HK1142492B - Use of a globin, a globin protomer or an extracellular hemoglobin for the preservation of organs, tissues, organ and tissue cells and cell cultures - Google Patents

Abstract

The invention relates to the use of at least one globin and/or at least one globin protomer and/or at least one naturally extracellular native hemoglobin for the preservation of organs, tissues, organ and tissue cells and cell cultures.

Description

The present invention describes the use of at least one globin, and/or at least one globin protomer, and/or at least one native, naturally extracellular hemoglobin, for the preservation of organs, tissues, or cells of organs or tissues, or for cell culture.

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The first images obtained on extracellular haemoglobins from Arenicoles (J. Roche, M. Bessis and J.P. Thiery, Biochim. Biophys. Acta 41 182-184 (1960); J. Roche, M.T. Bessis and J.P. Thiery, C. R. Soc. Biol. 154 73-80 (1960)) revealed hexagonal elements. Each haemoglobin molecule is made up of two superimposed hexagons (O. Levin, J. Mol. Biol. 2.95-101 (1963); J. Roche, Electron molecular microscope studies on high weight chlothrocruorins (in erybrate hemoglobins) and chlorocruorins of Oxford annelids., D.A. M. (Edagagag, M.E., 62-80, Pressday, 1965)) which is named after the bioluminescence hexagon and each hexagon is formed by the same six elements (E) in the form of a hexagonal hexagonal hexagonal (E) and is called the Permond.Err1:Expecting ',' delimiter: line 1 column 593 (char 592)a molecular mass of between 200 and 250 kDa constituting the functional unit of the native molecule. The structure of its extracellular haemoglobin is already known (F. Zal, B. N. Green, F. H. Lallier, S. N. Vinogradov and A. Toulmond, Eur. J. Biochem. 243 85-92 (1997)). WO01/92320 and Rousselot et al (Biotechnology Journal, vol.1, no.3, 2006, pp. 333-345) report on the use of haemoglobin from Arenicola marina as a blood substitute. DE10220990 describes the use of myoglobins and haemoglobins in the culture of cells. WO02007/856 reports on the use of haemoglobin from Arenicola marina to treat calcium-bound disorders.Badet al. (Use of preservative fluids in renal transplantation, Prog Urol, 2006, 16, 25-31) describes recommendations for the use of organ preservative fluids. Organ transplantation involves replacing a diseased organ from a patient with a healthy organ, called a graft, from a donor. The oxygen supply to all organs and tissues in the human body is provided by the hemoglobin present in the human or animal bloodstream. After the organ is taken from the donor, the organ is no longer supplied with oxygen and therefore, during the organ transplant process, the maintenance of life, as long as possible, of the organ outside the body during the time between the donor's taking of the organ,The first is the need to ensure that the patient is properly treated and that the patient is treated with the appropriate medical care. In normothermy (37°C), the interruption of vascularisation of an organ leads to rapid necrosis of the cells that constitute it. The solution of organ preservation intervenes in order to protect it. One of the main principles of organ preservation is to quickly lower its temperature from 37°C to 4°C. Indeed, the decrease in the temperature of the tissues leads to a decrease in the cellular metabolism, without stopping it (Belzer F.O., Southard J.H. Principles of solid-organ preservation by cold storage. Transplantation 1988; 45(4): 673-676.) Hypothermia and the composition of the preservative solution help to combat the deleterious effects of oxygen and nutrient deprivation induced by blood circulation arrest and delay cell death, which is responsible for tissue necrosis. During the collection phase, the graft will be infused with the preservative solution to rinse it and remove the donor blood ( rinse solution ).The organ is cooled following hypothermic infusion performed ex vivo or in situ depending on whether it is a live or cadaver donor sample. Washing also serves to balance the organ with the components of the solution. The period from the beginning of the implantation in the donor to the end of the implantation in the recipient is critical: this is the duration of total ischemia. This stage is the source of many of the observed deleterious effects. Ischemia can be defined as insufficient tissue blood supply, with loss of three important functions of blood flow: nutrient supply, oxygenation and waste disposal.

A distinction is made between the period of hot ischemia, when the organ is no longer infused with the donor's blood but not yet refrigerated, and the period of cold ischemia, when the organ is washed and refrigerated until it is revascularized in the recipient.

