend the pellet by pipetting in lysis buffer; make up to 50 ml with lysis buffer.

Centrifuge at 250 g for 15 minutes, resuspend the pellet with PBS, add PBS to 50 ml, centrifuge at 250 g for 15 minutes, and add PBS to 50 ml.

WBC measurement at Sysmex

1:37.100 WBC/µl

The experimental design for the plate is explained below (see Figure 2):

3 x 12 well MTP: WBC treated with compounds useful in the methods of the invention:

4x assay:

Row A: Introduce 200µl PBS, add 1 nmol of compound, mix, incubate for about 30min, wash 2xPBS,

800µl of PBS was introduced and 300,000 WBC (untreated) were added.

Row B: 800µl PBS was introduced and 300,000 WBC (untreated) were added.

Row C: 10 x 10^6 WBCs in 1 ml were incubated with 10 nmol of a compound useful in the method of the present invention for 10 min.

800µl PBS/well and 300,000 treated WBCs were introduced.

After 30 min, wash the first MTP plate 2x with PBS, cover with Höchst and incubate for 15 min. Measure > Cellavista (Operator s9s5).

-Measure the second plate after 90 minutes.

-Measure the third plate after 150 minutes.

The results of the calculations are shown in Figure 3. A graph representing these results is depicted in Figure 4. The experimental plate is shown in Figure 5.

The process of the present invention has been found to be significantly and surprisingly advantageous.

Claims (35)

