HK1091508B - Skin regeneration system - Google Patents

Description

Skin regeneration system

Technical Field

The present invention relates to cell cultures. More particularly, the present invention relates to media, systems and methods for proliferating keratinocytes for subsequent use in skin growth and regeneration. The invention also relates to compositions for the in situ growth and regeneration of skin.

Background

Insulin-like growth factors (IGF) IGF-I and IGF-II are mitogenic peptide growth factors involved in a variety of cellular processes including proliferation, DNA synthesis, differentiation, cell cycle progression and inhibition of apoptosis (Keiss et al, 1994, Hormone Research 4166; Wood & Yee, 2000, J.Mammary Gland Biology and Neoplasia 51; joints & Clemmons, 1995, Endocrine Rev.163). These effects are mediated by binding to their tyrosine-kinase linked cell surface receptor type I IGF receptors (IGF-IR). IGFs are also tightly regulated by a family of specific binding proteins called IGFBPs, whose main role is to bind free IGFs and thereby regulate their half-life, specificity and activity (Clemmons, 1998, mol.

Recently, Vitronectin (VN) has been shown to bind directly to IGF-II (Upton et al, 1999 Endocrinology 1402928-31), while IGF-I binds to VN in the presence of certain IGFBPs (International publication WO 02/24219; Kricker et al, 2003, Endocrinol.1442807-15). VN, an ECM structure and adhesion molecule, binds IGF-II with an affinity similar to that of IGF-II for its biologically relevant receptor IGF-IR (Upton et al, 1999, supra), revealing a specific physical link between IGF action in ECM and VN. In addition, IGF-II bound to VN and IGF-I bound to VN via IGFBP stimulate a synergistic functional response in vitro in a variety of cells including human keratinocytes (International publication WO 02/24219; Noble et al, 2003, supra; Kricker et al, 2003, supra).

Wounds, burns and ulcers are consumable and painful skin conditions that require elaborate and expensive treatment, which in many cases is only partially successful. For example, over 520000 australians are currently diagnosed with diabetes, and over 5% of them will develop foot ulcers. These wounds severely compromise the quality of life of the patient, often resulting in prolonged hospitalization, and may ultimately lead to amputation. In fact, most lower limb amputations performed are due to non-healing ulcers.

An increasingly preferred method of healing wounds, burns and ulcers is to replace dead or damaged skin with autologous or allogeneic keratinocytes cultured in vitro. Typically, keratinocytes are cultured in defined media in the presence of exogenous factors such as serum or bovine pituitary extract, and are usually cultured with feeder cells that optimize keratinocyte growth.

Disclosure of Invention

Typically, prior art in vitro cell culture systems are relatively expensive due to the inclusion of the aforementioned exogenous factors. In addition, exogenous factors of animal origin, such as serum and bovine pituitary extract, are relatively poorly defined (pororly defined) and may contain infectious agents that cause, for example, CJD, HIV and other diseases.

To this end, the inventors of the present invention found that protein complexes comprising IGF-II and VN or IGF-I and IGFBP and VN stimulate significant proliferative responses in ex vivo primary cell cultures in the absence of serum. More particularly, protein complexes comprising IGF-II and VN or IGF-I and IGFBP and VN may be used to enhance keratinocyte growth for skin replacement, burn and wound healing, and other therapeutic treatments requiring ex vivo skin growth.

Accordingly, in one aspect, the present invention provides a cell culture medium comprising:

(i) at least one IGF selected from IGF-I and IGF-II; and

(ii) serum in an amount that is serum-free or does not support cell growth in the absence of said at least one IGF.

In one embodiment, the medium comprises IGF-I and IGFBP.

In a second aspect, the invention provides a cell culture system comprising a culture vessel and the cell culture medium of the first aspect.

It will be appreciated that the medium and/or culture system of the invention may further comprise Vitronectin (VN) and/or Fibronectin (FN) or fragments thereof.

In a third aspect, the present invention provides a cell culture method comprising the step of culturing one or more cells in the cell culture medium of the first aspect and/or the cell culture system of the second aspect.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising one or more cells produced by culture according to the method of the third aspect and a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment, the pharmaceutical composition is suitable for aerosol delivery of keratinocytes or keratinocyte progenitor cells.

In a fifth aspect, the present invention provides a method of delivering keratinocytes or keratinocyte progenitor cells for skin regeneration in situ, the method comprising the step of delivering the pharmaceutical composition of the fourth aspect to an individual to promote skin regeneration.

A preferred embodiment of this aspect provides a method of regenerating skin in situ, the method comprising the steps of:

(i) spraying one or more keratinocytes or keratinocyte progenitor cells onto the skin of an individual;

(ii) growing the keratinocytes or keratinocyte progenitor cells to form a rejuvenated skin in situ.

Throughout this application, unless otherwise indicated, the use of "including," "comprising," and "containing" is open-ended and not closed-ended, such that a referenced entity or group of entities may include one or more other, non-referenced entities or groups of entities.

Brief Description of Drawings

FIG. 1: growth of keratinocytes in the presence of isolated protein complexes and feeder cells without serum. The mean growth of freshly isolated keratinocytes relative to the conventional method in which both fetal bovine serum and 3t3 cells were present was VitroGro (+3t3 cells). P0, P1 and P2 are relative to the secondary number of cells that have been harvested and replated (P0 — the performance of cells immediately after isolation from a skin sample). Data were obtained by MTT staining, which was converted to a colored substrate by the growing cells. Error bars are not shown due to the large differences between different donor tissues.

FIG. 2: keratinocyte morphology after growth in the presence of isolated protein complexes and feeder cells in the absence of serum. Cells were grown for 3 weeks in the presence of fetal bovine serum and mouse 3t3 cells (A) or in the presence of VitroGro (B; vitronectin, IGFBP5, and IGF-I) and mouse 3t3 cells and in the absence of serum for 3 weeks. The scale bar is illustrated as being about 200 micrometers (μm).

FIG. 3: relative activity of isolated protein complexes comprising IGFBP3 or IGFBP 5. Control-standard keratinocyte growth medium supplemented with 10% fetal bovine serum. VitroGro-3 ═ vitronectin, IGFBP3, and IGF-I (serum-free). VitroGro-5 ═ vitronectin, IGFBP5, and IGF-I (serum-free). All cultures were grown in the presence of gamma irradiated mouse 3t3 cells.

FIG. 4: the IGF protein complex supports the expansion of ex vivo keratinocytes. Keratinocytes seeded on IGF protein complex derived from adult skin survived and grew at a rate comparable to cells seeded on irradiated mouse 3T3 cells in the presence of fetal bovine serum. Cell growth was observed by the following method: (a) visual inspection of culture morphology/fusion; and (b) quantification using the MTT assay. (a) From left to right: feeding layer + bovine serum; no feeder or serum control; IGF-I + IGFBP5+ VN without feeder or serum; (b) from left to right: green culture medium, feeding layer and bovine serum; green medium + separate feeder layer; green medium-insulin + IGF-I, + IGFBP-3+ VN; green medium-insulin + IGF-I, + IGFBP-5+ VN. VN was present at 300 ng/well. IGF-I or IGF-II is present at 100 ng/well, while IGFBP is present at 300 ng/well.

FIG. 5: the IGF protein complex supplemented with additional growth factors further enhances the growth of keratinocyte cell cultures. Keratinocytes derived from adult skin are cultured on an IGF protein complex to which Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) are added, and³H]leucine incorporation assay protein synthesis. Cells seeded with the trimer IGF-I, IGFBP5 and VN protein complex or the dimer IGF-II and VN protein complex grew at a rate equivalent to that of Define Keratinocyte Media (DKM, Invitrogen). The IGF protein complex with additional EGF (100 ng/well) and bFGF (100 ng/well) significantly enhanced protein synthesis compared to DKM (p < 0.05). IGF-I or IGF-II is present at 100 ng/well, VN is present at 300 ng/well and IGFBP is present at 300 ng/well.

FIG. 6: effect of TISSOMAT on keratinocyte viability. Spray delivery of keratinocytes to cell distribution and growth after 150mm diameter collagen coating of the culture dish. Cells were sprayed at two different concentrations to determine the number of cells needed to cover the area of the spray. Cultures for nebulization were initially grown on control (with serum) or vitronectin (VitroGro) with IGFBP3 and IGF-I. All cultures were prepared in the presence of 3t3 cells. The cultures were then stained with crystal violet after 7 days of growth in the presence of serum on collagen coated plates designed to mimic conditions after delivery to the wound bed. The spray volume was 0.2ml, pressure 20psi, height 10 cm.