Hypothermia is the essential element of preservation. It reduces tissue metabolism, i.e. it slows down the catalytic enzymatic activity necessary for cellular viability. Metabolism is reported to be reduced by 12 to 13 times when the temperature goes from 37°C to 0°C (Belzer and Southard, Principles of solid-organ preservation by cold storage.Transplantation 1988; 454): 673 (((67).

The need to reduce the demand and consumption of oxygen and energy in the graft is due to the fact that the graft is in an ischemic state, which means that the tissue is deprived of oxygen, so that the synthesis of energy in the form of ATP is no longer provided by oxidative phosphorylation but by anaerobic glycolysis, which is much lower in efficiency.

The quality of the cold storage (∼4°C) will determine the success of reperfusion after implantation. The organ is simply immersed in the solution maintained at low temperature by ice ground under conditions of guaranteed asepsis: static cold storage.

The acceptable time to ensure the subsequent recovery of the transplant function varies from one organ to another. e.g. it is about 4-5 hours for the heart, 4-6 hours for the lung, 6 hours for the intestine, 10-16 hours for the liver, 24-35 hours for the kidney and 12-18 hours for the pancreas (Melle Delphine FORNAS thesis supported on 15 June 2001: Organ preservation solution: descriptive, regulatory status and registration in Europe; Claude Bernard University - I Lyon; Faculty of Pharmacy; Institute of Pharmaceutical and Biological Sciences).

Therefore, the development of conservation methods has been studied in particular.

Thus, US Patent 7,220,538 concerns a two-phase organ or cell preservation composition, comprising a first phase comprising a nutrient base medium and a second phase comprising nanoparticles containing a solution or suspension with a component capable of binding or providing oxygen which may be chemically modified haemolytic intracellular hemoglobin.

US Patent 6,994,654 relates to an organ and tissue preservative solution containing a high potassium electrolyte solution and an additive that may be PEG-Hemoglobin for a non-hemorrhagic delivery, preservation, transplantation and/or surgery process involving an organ or tissue. It specifies that oxygen-carrying molecules, used as an additive, are for thermal infusion . When the infusion is hypothermic, there is no presence of oxygen carrier at hypothermic temperatures, since vertebrate hemoglobin is derived from this molecule which does not have the same functional properties as hemoglobin at 37°C, particularly its affinity for oxygen, as its temperature depends on the affinity for oxygen.

US patent application 2006/0063142 concerns an organ infusion device and process for monitoring, maintaining and/or restoring organs and preserving organs during storage and/or transport.

The procedure consists of infusing an organ with a first medical fluid at a first temperature (preferably above 25°C) which may contain an oxygen carrier such as red blood cells or reticulated haemoglobin, after rinsing with a solution which may be VIASPANTM or other colloidal solutions containing dextran or HES (hydroxyethyl starch) or other equivalent compounds, and then infusing the organ with a second medical fluid which does not contain oxygen at a second temperature (preferably between 4 and 10°C) lower than the first.

US Patent 6,642,045 relates to a metabolic assist system comprising an organ or tissue that uses an infusion solution that may include an oxygen carrier such as hemoglobin, stabilized hemoglobin, haemoglobin polyoxyethylene conjugates or recombinant haemoglobin.

Application WO 01/01774 concerns a composition for the preservation of organs for transplantation containing bovine PEG-hemoglobin, one or more essential electrolytes, at least one soluble protein, at least one nutritional formulation and at least one cardiovascular agent.

In these papers, the hemoglobin used is of human or mammalian origin and is either in red blood cells or in liposomes, or it is cross-linked or cross-linked with polyethylene glycol (PEG-hemoglobin) to avoid oxidation because it is well known that when a hemoglobin is isolated from the red blood cell, it will oxidize due to the absence of the antioxidant activity of enzymes present in the red blood cell. (Savitsky JP, Doczi J, Black & JD serum (1978) A Thermal safety trial of stroma-hemoglobin. Clinlinlin Pharma 23, 73-80), (Channel, W.L., NL, Yim Lai, Tam (2000); This chemical exchange tends to be induced by the normal chemical exchange of stroma and red blood cells.

The use of human or mammalian haemoglobin also allows only normothermal temperatures for organ preservation because below this, red blood cell functioning is severely impaired (Jensen, FB, Wang T., Brahm, J., 2001, Acute and chronic influence of temperature on red blood cell anion exchange, 204, 39-45).