1. Use of a compound or a salt thereof for stabilizing animal cells, characterized in that the compound comprises two or more hydrophobic domains connected to a hydrophilic domain, wherein the two or more hydrophobic domains are covalently bonded to the hydrophilic domain, and wherein each of the two or more hydrophobic domains comprises myristic acid or cholesterol, and wherein the hydrophilic domain comprises a polyethylene glycol moiety. The hydrophilic domains described herein comprise compounds of formula (I): X1-[A1 -(L1) _n ] _k1 -Z - [A2 -(L1) _n ] _k2 - X2 (I), in Z is a linear polyethylene glycol moiety containing 1-50 -O- _CH2 - _CH2- portions, wherein the polyethylene glycol moiety optionally includes one or more spacer groups SP that connect two -O- _CH2 - _CH2- portions. And the linear polyethylene glycol portion is optionally included in the connector portion L3 at one or both ends. Each L1 is a connector portion that is selected independently from the others. Each n is either 0 or 1, and they are chosen independently. A1 and A2 are independently chosen difunctional or trifunctional components, provided that at least one A1 or A2 is trifunctional. The trifunctional moiety is selected from trifunctional moieties having 1-10 C atoms and containing at least one -OH, -SH and/or at least one -NH2 group. k1 and k2 are integers between 0 and 10, and are chosen independently of each other, provided that at least one of k1 and k2 is not 0. X1 and X2 are independently selected from hydrogen or protecting groups. L3 is independently selected from a straight-chain alkyl or alkenyl chain having 1-10 C atoms, which is optionally (i) interrupted by 1-3 N, O, or S atoms, and/or (ii) substituted with 1-4 hydroxyl, carbonyl, amino, or mercapto groups. The one or more spacer bases SP are independently selected from each other and are selected from phosphate esters and bifunctional moieties. and The hydrophilic domain is covalently bonded to the two or more hydrophobic domains via the trifunctional domain. 2. The use according to claim 1, wherein Z is a linear polyethylene glycol moiety containing 4-30 -O-CH ₂ -CH ₂ - portions. 3. The use according to claim 1, wherein the compound comprises two or more hydrophobic domains connected to a hydrophilic domain. 4. The use according to claim 1, wherein Z in formula (I) has the following structure: -(L3) _n2 - [[O-CH ₂ -CH ₂ ] _y - (SP) _n1 ] _m -[O-CH ₂ -CH ₂ ] _y1 -(L3) _n2 , in SP is as defined in claim 1. Each n1 is either 0 or 1, and each m part is chosen independently. Each n² is either 0 or 1, and they are chosen independently. m is an integer between 1 and 50. y is an integer from 1 to 50. y1 is an integer between 0 and 30. The condition is that y*m + y1 ≤ 50. L3 is independently selected from straight-chain alkyl or alkenyl chains having 1-10 C atoms, which are optionally (i) interrupted by 1-3 N, O or S atoms, and/or (ii) substituted by 1-4 hydroxyl, carbonyl or mercapto groups. 5. The use as claimed in claim 4, wherein m is an integer from 4 to 30. 6. The use as claimed in claim 4, wherein y is an integer from 4 to 30. 7. The use as claimed in claim 4, wherein y1 is an integer from 0 to 10. 8. The use as claimed in claim 7, wherein y1 is an integer from 0 to 4. 9. The use as described in claim 4, wherein (a) n1 is the same for m parts -[[O-CH ₂ -CH ₂ ] _y - (SP) _n1 ]-, and/or (b) y1 is 0, and/or (c) y is 4, 5, or 6, and n1 is 1, and/or (d) n2 are all 0, or (e) One or two n2=1, and L3 is an alkyl group having 1-10 C atoms, which optionally includes an amide group, a carbonyl group, a carbamate group, and/or an NH group. 10. The use as claimed in claim 1, wherein X1 or X2 is replaced by a hydrophobic structural domain. 11. The use as described in claim 1, wherein (a) Two, three, or four hydrophobic domains are covalently bonded to the hydrophilic domain, and/or (b) The two or more hydrophobic domains covalently bonded to the hydrophilic domain are different or the same. 12. The use of claim 11, wherein the two or three hydrophobic domains are covalently bonded to the hydrophilic domain, and/or the two or more hydrophobic domains covalently bonded to the hydrophilic domain are identical. 13. The use according to claim 1, wherein the hydrophobic structural domain (a) Composed of myristic acid or cholesterol, or (b) Containing myristic acid or cholesterol covalently bonded to the trifunctional moiety A1 via a linker portion L2, wherein L2 comprises: a phosphate ester group, a portion - [[O-CH ₂ -CH ₂ ] _y2 - (SP) _n ] _m1 -, Wherein SP and n are as defined in claim 1, y2 is an integer from 1 to 30, and m1 is an integer from 1 to 10, glycerol moiety, urethane group, amide group, straight-chain alkyl chain having 1 to 10 C atoms, wherein the alkyl chain contains a functional group at the terminal C atom, wherein the alkyl chain is optionally substituted with 1, 2, 3, 4 or 5 moieties R1, wherein R1 is independently a C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl, hydroxyl, mercapto, amino or carbonyl moieties. 14. The use according to claim 13, wherein L2 and below comprise: phosphate ester groups, partially - [[O-CH ₂ -CH ₂ ] _y2 - (SP) _n ] _m1 -, Wherein SP and n are as defined in claim 1, y2 is an integer from 1 to 30, and m1 is an integer from 1 to 10, glycerol moiety, urethane group, amide group, straight-chain alkyl chain having 1 to 10 C atoms, wherein the alkyl chain contains a functional group at the terminal C atom, the alkyl chain being optionally substituted with 1, 2, 3, 4 or 5 moieties R1, wherein R1 is independently a C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl, hydroxyl, mercapto, amino or carbonyl moieties. 15. The use according to claim 13, wherein n=0, and/or y2 is an integer from 3 to 10, and/or m1 is an integer from 1 to 3, and/or the functional group at the terminal C atom is independently selected from amine, carbonyl, hydroxyl, mercapto and carbonate groups. 16. The use as described in claim 13, wherein L2 is selected from phosphate esters, amides, carbamates, ester groups and part of -[[O-CH ₂ -CH ₂ ]y ₂ - (SP) _n ] _m1 -, wherein SP and n are as defined in claim 1, y2 is an integer from 1 to 30 and m1 is an integer from 1 to 10. 17. The use as claimed in claim 16, wherein n=0, y2 is an integer from 3 to 10, and m1 is an integer from 1 to 3. 18. The use of claim 12, wherein the myristic acid or cholesterol is bound to the trifunctional moiety A1 via the connector portion tetraethylene glycol (TEG) or phosphate ester. 19. The use as described in claim 1, wherein (a) k1 is 1, 2, 3, 4 or 5, and/or (b) The hydrophobic domain is covalently bound to the hydrophilic domain only through the trifunctional A1 region, and/or (c) k2 is 1, 2, 3, 4, 5 or 6. 20. The use as claimed in claim 19, wherein k1 is 2 or 3, and/or wherein k2 is 1, 2 or 3. 21. The use according to claim 1, wherein the compound further comprises a labeling portion and/or a linking group. 22. The use of claim 21, wherein the marking portion and/or the linking group are covalently bonded via the trifunctional portion A2. 23. The use according to claim 22, wherein one or more portions A2 are marking portions or linking groups. 24. The use as claimed in claim 23, wherein part A2 is a portion containing nucleobases. 25. The use as claimed in claim 24, wherein a portion of A2 is dT. 26. The use of claim 22, wherein the marking portion is a fluorescent marker and/or wherein the linking group is biotin. 27. The use as claimed in claim 1, wherein (a) Connector L1 is independently selected from phosphate esters, amides, carbamates and ester groups, and/or (b) Parts A1 and A2 are independently selected from the following bifunctional groups: phosphate ester group, carbamate group, amide group, moiety containing a nucleobase, and straight-chain alkyl group having 1-10 carbon atoms, wherein the alkyl chain contains a functional group at the terminal carbon atom, and trifunctional moiety having 1-10 carbon atoms and containing at least one -OH, -SH and/or at least one -NH2 group, and/or (c) Connector L2 is independently selected from phosphate esters, amides, carbamates, ester groups and some - [[O-CH ₂ -CH ₂ ] _y2 - (SP) _n ] _m1 -, in SP and n are as defined in claim 1. y2 is an integer from 1 to 30, and m1 is an integer from 1 to 10. 28. The use according to claim 27, wherein the nucleobase is dT, and/or wherein the functional group at the terminal C atom is selected from amine, carbonyl, hydroxyl, mercapto, and carbonate groups, and/or wherein the trifunctional moiety is selected from lysine, serine, serine, -O-CH2-CH((CH2)4-NH2)-CH2-, glycerol, and 1,3-diaminoglycerol moiety. 29. The use as claimed in claim 27, wherein n=0, and y2 is an integer from 3 to 10, and m1 is an integer from 1 to 3. 30. A method for stabilizing animal cells, the method comprising: (a) Providing a compound as defined in any one of claims 1-29; and (b) Stabilizing animal cells by contacting the compound with the animal cell membrane under conditions that allow the compound to interact with the animal cell membrane, and (c) Optionally, apply shear force to the animal cells. The characteristic feature is that in step (a), a compound as defined in any one of claims 1-29 is provided. 31. The method of claim 30, wherein shear force is applied to animal cells via centrifugation, large-scale cell culture, flow cytometry, fluorescence-activated cell sorting, and/or bead-based cell separation. 32. The use according to claim 1 or the method according to claim 30 or 31, wherein the animal cells are suspension cells. 33. The use or method of claim 32, wherein the animal cell is a vertebrate cell. 34. The use or method of claim 33, wherein the vertebrate cell is a mammalian cell. 35. The use or method of claim 34, wherein the mammalian cell is a human cell.