FIG. 7: effect of TISSOMAT on keratinocyte viability. Cultures were established using conventional media supplemented with serum. (A) Trypan blue exclusion test was performed within minutes after the sprayed cells entered the collection tube. The dye is impermeable to viable cells. (B) MTT conversion data is a measure of viability that provides an indication of metabolic activity 24 hours after nebulizing cells.

Detailed Description

The present invention is proposed from the following findings: culture media comprising IGF-II and VN or IGF-I and IGFBP and VN produce significant proliferative responses in serum-free presence ex vivo primary keratinocyte cultures, serum being generally required for ex vivo keratinocyte growth.

In addition, the absolute need for feeder cells can be at least partially eliminated, particularly in the late phase of cell culture after the initial establishment of the cell culture.

The present invention thus provides techniques that improve the best practices of current clinical ex vivo skin regeneration. In addition, the present invention also provides for the source and establishment of keratinocytes from tissue biopsies. In a preferred form, the present invention provides keratinocyte cell culture media and systems that utilize autologous vitronectin isolated or recombinantly produced from the patient's own serum, thereby further minimizing the use of xenogeneic or allogeneic support systems and avoiding the use of less well-defined (pororly-defined) supplemental products. This would therefore provide an autologous cell-based tissue engineering system that could be converted into an approved therapeutic application.

For the purposes of the present invention, "isolated" refers to a substance that has been isolated from its natural state or otherwise manipulated by man. An isolated substance may be substantially free of components normally associated with its natural state, or may be manipulated to place it in an artificial state, coexisting with components normally associated with its natural state. Isolated material may be in natural, chemically synthesized, or recombinant form.

As used herein, "synthetic" means not naturally occurring, but produced by the intervention of artificial techniques. In synthetic proteins and nucleic acids, this includes molecules produced by recombinant or chemical synthesis and combinatorial techniques well known in the art.

"protein" refers to an amino acid polymer. The amino acids may be natural or unnatural amino acids, D-or L-amino acids, as are well known in the art.

"peptide" refers to a protein having less than fifty (50) amino acids.

"polypeptide" refers to a protein having fifty (50) or more amino acids.

In particular aspects, the invention provides cell culture media and cell culture systems comprising at least IGF-I and/or IGF-II such that exogenous, animal-derived factors, such as serum, are not required or are required at substantially low levels, while cell growth and/or viability is maintained.

It is to be understood that the present invention is applicable to any mammalian cell type that is responsive to IGF-I and/or IGF-II.

Typically, such cells are mesoderm-derived cells such as epithelial cells, myoblasts and their progenitors, bone marrow and dendritic cells.

In a preferred embodiment, the invention is applicable to epithelial cells, including skin epithelial cells such as keratinocytes, keratinocyte progenitor cells, and corneal epithelial cells. In fact, both skin and corneal epithelial cells can be considered "keratinocytes" because they both produce keratin.

Keratinocytes and/or their progenitors may be derived from normal skin, such as, but not limited to, skin biopsies from wound or ulcer or from Outer Root Sheath (ORS) cells of hair follicles.

It will therefore be appreciated that the culture medium, method and system of the present invention may potentially be used to engineer replacement of any tissue in which epithelial cells are present, such as oral and respiratory mucosa (lining of the mouth, nose, trachea and oesophagus) and genito-urological tissue (e.g. vagina, bladder). These tissues may also be damaged by burns and other wounds, and may likewise be treated with culture grafts cultured in a manner similar to skin biopsies.

The invention is also applicable to human embryonic stem (hES) cells which also typically require serum in culture.

Thus, it is understood that "serum free of serum or in an amount that does not support cell growth in the absence of said at least one IGF" refers to an amount or concentration of serum that is free of serum or is much less than that typically required for optimal cell growth and/or development in vitro.

"serum" refers to a fraction derived from blood that contains various macromolecular, lipid-like and trace element carrier proteins, cellular appendages and spreading factors, low molecular weight nutrients, hormones, and growth factors. Operationally, serum may be defined as the blood protein, non-cellular fraction remaining after removal of red blood cells, platelets, and coagulated plasma components. The most widely used animal serum for cell culture is fetal bovine serum FBS, although adult bovine serum, horse serum and protein fractions thereof (e.g., serum albumin fraction V) may also be used.

Typically, mammalian cells require 5-10% serum, depending on the cell type, duration of culture, presence or absence of feeder cells, and/or other components of the culture system and other factors that will be apparent to those skilled in the art.

Thus, in a preferred embodiment, the invention contemplates less than 5% serum, more preferably less than 2% serum, even more preferably less than 1% serum or advantageously no more than 0.5%, 0.4%, 0.3% or 0.2% serum (v/v).

In a particularly advantageous embodiment, the invention contemplates no serum or no more than 0.1% or 0.05% serum (v/v).

In embodiments where IGF-I is present, preferably IGF-I is a component of a protein complex further comprising IGFBP and Vitronectin (VN).

IGFBPs are selected from IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5 and IGFBP 6.

Preferably, the IGFBP is IGFBP3 or IGFBP 5.

More preferably, the IGFBP is IGFBP 5.

In one embodiment where IGF-II is present, preferably IGF-II is a component of an isolated protein complex further comprising Vitronectin (VN).

It is also understood that Vitronectin (VN) can be in monomeric or multimeric form.

In a particular embodiment, the invention comprises purified autologous VN.

Preferably, the keratinocytes are cultured in a culture vessel commonly used in the art. It will therefore be appreciated that the respective amounts of IGF, VN and IGFBP present during culture will depend on factors such as the size of the culture vessel, the amount of liquid medium present in the vessel, cell density and other factors known in the art.

For guidance, at 1.9cm²In the pores, preferred amounts are as follows:

VN: 50-5000ng, more preferably 100-500ng or advantageously 250-350 ng;

IGF: 0.1-1000ng, more preferably 10-200ng or advantageously 50-150 ng; and

IGFBP: 1-1000ng, more preferably 30-700ng or advantageously 300-500 ng.

Suitably, the medium of the invention comprises other defined components. Non-limiting, in some cases optional ingredients include well-known basal media such as DMEM or Ham media, antibiotics such as streptomycin or penicillin, Human Serum Albumin (HSA), phospholipids (e.g., phosphatidylcholine), amino acid supplements such as L-glutamic acid, antioxidants such as beta-mercaptoethanol, transferrin, buffers such as carbonate buffer, HEPES, and a source of carbon dioxide typically provided by a cell culture incubator.

The present invention also contemplates the use of other biologically active proteins that regulate Cell growth, differentiation, survival and/or migration, such as epidermal growth factor (EGF; Heldin et al, 1981, Science 41122-1123), fibroblast growth factor (FGF; Nurcombe et al, 2000, J.biol. chem.27530009-30018), basic fibroblast growth factor (bFGF; Tarabboletti et al, 1997, Cell growth. Differ.8471-479), osteopontin (Nam et al, 2000, Endocrinol.1411100), thrombospondin-1 (Nam et al, 2000, supra), tenascin-C (Arai et al, 1996, J.biol. chem.2716099), PAI-1(Nam et al, 1997, Endocrinol.13872), plasminogen (Camebel et al, 1998, amino.J.PhysE.275), fibronectin (Campbell et al, Campbell.1999, Metrinol. 1999, J. 27430215, Metrinol et al, Metrinol J. 27430215).

Preferred additional biologically active proteins are EGF and bFGF.

Other biologically active proteins such as EGF and bFGF can be administered at 1.9cm per day²Culture well 0.1-1000ng or advantageously 1 to 100 ng.

In a specific embodiment, the invention contemplates the use of any growth factor having a heparin-binding-like domain.

In another specific embodiment, the present invention contemplates the use of LIF and/or other agents that inhibit cell differentiation in addition to isolating protein complexes.

In yet another specific embodiment, the present invention contemplates the use of one or more of poly-L-lysine and poly-L-arginine and secreted cellular material that interacts with vitronectin, such as collagen, fibronectin, mucopolysaccharide/proteoglycan, laminin, sialoprotein, and/or polymers of mucin in the media, systems, and/or methods of the invention.

It is also contemplated that the invention may facilitate cell culture in the absence of feeder cells at least after the initial establishment phase of the cell culture.