However, improving the survival of the transplanted person during ischemia over a longer period will allow better study of immunology and operating conditions, thereby promoting successful transplantation.

Similarly, improved oxygenation of the graft and hence its quality will allow a faster recovery.

Therefore, one of the objects of the invention is to provide globins, and/or globin protomers and/or extracellular haemoglobins in combination with an organ preservation medium to provide a composition for preserving organs, tissues, or cells of organs or tissues, or for cell culture, as defined in claim 1.

Another object of the invention is to provide a preservative composition of organs, tissues, or cells of organs or tissues, as defined in claim 1, without the need for blood typing.

Another aspect of the invention is to provide an organ, tissue or organ or tissue cell preservative composition, as defined in claim 1, that allows for prolonged use over time and can operate at hypothermic temperatures.

A cell culture composition capable of functioning at hypothermic temperatures is also described.

Another object of the invention is to provide a composition comprising at least one globin and/or at least one globin protomer and/or at least one extracellular hemoglobin in an organ preservation medium, as defined in claim 1, allowing the preservation of organs, tissues or cells of organs or tissues or the culture of cells.

The invention also relates to the provision of a method for the preservation of organs, tissues or cells of organs or tissues, as defined in claim 8.

A procedure for organ infusion is also described.

A cell culture method is also described.

Therefore, the invention describes the use of at least one globin and/or at least one globin protomer and/or at least one naturally occurring extracellular haemoglobin from an invertebrate animal selected from the phylum of annelids and in particular at least one globin and/or at least one globin protomer and/or at least one extracellular haemoglobin from sea worms such as Arenicola marina, at a concentration, relative to the final volume, of 0,625 mg/ml to 100 mg/ml, preferably 0,625 mg/ml to 20 mg/ml, preferably 0,625 mg/ml to 5 mg/ml, in particular 1,25 mg/ml, in combination with an organ or cell culture medium, or a culture medium for the preservation of organs, tissues, tissues, organs, tissues, organs, or tissue, or for the perfusion of organs or cells.

globin protomer , the twelfth or dodecamere of globin.

native means a globin, or a globin protomer or a hemoglobin originating from that invertebrate animal.

By naturally extracellular , we mean a globin or globin protomer or hemoglobin that is naturally not contained in a cell and can therefore circulate freely in the circulatory system without chemical modification to stabilize and function.

Arenicola marina extracellular hemoglobin is a giant biopolymer with a mass of about 3 to 4 million daltons and is composed of about 200 polypeptide chains of two types. Three quarters are globin-like chains capable of reversible fixation of oxygen (O2) and the remaining quarter are chains of structures ( linkers) that maintain the quaternary structure and are thought to be responsible for the antioxidant activity of this molecule.

Therefore, extracellular hemoglobin, or globin protomer or invertebrate globin can be made up of a single polypeptide chain up to about several hundred polypeptide chains, or a molecular weight ranging from about 15,000 Daltons to about 8 million Daltons.

The use of at least one globin and/or at least one globin protomer and/or at least one extracellular native haemoglobin can affect the intrinsic superoxide dismutase (SOD) activity (determined by the method of Flohé & Ötting; Flohé L, Otting F. Methods Enzymol (1984), 105, 93-104) of that haemoglobin or globin or globin protomer, thus providing intrinsic antioxidant activity and therefore no antioxidant required to function, in particular when using mammalian haemoglobin for which the anti-oxidant molecules inside the red globulin and not bound to the protective globulin are not required to function, or in the case of human globulin, in particular, the extracellular globulin, or globulin, are not required to function.

An invertebrate is an animal that does not have a spine, such as jellyfish, sponges, insects, crustaceans, molluscs, or an animal that is a member of the annelid family.

The haemoglobin concentration in the sea worm Arenicola marina is between 100 and 170 g/L of blood (Toulmond, A. (1975). Research on the respiratory physiology of the Annelid Polychthes Arenicola marina (L.).

The use of at least one globin and/or at least one globin protomer and/or at least one extracellular native hemoglobin avoids the necessary osmotic pressure control with the use of red blood cells. Globin, globin protomer and extracellular hemoglobin without blood type also avoid any immune response problems encountered with the use of mammalian red blood cells such as human or bovine hemoglobin contained in blood cells that have different types of glycan responsible for blood type on their surface.

The organs and tissues are derived from animals, including humans, mammals, birds, reptiles, fish or insects.

A tissue is a collection of identical cells, or at least of the same origin, that share a common function.

The term preservative means any medium capable of protecting organs and/or cells from the deleterious effects of reperfusion ischemia by meeting the minimum metabolic requirements of the organs and/or cells.

The preservative media are aqueous solutions containing electrolytes such as potassium, sodium, magnesium, calcium, chloride, sulphate, containing where appropriate impermeants such as mannitol, raffinose, sucrose, glucose, fructose, lactobionate or gluconate, and may also contain colloids such as abumin, hydroxyethyl starch, polyethylene glycol or dextran 40.

Cell culture media are commercially available and very varied, for example, but not limited to, the following media are available from Invitrogen: D-MEM, D-MEMIF-12, MEM, RPMI 1640, or 199... or any similar medium.

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All these organ preservation media are commercial products.

The cell culture described above may be that of vertebrate cells.

A vertebrate is a mammal, reptile, amphibian, bird, or fish.

Vertebrate cells correspond to all types of cells belonging to a vertebrate animal and may be, for example, but not limited to, kidney, liver, pancreatic, heart, lung, intestinal, stomach, colon cells.

The cell culture defined above may correspond to cells from invertebrates.

An invertebrate is an animal that has no spine, such as insects, molluscs, annulars, cnidarians, sponges, etc.

Invertebrate cells correspond to all types of cells belonging to an invertebrate animal and may be, for example, but not limited to, perivascular/haematopoietic tissue cells or others.

In a preferred method of manufacture, the temperature of the organ or cell preservation compositions or cell cultures defined above is between 4°C and 37°C, preferably 4°C and 25°C, more preferably 4°C and 15°C, in particular 4°C.

Thus, the use of at least one globin and/or at least one globin protomer and/or at least one extracellular native hemoglobin allows a composition to be formed that is able to work under hypothermic conditions, which is essential for conservation, since hypothermia reduces tissue metabolism, i.e. it slows down the catalytic enzymatic activity necessary for cellular viability.

The use of at least one globin and/or at least one globin protomer and/or at least one extracellular native hemoglobin also allows the culture of marine invertebrate cells, which is not currently possible. Primary cultures of marine invertebrates are carried out on some types of invertebrates (some are maintained for several months) but no cell line has been established. (Rinkevich B, Mar Biotechnol (NY). 2005 Sep-Oct;7(5):429-39).

In another method, the temperature of the organ infusion or cell culture composition is between 4°C and 37°C, preferably around 15°C to around 37°C, more preferably between 25°C and 37°C, especially 37°C.

When an organ is infused, the infusion temperature must generally be normothermal, i.e. close to physiological temperature.

In another respect, the invention relates to a composition as defined in claim 1 comprising at least one globin and/or at least one globin protomer and/or at least one haemoglobin, extracellular native, from an invertebrate animal selected from the phylum annelids and including at least one globin and/or at least one protomer and/or at least one extracellular haemoglobin belonging to marine animals such as Arenicola marina, at a concentration, relative to final volume, of 0,625 mg/ml to 100 mg/ml, preferably 0,625 mg/ml to 20 mg/ml, more preferably 0,625 mg/ml to 5 mg/ml, in particular 1,25 mg/ml; and an organ preservation medium.

The composition allows for the preservation of organs or tissues, or cells of organs or tissues, or the perfusion of organs or tissues.

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In another respect, a composition is described comprising at least one globin and/or at least one globin protomer and/or at least one haemoglobin, extracellular native, from an invertebrate animal selected from the phylum annelids and including at least one globin and/or at least one globin protomer and/or at least one extracellular haemoglobin from marine worms such as Arenicola marina, at a concentration, relative to final volume, of 0,625 mg/ml to 100 mg/ml, preferably 0,625 mg/ml to 20 mg/ml, preferably 0,625 mg/ml to 5 mg/ml, in particular 1,25 mg/ml; and a cell culture medium.

The invention also relates to a method of preserving an organ or tissue, or cells of an organ or tissue, which includes a static or dynamic preservation step of such an organ in a composition as defined in claim 1.