In keratinocytes and/or keratinocyte progenitor cells, feeder cells (e.g., irradiated 3t3 feeder cells) may be present during the first 6-7 days of serum-free culture, after which the feeder cells may be absent for up to two passages.

According to the foregoing, while not wishing to be bound by any particular theory, it is believed that IGF-I forms a separate protein complex with IGFBP and VN, while IGF-II forms a complex with VN, thereby exerting a biological effect in cell culture.

The term "isolated protein complex" is herein consistent with that used in International publication WO 02/24219 and International application PCT/AU 2004/000117.

The isolated protein complex may be preformed and contained in the medium of the present invention or may be formed in a culture vessel.

Typically, vitronectin and/or fibronectin is bound, immobilized, coated or otherwise associated on the culture vessel. The added IGF and optional IGFBP form a complex with vitronectin and/or fibronectin bound, immobilized, coated, or otherwise associated with the culture vessel.

As described in International application PCT/AU 2004/000117, the isolated protein complexes of the invention may comprise growth factors (e.g., IGF-I and IGF-II) or at least one domain of a growth factor capable of binding to an associated growth factor receptor (e.g., the IGF1 type receptor).

Herein, "domain" refers to at least that portion or region of a growth factor that is capable of binding to the associated growth factor receptor. Typically, although not exclusively, the cognate growth factor receptor is expressed by a cell, and binding or association of at least one domain of the growth factor with the cognate growth factor receptor triggers a cellular response such as cell growth, differentiation, survival and/or migration.

With particular reference to IGF-I, the domain suitably comprises amino acid residue 24, which is not a leucine residue.

Typically, the residue is tyrosine.

With particular reference to IGF-II, the domain suitably comprises amino acid residue 27, which is not a leucine residue.

Typically, the residue is tyrosine.

With particular reference to IGF-I, in one embodiment the domain comprises or consists of residues 1-70 of IGF-I.

In another embodiment, the domain comprises or consists of residues 4-70 of IGF-I.

It is also understood that another component of the isolated protein complexes of the invention is at least one integrin binding domain of vitronectin or fibronectin.

This includes and encompasses VN or FN binding alpha_vAny domain of integrins.

More preferably, the integrin is α_vβ₃Integrins or alpha_vβ₅Integrins.

As described in international application PCT/AU 2004/000117, the heparin-binding domain (HBD) of VN (and similar FNs) is not required for full biological activity of the isolated protein complex.

With respect to VN, the polyanionic region of VN (and similar FNs) is required for the most likely interaction with IGF-II or IGF-I/IGFBP complex.

The polyanionic region is amino acid residues 53-64 of the mature VN sequence.

In light of the foregoing, embodiments of synthetic chimeric proteins that do not comprise a polyanionic region of HBD and/or VN or FN are contemplated by the present invention.

With respect to VN proteins and amino acid sequences thereof that do not comprise HBDs and/or polyanionic regions, these may be native proteins such as 54kDa chicken yolk VN (lacking HBDs) or may be engineered by deletion, mutation or truncation of the VN protein or amino acid sequence such that HBDs and/or polyanionic regions are absent or at least substantially non-functional.

It will be readily appreciated from the foregoing that the isolated protein complexes of the invention may be in the form of non-covalently bound oligo-protein complexes, covalently cross-linked (reversible or irreversible) oligo-protein complexes, or in the form of synthetic chimeric proteins.

Thus, in a particular aspect, the invention provides an isolated protein complex in the form of a synthetic chimeric protein.

As used herein, a "chimeric protein" comprises a contiguous amino acid sequence derived from an integrin receptor binding domain of VN or FN and a growth factor or at least one receptor binding domain of a growth factor.

While not wishing to be bound by any particular theory, it is believed that synthetic chimeric proteins may be able to co-link and co-activate the cognate receptor for the growth factor and the integrin receptor for VN or FN, thereby stimulating, inducing, increasing or promoting cell migration.

The advantages of the chimeric proteins according to the invention are that they are easy to produce by chemical synthesis or recombinant methods and are expected to be more stable in vivo, since they do not rely on the protein-protein interactions required in non-covalent oligo-protein complexes.

In this regard, while isolated protein complexes comprising the receptor binding domain of IGF-I also comprise IGFBPs, it is believed that IGFBPs are preferably absent from the IGF-I/VN synthetic chimera in accordance with the aforementioned mode of action.

Preferably, the chimeric protein further comprises a "linker sequence" located between and adjacent to the growth factor sequence and the VN or FN amino acid sequence.

In one embodiment, the linker sequence comprises one or more glycine residues and one or more serine residues.

Specific examples of linker sequences may be selected from: gly₄ Ser；Gly₄ Ser₃And (Gly)₄ Ser)₃But is not limited thereto.

In another embodiment, the linker sequence comprises a plasmin cleavage recognition site, e.g., according to the sequence: leu Ile Lys Met Lys Pro are provided.

In yet another embodiment, the linker sequence comprises a collagenase-3 cleavage recognition site, e.g., according to the sequence: gln Pro Gln Gly Leu Ala Lys are provided.

The foregoing are examples of growth factors, growth factor binding proteins, and/or biologically active fragments of vitronectin/fibronectin.

In one embodiment, the "biologically active fragment" has no less than 10%, preferably no less than 25%, more preferably no less than 50% and even more preferably no less than 75%, 80%, 85%, 90% or at least 95% of the biological activity of the "full-length" protein.

Variant growth factors, growth factor binding proteins and/or vitronectin/fibronectin and/or encoding nucleic acids that may be used in accordance with the invention are also contemplated.

In one embodiment, a "variant" has one or more amino acids that have been substituted with a different amino acid. It is well known in the art that some amino acids can be changed to amino acids with widely similar properties without changing the active properties of the protein (conservative substitutions).

In one embodiment, the variant has at least 70%, preferably at least 80%, more preferably at least 90% and advantageously at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence described herein.

Preferably, sequence identity is determined over at least 60%, more preferably at least 75%, even more preferably at least 90% and advantageously over substantially the entire length of the synthetic protein of the invention.

To determine percent sequence identity, optimal alignments of amino acid and/or nucleotide sequences can be performed by Computer execution of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, Science DriveMadison, Wis., USA, incorporated herein by reference) or by examining the optimal alignment produced by any of the various methods chosen (i.e., the highest percent homology produced compared to the comparison window). Reference may also be made to the BLAST program family as disclosed in Altschul et al, 1997, Nucl. acids Res.253389, which is incorporated herein by reference.

In another example, "sequence identity" may be understood to refer to the "percent match" calculated by a DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA).

A detailed discussion of sequence analysis can be found IN Unit 19.3 of Current promoters IN MOLECULAR BIOLOGY tools, Ausubel et al (John Wiley & Sons Inc NY, 1995-1999).

The present invention also contemplates derivatives of growth factors, growth factor binding proteins, and/or vitronectin/fibronectin.

As used herein, a "derivative" is altered, for example, by addition, conjugation, or complexation with other chemical groups as is well known in the art, or by post-translational modification techniques.

"addition" of amino acids may include fusion with other peptides or polypeptides. Other peptides or polypeptides may, for example, aid in the purification of the protein. These include, for example, poly-histidine-tag, maltose-binding protein, Green Fluorescent Protein (GFP), protein A or glutathione S-transferase (GST).

Other derivatives contemplated by the present invention include, but are not limited to, side chain modifications, incorporation of unnatural amino acids and/or derivatives thereof in protein synthesis, and other methods of using cross-linking agents and imposing restrictions on protein conformation. Non-limiting examples of side chain modifications contemplated by the present invention include the following amino modifications: for example by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methyliminotetraacetate; carbamoylation of the amino group with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by NaBH₄Reduction; reductive alkylation by reaction with an aldehyde followed by NaBH₄Reduction; trinitrobenzene conversion of the amino group was carried out with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS).

The mercapto group can be modified by the following method: for example, performic acid to cysteine; forming a mercury derivative using 4-chloromercuriylbenzenesulfonic acid, 4-chloromercuribenzoic acid, 2-chloromercuriyl-4-nitrophenol, phenylmercuric chloride, and other mercury agents; and other thiol compounds to form mixed disulfides; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

The imidazole ring of a histidine residue may be modified by N-carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

Examples of the incorporation of unnatural amino acids and derivatives in peptide synthesis include, but are not limited to, the use of 4-aminobutyric acid, 6-aminocaproic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienylalanine, and/or the D-isomer of an amino acid.

Further examples of chemical derivatives of PROTEINs are provided IN chapter 15 of Current promoters IN PROTEIN SCIENCEs eds. Coligan et al, John Wiley & Sons NY (1995-2001).

According to the present invention, proteins may be prepared by any suitable method known to those skilled in the art.

In one embodiment, the protein may be in a substantially pure native form.

A specific example is purified autologous vitronectin.

In another embodiment, the protein may be prepared by chemical synthesis. Chemical synthesis techniques are well known IN the art, but one skilled IN the art can refer to an example of a suitable methodology IN chapter 18 of CurentProtocols IN PROTEIN SCIENCE Eds.Coligan et al, John Wiley & Sons NY (1995-2001).

In yet another embodiment, the protein may be prepared as a recombinant protein.

Recombinant proteins are well known in the art and the skilled person can refer to, for example, Sambrook et al, MOLECULAR CLONING.A. Laboratory Manual (Cold spring harbor Press, 1989), especially parts 16 and 17; curent PROTOCOLSIN MOLECULAR BIOLOGY eds. Ausubel et al, (John Wiley & Sons, Inc.1995-1999), especially chapters 10 and 16; and CURRENT promoters in SCIENCE eds. Coligan et al, (John Wiley & Sons, Inc.1995-1999), especially chapters 1, 5 and 6, which are incorporated herein by reference.

The recombinant protein may further comprise a fusion partner (partner).

Well-known examples of fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, Maltose Binding Protein (MBP), and Hexahistidine (HIS)₆) They are particularly useful for the separation of fusion proteins by affinity chromatography. For purification of the fusion protein by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-and nickel-or cobalt-conjugated resins, respectively. Many such matrices are available in "kit" form, e.g. for use in (HIS)₆) QIAexpress of fusion partner^TMSystem (Qiagen) and Pharmacia GST purification system.

In some cases, the fusion partner also has a protease such as factor X_aOr a thrombin cleavage site which allows the relevant protease to partially digest the fusion protein of the invention, thereby releasing the recombinant protein therefrom. The released protein is then separated from the fusion partner by subsequent chromatographic separation.

The fusion proteins according to the invention also include within their scope "epitope tags", which are typically short peptide sequences from which specific antibodies are available. Well-known examples of epitope tags from which specific monoclonal antibodies are readily available include c-fungus, haemagglutinin and FLAG tags.

Suitable host cells for expression may be prokaryotic or eukaryotic cells, such as E.coli (e.g., DH 5. alpha.), yeast cells, Sf9 cells using baculovirus expression systems, CHO cells, COS, CV-1, NIH3T3 and HEK293 cells, but are not limited thereto.

The invention further contemplates the use of cells, such as keratinocytes or keratinocyte progenitor cells, capable of expressing at least one recombinant protein selected from the group consisting of:

(i) recombinant IGF;

(ii) recombinant IGFBP;

(iii) recombinant vitronectin;

(iv) a recombinant chimeric protein as described above; and

(v) other biologically active proteins such as EGF or bFGF.

According to a particular embodiment, paracrine/autocrine expression of IGF, VN and/or IGFBP may enable keratinocytes or keratinocyte progenitor cells to be cultured in serum-free medium without the need to add one or more growth factors, IGFBP and/or vitronectin to the medium.

Recombinant protein expression can be obtained by introducing an expression construct into keratinocytes or keratinocyte progenitor cells.

Typically, the expression construct comprises an expression nucleic acid (encoding a recombinant protein) operably linked or linked to a promoter.

Promoters may be constitutive or inducible.

Constitutive or inducible promoters include, for example, tetracycline-repressible, ecdysone-inducible, ethanol-inducible, and metallothionein-inducible promoters. The promoter may be a naturally occurring promoter (e.g., alpha crystal promoter, ADH promoter, phosphoglycerate kinase (PGK), human elongation factor alpha promoter, and viral promoters such as SV40, CMV, HTLV derived promoters) or a synthetic hybrid promoter combining more than one promoter element (e.g., SR alpha promoter).

In a preferred embodiment, the expression vector comprises a selectable marker gene. Selectable markers are useful for either selection purposes for transformed bacteria (e.g., bla, kanR, and tetR) or transformed mammalian cells (e.g., hygromycin, G418, and puromycin).

The expression construct can be introduced into mammalian cells such as keratinocytes or keratinocyte progenitor cells by well-known methods such as electroporation, particle bombardment, virus-mediated gene transfer, calcium phosphate precipitation, DEAE-glucose, cationic liposomes, lipofectin (lipofectamine), lipofectamine (lipofectamine), and the like, but the method is not limited to the above-mentioned methods.

For non-limiting specific examples of methodologies potentially applicable to recombinant growth factor protein expression in keratinocytes, reference may be made to Supp et al, 2000, J.Invest.Dermatol.1145 and Supp et al, 2000, Wound Repair Regen.826-35.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising one or more cells produced using the culture medium and/or culture system of the invention, such as, but not limited to, keratinocytes, and a pharmaceutically acceptable carrier, diluent, or excipient.

The pharmaceutical compositions of the present invention may be used to promote or facilitate cell migration, tissue regeneration and wound healing.

In general, the compositions of the present invention may be used in therapeutic or prophylactic treatments as desired. For example, the pharmaceutical composition may be applied in the form of a therapeutic or cosmetic formulation for skin repair, wound healing, burn healing and other dermatological treatments.

Preferably, the pharmaceutically acceptable carrier, diluent or excipient is suitable for administration to a mammal, preferably to a human.

In a specific embodiment, the pharmaceutical composition comprises autologous or allogeneic keratinocytes cultured according to the invention.

By "pharmaceutically acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating material that is safe for systemic administration. Depending on the particular route of administration, various carriers well known in the art may be used. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline and salts such as inorganic acid salts including hydrochloride, hydrobromide and sulfate, organic acid salts such as acetate, propionate and malonate and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing co.n.j.usa, 1991), which is incorporated herein by reference.

In particular embodiments, the pharmaceutical composition further comprises a propellant.

Any safe route of administration may be used to provide the patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, transdermal, and the like may be used.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injected or implanted controlled release devices specifically designed for this purpose or other implant forms modified to act in this manner.

Controlled release formulations can be obtained, for example, by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. Controlled release may be achieved by using other polymer matrices, liposomes and/or microspheres. Non-limiting examples of controlled release formulations and delivery devices include osmotic pumps, polylactide-co-glycolide (PLG) polymer-based microspheres, hydrogel-based polymers, chemically cross-linked dextran polymersSugar gels such as Ocodex^TMAnd dextro lactate-HEMA.

The compositions described above can be administered in a manner and in a pharmaceutically effective amount compatible with the dosage form. The dose administered to a patient in the present invention should be sufficient to produce a beneficial response in the patient over a suitable period of time. The amount of active agent administered may be determined by factors which will depend upon the age, sex, weight and general health of the subject to be treated, and the judgment of the physician.

For pharmaceutical compositions for wound healing, see in particular U.S. Pat. No. 5,936,064 and International publication WO 99/62536, both of which are incorporated herein by reference.

In a particular embodiment, the compositions of the present invention are suitable for in situ spray delivery.

The term "spray" encompasses and includes terms such as "aerosol" or "fog" or "condensing agent" which generally describe liquid suspensions in the form of droplets.

According to the present invention, although optionally, the spray or aerosol composition may further comprise at least one IGF selected from IGF-I and IGF-II or in a particular embodiment an isolated protein complex comprising IGF, VN and IGFBP to promote in situ growth and migration of skin cells. Other biologically active proteins such as EGF and/or bFGF may also be included.

While not wishing to be bound by any particular theory, the present invention contemplates that the inherent "viscosity" of VN in the IGF complex present in the spray composition may facilitate delivery of IGF-I, IGF-II and other growth factors such as EGF and bFGF.

Typically, the spray composition of the present invention will be delivered by means such as a sealed canister equipped with a delivery outlet.

An example of an aerosolized keratinocyte delivery system, such as for wound healing in a pig model, has been provided by Navarro et al, 2000, J Burn Care Rehabil 21513. See also Grant et al, 2002, Br J plant Surg 55219, which describes the use of aerosolized keratinocytes and fibrin glue for wound healing in pig models.

Preferably, the spray composition of the present invention is substantially serum free.

In a particular embodiment, the skin spray composition of the present invention comprises(Baxter Healthcare) which facilitates spray application of fibrin glue by aerosolizing a liquid by delivery into a regulator-controlled stream of compressed medical grade gas. Pressures between 10-30psi are suitable, although a decrease in viability is observed with increasing pressure. The concentration of sprayed cells may be 50-150 million per ml. 0.2ml of cell suspension was applied at 20psi sufficient to cover an area of about 25 square centimeters (based on a measurement of cell coverage surface area after 7 days of in vitro growth). The cells are preferably delivered in serum-free growth medium, but may also be suspended in fibrin glue such as commercially available Tisseel/Tissucol (Baxter healthcare).

It is also contemplated that similar efficacy may be achieved using a syringe (e.g., a syringe equipped with a spray cap) to deliver the compositions of the present invention.

Therapeutic applications

In a particular aspect, the present invention provides methods of treating burns, wounds and ulcers and methods related to cosmetic skin treatments to improve or enhance skin quality or skin appearance.

These methods are particularly directed to the treatment of mammals, more particularly humans. However, it is also to be understood that the present invention may have veterinary applications for the treatment of domestic, livestock and work animals.

In a preferred embodiment, the invention provides media, systems and methods for propagating isolated primary keratinocytes, which cells can be administered to an individual according to the invention.

In a particular embodiment, the keratinocytes are autologous or allogeneic keratinocytes cultured according to the invention.

These methods include administration of a pharmaceutical composition as described above and may be by injection through a microneedle into a specific tissue site as described in us patent 6,090,790, by topical ointment, lotion or sealed dressing applied to a wound, burn or ulcer as described in us patent 6,054,122, or by an implant which releases the composition as described in international publication WO 99/47070.

There are also methods by which skin cells can be genetically modified for the purpose of producing skin substitutes, for example by genetically engineering the expression of the required growth factors (Supp et al, 2000, j. invest. dermotol.1145). Examples of reviews in this area are provided by Bevan et al, biotechnol.

It is also contemplated to "inoculate" a recipient with transfected or transformed cells, as described in International publication WO 99/11789.

These methods are useful for stimulating cell migration and thereby facilitating or promoting wound and burn healing, repair of skin injuries such as ulcers, tissue replacement and transplantation such as by culturing autologous skin in vitro, regeneration of epidermal cells of internal organs such as kidney and lung, and repair of damaged neural tissue.

Skin replacement therapy is well known in the art and co-cultured epithelial/keratinocyte cell lines may be used, such as described by Kehe et al, 1999, Arch. Dermatol. Res.291600, or primary (usually autologous) epidermal, dermal and/or keratinocyte cells cultured in vitro. These techniques may also utilize engineered biomaterials and synthetic polymer "scaffolds".

General examples of reviews in the art are provided by Terskikh & Vasiliev, 1999, int.Rev.Cytol.18841 and Eaglestein & Falanga, 1998, Cutis 621.

More specifically, Izumi et al, 2000, j.dent.res.798, describe the preparation of alternative oral mucosa useful in craniofacial surgery. Fetal keratinocytes and dermal fibroblasts can be expanded in vitro to produce skin for transplantation to treat skin lesions as described by Fauza et al, j.pediatr.surg.33357, while skin substitutes from dermal and epidermal components cultured on hyaluronic acid-derived biomaterials have shown potential application in the treatment of burns (zachi et al, 1998, j.biomed.mater.res.40187).

The present invention also contemplates the use of polymeric scaffolds to facilitate alternative skin engineering, as described by Sheridan et al, 2000, J.Control Release 1491 and Fauza et al, 1998, supra, as well as the use of microsphere formulations for the delivery of skin cells to the site of wounds and burns (LaFrance & Armstrong, 1999, Tissue Eng.5153).

The keratinocyte sheets, usually prepared for therapeutic use, are responsible for the eventual closure of burn wounds. The patch graft technique is applicable to all partial cortical burns and is most useful in treating large surface area wounds where earlier permanent closure of the wound and donor site is almost impossible without external assistance. This is the type of injury that causes death in burn patients in the recent Bali island explosion.

Currently, it is possible to biopsy a piece of skin 50 cent-sized from a patient's skin to grow enough to cover the entire adult's skin. This cultivation took 17 days.

However, there is an urgent need for earlier skin replacements to reduce patient trauma, risk of infection, scarring and the current need for expensive temporary skin replacements prior to permanent skin grafts. In addition, the cultured skin sheets contain many skin cells, some mature and some immature. The simple act of allowing cultured keratinocytes to reach confluence, which is necessary for the production of skin sheets, causes the cells to prematurely lose their original characteristics, i.e. differentiation. When a cultured skin patch is applied, only immature cells are able to attach to the patient and grow. Because only a small area adheres, these patches are easily damaged by patient friction or movement and sometimes result in the loss of the entire graft. In addition, in sheet transplantation, the more mature cells in the sheet, the more likely it is that transplantation will not occur and the cells themselves will not proliferate and migrate on the wound surface itself. It is thus clear that earlier application of immature skin cells will result in better engraftment and reduced scarring.

The present invention thus provides a spray or aerosol delivery method for delivering ex vivo cultured skin cells onto the skin of a burn, ulceration or wound in a patient that is capable of covering a larger surface area of the patient's body with immature skin cells much earlier than prior patch grafting techniques. This may be as early as only 7 days. This will also significantly reduce scarring, shock and heat loss and will enable faster recovery of skin function for partial and full skin burns.

According to the present invention, although optionally, the spray or aerosol administered may further comprise an isolated protein complex comprising IGF, VN and IGFBP and EGF and/or bFGF to promote growth and migration of skin cells in situ.

Patient's own skin cells (autologous skin) and donor skin cells (allogeneic or xenogeneic skin) can be cultured and used for early burn closure. The donor cells do not express the transplantation antigen and therefore they do not elicit an immune response in the patient. But the donor skin cells are eventually replaced by the patient's own skin cells.

Although autologous cells are preferred, the use of allogeneic or xenogeneic cells in a skin spray may allow for immediate administration to a patient in need thereof. Alternatively, sufficient autologous skin cells can be cultured for a therapeutic spray within about seven days.

Another treatment contemplated by the present invention is the treatment of burn patients to achieve early closure of a full-cortical wound, since cultured skin is not readily available on a wound surface with the skin surface (epidermis) and deep layers (dermis) removed. The present invention contemplates the use of dermal substitutes in combination with a skin spray to achieve early permanent closure of these most severe lesions. Contemplated alternatives are biological and synthetic dermal substitutes. For example, a de-epidermal, decellularized dermal scaffold derived from a cadaver comprising the isolated protein complex of the present invention may be covered with a synthetic epidermis (dressing). After about 7 days, the dermis the present inventors hypothesized that the dermis will be highly infiltrated by autologous endothelial cells. At this point, the synthetic dermis will be removed and the patient's own ex vivo expanded fibroblasts and keratinocytes will be applied to the allo-dermis (allo-dermis).

It is expected that a skin spray will be successful rather than an epidermal sheet, as the dermal substitute will promote migration and anchorage of skin cells and other important cells typically present in the skin as a nutrient-stable scaffold. This will result in improved acquisition of cultured skin cells in full-thickness skin lesions.

In order that the invention may be more readily understood and put into practical effect, reference will now be made by those skilled in the art to the following non-limiting examples.

Examples

Example 1

Growth of human primary keratinocytes in the absence of serum

Materials and methods

Growth factor concentration/Pre-adsorption to cultured Plastic

The standard method of addition of VN, IGF and IGFBP was used in all studies. Culturing the plastic by mixing with 150ng/cm of serum-free medium at 37 deg.C²Was incubated for 2 hours. The VN solution was then removed and the supernatant was washed with a solution containing IGFBP (250 ng/cm)²)、IGF-I(50ng/cm²) And EGF (50 ng/cm)²) The serum-free medium of (1) is replaced. Growth factors were adsorbed onto VN treated plastic overnight at 4 ℃ (refrigerator). The next day, the growth factor solution was removed and replaced with growth medium (defined below) containing 50ng/ml VN, 50ng/ml IGFBP, 15ng/cm IGF-I and 15ng/cm EGF.Cells were added at the densities given below. The medium is typically changed every 3 days. Each culture was cultured for about 6 passages after nature: i.e., about 6 days between generations.

Growth medium

The basal medium was a 3: 1 mixture of Dulbecco's Modified Eagle Medium (DMEM) and Ham's F12 medium, routinely supplemented with L-glutamine (2mM), cholera toxin (0.1. mu.g/ml), adenine (180. mu.M), hydrocortisone (0.4. mu.g/ml) and a non-essential amino acid mixture (1% v/v).

The positive control medium contained additional 10% fetal bovine serum, insulin (5. mu.g/ml) and epidermal growth factor (EGF, 10 ng/ml).

Density of inoculation

Cultures were grown at 25X 10⁴/cm²The density of growth-arrested mouse 3t3 cells was cultured. 3t3 cells were "growth arrested" by irradiation with gamma immediately prior to use.

Keratinocytes were seeded at two different densities depending on the number of passages. The initial culture (P0) was inoculated by 3.8X 10⁴/cm²Cells are established. The subsequent cultures (P1, P2, etc.) were re-inoculated by 6.4X 10³/cm²Density of harvested cells. The higher seeding density used for the P0 culture was because only a fraction of the freshly harvested cells would show ongoing proliferation in culture. Thus, culturing these cells can expand the proliferative subpopulation.

Results

Comparison with culture cultured with conventional growth Medium containing serum

Referring to FIG. 1, this figure shows the average growth of newly isolated keratinocytes with VitroGro (+3t3 cells) relative to a conventional method in which both fetal bovine serum and 3t3 cells are present. P0, P1 and P2 are relative to the secondary number of cells that have been harvested and reseeded (P0 — the performance of cells immediately after isolation from a skin sample). Data were obtained by MTT staining. The data show that cultures in the presence of the isolated protein complex in the absence of serum consistently achieved at least 90% of the cell growth achieved in the presence of 10% serum.

Referring to fig. 2A and 2B, skin cells grown on VitroGro showed similar appearance to those grown in the presence of fetal bovine serum (fig. 2A). More detailed comparisons based on the presence of molecular markers are currently being made to determine this conclusion. The techniques employed will include immunocytochemistry, Fluorescence Activated Cell Sorting (FACS) analysis, western blotting, and Polymerase Chain Reaction (PCR) methods. The use of state-of-the-art proteomics (proteomics) and gene array technology is also contemplated. The primary markers studied will include cytokeratins (CK1 and CK10, CK6, CK14 and CK19) and putative keratinocyte progenitor markers (e.g., p63, α 9-integrin, α 6-integrin^bri/CD71^dim). Attention should be paid particularly to comparing the expression of putative progenitor markers, as they are likely to be related to the clinical efficacy of the post-transplant culture. In addition, the reaction (adhesion, migration, proliferation) of the cells can be detected by performing a conventional in vitro functional assay.

Referring to fig. 3, it is clear that the isolated protein complex comprising IGFBP5 is more efficient in keratinocyte production than the complex comprising IGFBP 3.

Example 2

Growth of human Primary keratinocytes without feeder cells or in the absence of serum

Materials and methods

Primary keratinocyte culture

Keratinocyte cells were isolated from adult skin using essentially the same standard methods as originally reported by Rheinwald & Green, 1977, Nature 265421. Briefly, it involves digesting a skin sample in Dispase II solution for one hour at 37 ℃. The recovered epithelial cells were then re-digested with 0.25% trypsin/0.02% EDTA at 37 ℃ for 10 minutes to detach the cells. Residual trypsin activity was inactivated, and the recovered cells were then washed and co-plated in tissue culture dishes with or without lethal dose of irradiated 3T3 mouse fibroblasts. "control" cells cultured using these standard conditions were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum, 0.1% penicillin-streptomycin solution, 0.4. mu.g/ml hydrocortisone, 0.1. mu.g/ml cholera toxin, 10ng/ml human recombinant Epidermal Growth Factor (EGF), 5. mu.g/ml insulin, 5. mu.g/ml transferrin, and 2nM triiodothyronine, whereas cells treated with the isolated growth factor complex used the same medium except that it did not contain insulin. Insulin is excluded in the media used in combination with the treatment with the isolated protein complex to minimize competitive binding of insulin to the type 1 IGF receptor. Isolation of cells cultured on growth factor complex coated dishes also differs from cells cultured according to standard methods in that these cells will be seeded on plates without irradiated mouse fibroblasts.

Protein synthesis assay

Keratinocytes were derived from adult skin biopsies and expanded up to passage 2 using standard methods of adding Green medium, serum and feeder cells. These cells were then evaluated for stimulation of protein synthesis in the presence or absence of the IGF + VN complex. Here, 24-well plates were coated with 300ng vitronectin for 2 hours, and then washed to remove unbound vitronectin. The wells are then incubated with growth factors to be detected, i.e.: epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-I and insulin-like growth factor-II, were added to the wells and allowed to bind to vitronectin overnight. The wells were washed twice the next day to remove any unbound growth factor and the plates were allowed to air dry. Keratinocytes were then harvested and cultured at 1X 10⁵The density of each cell/well is equal to 1 uCi/well³H]Leucine together in serum-free Dulbecco's Modified Eagle's Medium (DMEM). In selected wells, cells were seeded in Define Keratinocytocyte Media (DKM) (Invitrogen)a is a commercially available product for serum-free culture of keratinocytes. The plate is then incubated for 48 hours and then washed to remove any unincorporated [ 2 ]³H]-leucine. Measurement by beta-scintillation counting from a sample of the dissolved protein precipitate³H]Incorporation of leucine into the newly synthesized protein.

MTT-Esta assay

Human keratinocytes were isolated and cultures established using standard culture techniques with a fully supplemented Green medium with a lethal dose irradiated mouse 3T3 cell feeder layer. Cells were expanded to passage 3 and seeded in 24-well plates in Green medium with or without Fetal Calf Serum (FCS) and 3T3 cells. In the selected treatment, the wells are coated with the protein isolate complex. The wells were incubated with 300ng vitronectin for 2 hours and then aspirated before IGF-I and IGFBP3 or IGFBP5, or IGF-II were added. Plates were incubated overnight and aspirated before seeding with cells. The metabolic activity of the cultures was assessed using the MTT-esta assay previously described (Ealey et al, 1988, J Mol Endocrinol 1: R1-R4).

Results

In view of the significantly enhanced functional response obtained with isolated growth factor complexes in cell lines (International publication WO 02/24219; Noble et al, 2003, supra; Kricker et al, 2003, supra), we have recently extended our research to cultures of adult skin-derived keratinocytes. In particular, we examined the potential of isolated growth factor complexes to replace serum and feeder cells in the best clinical practice of ex vivo expansion of keratinocytes currently used for stratified autologous transplantation. Although this method is a significant advance treatment available to burn patients, the culture of patient-derived keratinocytes is performed in the presence of Fetal Bovine Serum (FBS), a semi-defined (semi-defined) xenobiotic product that is a potential pathogen. In addition, in the early stages of keratinocyte derivation and establishment, feeder layers derived from another substance, mouse 3T3 fibroblasts, were used as a source of cytokines and matrix components to promote cell attachment and growth. FBS also contributes to these effects.

Because (i) IGF accounts for a large portion of the cytokines secreted by feeder cells; (ii) we have determined that VN, instead of any need for serum, promotes the attachment of primary cultured keratinocytes seeded at low density to plastic ware; (iii) we obtained comparable results with the keratinocyte cell lines cultured on isolated growth factor complexes to those obtained with media containing 10% FBS, so we hypothesized that media supplemented with isolated growth factor complexes have the potential to provide better engineering applications of autologous keratinocytes. This hypothesis is supported by the fact that IGF is a key mitogen that stimulates keratinocyte proliferation, while keratinocytes themselves do not secrete IGF-I. Despite the absence of serum media such as KGM^TM(Clonetics) and EpiLife^TM(Sigma-Aldrich) has been developed commercially for the expansion of keratinocytes, but these media require the addition of bovine pituitary extract, which is also an undefined xenobiotic and potentially pathogenic agent, or require the addition of expensive supplements. In addition, the current use of most serum-free keratinocyte culture media requires very high seeding densities, which defeats the purpose of attempting to rapidly culture large numbers of keratinocytes and has led to these practices being rarely adopted by routine clinical use.

We examined our hypothesis directly and the results are illustrated in fig. 4. In this experiment, keratinocytes were derived from adult skin and established using conventional methods, and grown for 7 days. The cells were then passaged by trypsinization and at low density (8500 keratinocytes/cm)²) Seeded on tissue culture plastic coated with isolated growth factor complexes and cultured for an additional 7 days without feeder cells, with FBS and insulin removed (figure 4). Cells cultured under these conditions were found to expand more rapidly than cells cultured using only the current best clinical practice (i.e., in the presence of FBS and 3T 3; FIG. 4). The edges of colonies grown in the presence of the isolated protein complex indicate that keratinocytes are mobile, healthy and proliferating in appearance. The innermost cells shown in fig. 4 show the typical flattened morphology observed in near confluent keratinocyte cultures, and in this case only 7 days of resulting fusion. These results were confirmed by quantification of keratinocyte proliferation in the presence of these protein complexes by the MTT assay (figure 4B).

Later data tend to indicate that the ability of keratinocytes to grow well in the absence of feeder cells (also serum-free) is limited to later stages of cell culture, as feeder cells appear to be important for the establishment of cultures from initial biopsies.

The effects of the other growth factors EGF and bFGF are shown in figure 5. We examined generation 3 human skin keratinocytes (from adult skin biopsies) and evaluated the stimulation of protein synthesis by supplementation with IGF + VN complex for 48 hours. These treatments were tested in parallel with cells cultured in established keratinocyte-SFM (dkm) (invitrogen), a commercially available product for serum-free culture of keratinocytes containing indeterminate amounts of insulin, EGF and bFGF. The increase in DKM-stimulated protein synthesis was found to be 148% higher than that of the control well (-VN), which was significantly higher (p < 0.05) than the effect of VN (+ VN) or no VN and growth factor (-VN) alone. The dimer IGF-II + VN complex and the trimer IGF-II + VN + IGFBP-5 complex also significantly stimulated an increase in protein synthesis of 134% and 161%, respectively (p < 0.05). There was virtually no significant difference in the observed stimulation of protein synthesis for DKM, dimeric and trimeric complexes (p > 0.05), indicating that these two complexes are equivalent in stimulating keratinocyte protein synthesis compared to the commercially available DKM.

When EGF, bFGF or a combination of the two growth factors were added to the trimeric complex, 216%, 248% and 213% increase was observed. All these reactions were significantly higher than DKM (p < 0.05). In addition, when EGF or EGF and bFGF were added to the dimeric complex, significant increases of 192% and 198%, respectively, in protein synthesis were obtained, which were also significantly higher than DKM (p < 0.05). These results highlight that the addition of EGF and bFGF to the protein complex stimulates higher protein synthesis than commercially available products for serum-free and feeder-free culture of keratinocytes.

Example 3

Skin spray technology

Materials and methods

This is emphasized by two factors. First, a sufficient number of cells were generated on VitroGro to support the administration of a nebulized cell suspension for one week. Therefore, this technique is comparable to the commercial technique (Clinical Cell Culture Ltd), but has the advantage of being serum-free. Second, cells grown on VitroGro remained viable after nebulization. The delivery system we used is(Baxter Healthcare). The Tissomat delivery system is designed for spray application of fibrin glue by aerosolizing a liquid by delivery into a regulator-controlled stream of compressed medical grade gas. However, we also contemplate that similar results can be obtained using other spray methods (syringes equipped with spray caps). Pressures between 10-30psi are suitable, although a decrease in viability is observed with increasing pressure. The concentration of sprayed cells may be 50-150 million per ml. Administration of 0.2ml of cell suspension at 20psi was sufficient to cover an area of approximately 25 square centimeters (based on a measurement of cell coverage surface area after 7 days of in vitro growth). Cells may be delivered in serum-free growth medium, but may also be suspended in fibrin glue, such as commercially available Tisseel/Tissucol (Baxter healthcare). Our studies indicate that the fibrin glue should be adjusted before use by diluting to an isotonic state with sterile water for injection and further adjusting the final fibrin glue composition to 1-10mg/ml fibrinogen and 10-100 units/ml thrombin with sterile saline.

Results

Figure 6 shows cell distribution and growth after spray delivery of keratinocytes to collagen coated culture dishes 150mm in diameter. Importantly, the cells grown on VitroGro showed good viability after spraying. Cells were sprayed at two different concentrations to determine the number of cells needed to cover the area of the spray. Cultures for nebulization were initially grown on control (with serum) or vitronectin (VitroGro) with IGFBP3 and IGF-I. All cultures were prepared in the presence of 3t3 cells. After spraying, cells were grown in the presence of serum to mimic conditions that might be experienced on the wound surface. The cultures were stained with crystal violet to show cell distribution.

As shown in fig. 7, the effect of spraying cultured keratinocytes with the Tissomat delivery system can be seen. For these preliminary experiments, cultures were established using conventional media supplemented with serum and feeder cells. In fig. 7A, the trypan blue exclusion assay was performed within minutes after the sprayed cells entered the collection tube, based on the principle that the dye was unable to penetrate the viable cells. As shown in fig. 7B, the MTT conversion data is a more robust measure of viability as it provides an indication that the cells are metabolically active 24 hours after nebulization.

In FIGS. 7A and 7B, the optimal delivery pressure can be seen to be 10-20psi, although viability is still acceptable at a delivery pressure of 30 psi.

Example 4

Clinical test for skin spray

Obtaining skin biopsies

The appropriate donor site is selected and prepared by shaving and wiping with a disinfectant. Approximately 10 square centimeter area of the delaminated skin graft was removed in the operating room under local anesthesia. The biopsy was placed in sterile saline solution containing antibiotics and immediately sent to a skin culture laboratory for processing. The donor site will be applied with Opsite or other dressing according to the judgment of the referring surgeon.

Isolation and culture of keratinocytes

When the skin culture is delivered, each patient's biopsy will be aseptically bufferedWashed in solution and incubated for 1 hour at room temperature in antibiotics to reduce the possibility of contamination during subsequent incubation. The epidermal and dermal layers will be separated by trypsinization. The opposite side of the isolated tissue was scraped and the scraped cells (mainly basal keratinocytes) were washed and resuspended in serum-free medium containing soybean trypsin inhibitor. The final cell suspension was seeded on mouse 3t3 fibroblasts (2.5X 10) containing growth arrest⁴/cm²) And 5ml of DMEM/F12 medium 25cm²The DMEM/F12 medium was supplemented with vitronectin (VN, 50 ng/cm)²) Insulin-like growth factor I (IGF-I, 15 ng/cm)²) Insulin-like growth factor binding protein 5(IGFBP5, 50 ng/cm)²) Epidermal growth factor (EGF, 15 ng/cm)²) Adenine (180. mu.M), cholera toxin (0.1. mu.g/ml), L-glutamine (2mM), hydrocortisone (0.4. mu.g/ml) and non-essential amino acids (1% v/v). VN (300 ng/cm) for culture flask²)、IGF-I(100ng/cm²)、IGFBP5(500ng/cm²) And EGF (100 ng/cm)²) Pre-treatment to promote pre-adsorption of the protein complex. Fresh medium was used after 3 days of culture. After 6 days of culture, 3t3 cells were removed by incubation in buffered saline containing EDTA. The remaining keratinocytes were harvested by further incubation with trypsin/EDTA and washed in buffered saline containing soybean trypsin inhibitor. Adjusting the recovered cells to 2X 10⁶At a concentration of/ml and delivered to the operating room in buffered saline containing 0.2% human serum albumin.

Preparation and delivery of keratinocyte suspension

TISSEEL Duo according to the manufacturer's instructionsThawing at 37 deg.C. Once thawed, the fibrinogen and thrombin syringes were removed from their respective holders and applied to sterile plastic tubing. The fibrinogen component was diluted 1: 1 with sterile water for injection and then further diluted with a stock patient cell suspension (2X 10)⁶Individual cells/ml, prepared as in step 2) Diluting at a ratio of 1: 4. The thrombin component will be diluted 1: 0.25 (i.e., 4: 1) with sterile water for injection. Equal volumes of each modified component (fibrinogen + cells and thrombin) were loaded into separate 1ml syringes and connected to the TISSOMAT via a Duploject spray nozzle. In application, two syringes were equally depressurised to allow further 1: 1 mixing of the ingredients. Thus, the final concentration in the spray product is: 0.8X 10⁶Individual cells/ml, 170IU/ml thrombin and 4.7mg/ml fibrinogen. Approximately 0.5ml of the mixed solution will be delivered continuously from a height of 10cm to 20cm each at 20psi²On the stratified wound surface. Thus the average seeding density of the administered cells will be 0.2X 10⁵/cm². The height and distance (interval) of each spray is similar to the hand width (height) and the total width (distance) of the three fingers. In the course of performing a conventional stratified autograft (burn treatment or contracture relief), treatment of a wound is performed. Approximately half of each wound in the spray was covered with a sterile hood for use as an untreated control. Two donor sites can be used: one treated and the other untreated. Each wound will be photographed before and after application of the cell suspension. The treated wound will be covered with Opsite silicone dressing.

Clinical care and evaluation after surgery was performed according to established protocols.

Example 5

Growth and migration of ORS-derived cells

Primary Outer Root Sheath (ORS) cultures were derived from anagen follicles obtained from the scalp of consenting diabetic patients and cultured as described by Limat & Hunziker, 2002, Cells Tissues Organs 17279-85 and International publication WO 01/59442. Cells will be expanded ex vivo using a preformed human postmitotic dermal fibroblast feeder layer and the above described medium supplemented with fetal bovine serum for skin-derived keratinocytes. The cultures will be maintained in a sub-confluent state for maximum three passages and for morphological and functional assessment of cell growth in the presence of isolated protein complexes without serum and feeder cells.

The specific complex identified as optimal for the growth of skin-derived keratinocytes was tested.

Growth and migration of ex vivo expanded ORS-derived keratinocyte progenitor cells in the presence of isolated protein complexes was established, and then it was determined whether initial derivation of cells from a growing ORS and subsequent primary culture could also be performed under serum-free and feeder cell-free conditions. Thus, ORS of anagen follicles will be explanted onto the microporous membrane of the cell culture insert, and instead of coating the underside of the membrane insert with a postmitotic dermal fibroblast feeder layer, the underside will be coated with an isolated protein complex. Cells will only grow in serum-free medium or in autologous serum-supplemented medium obtained from the patient or medium containing the isolated protein complex. The growth rate of ORS-derived cells cultured in the presence of the isolated protein complex was compared to cells cultured on the insert using standard methods.

Epidermal equivalents (equivalents) will also be prepared by exposing cells to air as described by Limat & Hunziker, 2002, supra, and characterized by histological, ultrastructural (e.g., basement membrane-like structures, keratohyaline granules, keratosomes) and immunohistochemical (e.g., keratin, integrin, gp80, endothelial, silk keratin) standards. If successful, the use of ORS-derived progenitor cells and this growth factor + VN technology will not only significantly reduce production costs, but will also increase safety, thereby speeding up regulatory issues associated with the approval of cell-based therapies.

Example 6

Preparation of pure vitronectin

Autologous VN (typically present at 0.4 mg/ml) purified from the patient's blood will be used to support the ex vivo growth of the patient's own keratinocytes. We will evaluate monoclonal antibodies raised against vitronectin, which have been successfully used to purify vitronectin from human serum (Underwood et al, 2001, J immunological methods.247217-24). SelectingMonoclonal antibodies selected for evaluation will be conjugated to the supporting purification matrix using methods similar to those described for purification of VN from serum. At this point, we estimate that 0.25mg VN will be required to culture 1m²Should be readily available from 20 ml of patient blood. The purification method of Underwood et al, 2001, supra, will be improved with emphasis on minimal operability and simplicity: the aim was to develop a disposable affinity purification matrix which ideally requires only 2-3 washing steps. As VN will be from the patient himself, the need for pure VN will be reduced as long as the VN obtained is still effective in promoting cell growth. VN purified using a developed protocol will therefore be evaluated for efficacy in promoting keratinocyte growth, as well as by standard biochemical assays such as SDS-PAGE, N-terminal protein sequencing, electrospray mass analysis, IGF-and IGFBP-binding, and compared to VN purchased from Promega pty.

The purpose served by the specification is to describe the preferred embodiments of the invention, not to limit the invention to any one embodiment or specific set of features. Thus, those skilled in the art will appreciate that, in light of the present disclosure, various modifications and changes may be made in the specific embodiments illustrated without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Claims (30)

1. A mammalian epithelial cell culture medium comprising:

(i) at least one IGF selected from IGF-I and IGF-II, wherein said medium comprises an insulin-like growth factor binding protein (IGFBP) selected from IGFBP3 and IGFBP5 when IGF-I is present as a non-IGF-I/VN synthetic chimera;

(ii) vitronectin (VN) or an integrin binding domain thereof;

(iii) epidermal Growth Factor (EGF) and/or basic fibroblast growth factor (bFGF); and

(iv) serum in an amount that is serum-free or does not support epithelial cell growth in the absence of said at least one IGF.

2. The mammalian epithelial cell culture medium of claim 1, wherein serum is absent or present at a concentration of no more than 1% v/v.

3. The mammalian epithelial cell culture medium of claim 2, wherein the serum is present at a concentration of no more than 0.5% v/v.

4. The mammalian epithelial cell culture medium of claim 3, wherein the serum is present at a concentration of no more than 0.1% v/v.

5. The mammalian epithelial cell culture medium of claim 1, wherein serum is absent.

6. The mammalian epithelial cell culture medium of claim 1, wherein said IGF is IGF-II.

7. The mammalian epithelial cell culture medium of claim 1, wherein said IGF is IGF-I.

8. The mammalian epithelial cell culture medium of claim 1, wherein said IGFBP is IGFBP 3.

9. The mammalian epithelial cell culture medium of claim 1, wherein the VN domain does not comprise a Heparin Binding Domain (HBD).

10. The mammalian epithelial cell culture medium of claim 9, wherein the VN domain comprises a polyanionic region.

11. The mammalian epithelial cell culture medium of claim 10, whereinThe VN domain is capable of binding to alpha_vIntegrin receptors.

12. The mammalian epithelial cell culture medium of claim 11, wherein the VN domain is capable of binding to a selected from the group consisting of_vβ₃Integrins or alpha_vβ₅Integrin receptors for integrins.

13. The mammalian epithelial cell culture medium of claim 1, wherein Vitronectin (VN) is purified autologous Vitronectin (VN).

14. The mammalian epithelial cell culture medium of claim 1 comprising IGF-I, IGFBP and vitronectin or an integrin binding domain thereof in the form of an isolated protein complex.

15. The mammalian epithelial cell culture medium of claim 1 comprising IGF-II and vitronectin or an integrin binding domain thereof in the form of an isolated protein complex.

16. The mammalian epithelial cell culture medium of claim 13 or claim 14, wherein the isolated protein complex is a synthetic chimeric protein.

17. The mammalian epithelial cell culture medium of claim 1, for culturing epithelial cells.

18. A mammalian epithelial cell culture system comprising a culture vessel and the mammalian epithelial cell culture medium of any one of claims 1-17.

19. The mammalian epithelial cell culture system of claim 18, comprising vitronectin and/or fibronectin or integrin binding domains thereof immobilized, bound or associated to the culture vessel.

20. A mammalian epithelial cell culture method comprising the step of culturing one or more mammalian epithelial cells in the mammalian epithelial cell culture system of claim 18 or claim 19.

21. The method of claim 20, wherein feeder cells are absent for at least a portion of the duration of the culture.

22. The method of claim 20 or claim 21, wherein the one or more mammalian epithelial cells are keratinocytes or keratinocyte progenitor cells.

23. The method of claim 20 or claim 21, wherein the one or more mammalian epithelial cells are corneal cells.

24. A method of preparing a pharmaceutical composition for aerosol delivery of keratinocytes or keratinocyte progenitor cells comprising the steps of: culturing one or more keratinocytes according to the method of any one of claims 20-22; and combining the cultured keratinocytes with a pharmaceutically acceptable carrier, diluent or excipient.

25. The method of claim 24, wherein the pharmaceutical composition further comprises a propellant.

26. The method of claim 25, the pharmaceutical composition further comprising a fibrin glue.

27. The method of claim 26, the pharmaceutical composition further comprising at least one IGF selected from IGF-I and IGF-II.

28. The method of claim 27, wherein the pharmaceutical composition comprises IGF-I, IGFBP and vitronectin or a fragment thereof in the form of an isolated protein complex.

29. The method of claim 27, wherein the pharmaceutical composition comprises IGF-II and vitronectin, or a fragment thereof, in the form of an isolated protein complex.

30. Use of a pharmaceutical composition prepared according to the process of any one of claims 24-29 in the preparation of a medicament for the in situ regeneration of skin.