In a preferred method of manufacture, the process of preserving an organ or tissue, or cells of an organ or tissue, as defined above, shall include the following steps: removal of the organ or tissue, or of the cells of the organ or tissue;rinsing of the organ or tissue, or of the cells of the organ or tissue, at a temperature of 4°C to 37°C, preferably 4°C to 25°C, more preferably 4°C to 15°C, in particular 4°C, with a composition as defined above;static or dynamic preservation by perfusion of the organ or tissue, or of the cells of the organ or tissue, at a temperature of 4°C to 37°C, preferably 4°C to 25°C, more preferably 4°C to 15°C, in particular 4°C, for a specified period of time, or the tissue, or cells of the organ or tissue, in a composition as defined above.

The term time specified, depending on the organ or tissue, or on the cells of the organ or tissue means a storage time which is specific and depends on the organ used.

The acceptable time for the transplant to return to function varies from one organ to another. For example, it is about 4-5 hours for the heart, 4-6 hours for the lung, 6 hours for the intestine, 10-16 hours for the liver, 24-35 hours for the kidney and 12-18 hours for the pancreas. All these values are well known to the professional and can be found, for example, in the thesis of Ms Delphine Fornas (Thesis of Ms Delphine Fornas supported on 15 June 2001: Organ preservation solution: descriptive, regulatory status and registration in Europe; Claude Bernard University - Lyon I; Faculty of Pharmacy; Institute of Pharmaceutical and Biological Sciences).

A process for culturing organ cells or tissue from organs of vertebrates or invertebrates including a cell culture in a composition as defined above.

Depending on the preferred method of production, the procedure for preserving an organ, as defined above, includes the following steps: collection of the organ;rinsing of the organ or tissue or of cells of the organ or tissue at a temperature of 4°C to 37°C, preferably 15°C to 37°C, preferably 25°C to 37°C, with a composition as defined above;static or dynamic perfusion of the organ at a temperature of 4°C to 37°C, preferably 15°C to 37°C, preferably 25°C to 37°C, in particular 37°C, for a specified time, in The Commission shall be responsible for the function of that body, in the composition defined above.

The term time determined according to the organ means a storage time which is specific and depends on the organ used, as indicated above.

A process of culture of vertebrate cells, including the following steps: sampling of these cells from a vertebrate;culture at a temperature of about 4°C to about 37°C, preferably about 15°C to about 37°C, more preferably about 25°C to about 37°C, in particular about 37°C, for a specified time or not, depending on the cells, in a composition defined above;harvesting of cells by rinsing and lysing of the cell mat.

The expression time determined, depending on the cells refers to a culture time that is specific and depends on the type of cells used, the cell line used.

For example, it is usually from a few hours to about a week for cell cultures such as blood cells such as neutrophil polynuclear cells.

These cells cannot usually be kept in culture indefinitely, in particular because of their limited number of divisions (Hayflick limit).

For primary cultures, culture of cells that come directly from a tissue, it is usually from a few days to several weeks.

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Finally, in the case of immortal cells, this time is not determined and theoretically can be infinite.

A process for the culture of invertebrate cells is also described, including the following steps: the collection of these cells from an invertebrate;the culture at a temperature of about 4°C to about 37°C, preferably about 4°C to 25°C, more preferably about 4°C to about 15°C, in particular about 4°C, for a specified time, depending on the cells, in a composition defined above;the collection of cells by rinsing and lysing of the cell mat.

The expression time determined, depending on the cells refers to a conservation time that is specific and depends on the type of cells used, the cell line used.

The following is a description of the figures:

Figure 1 shows the kinetics of dissociation of haemoglobin from Arenicola marina (HbAm) in four organ storage media (white square: UW, black circle: IGL1, cross: Celsior, white triangle: BMPS Belzer). The grey square represents the stability of Arenicola marina haemoglobin in a buffer used to purify the molecule.Figure 2 shows the functional properties of Arenicola marina haemoglobin (HbAm) in different organ conservation environments. For each medium, the measure of P50 (full traces) representing the affinity of Arenicola marina haemoglobin for oxygen and the measure of n50 (dotted traces) representing cooperation are shown.Figure 3 shows the percentage of lactate dehydrogenase (LDH) released into the preservative by porcine renal tubular cells LLC PK1 (axis of orders) after 2 hours at 4°C based on the haemoglobin concentration of Arenicola marina (1.25 mg/ml to 20 mg/ml) in an organ preservation medium (ViaSpan®, Bristol-Myers-Squibb). The black columns represent the results obtained for the different concentrations of HbAm, the grey columns represent the results obtained with the buffer alone in ViaSpan®, in the absence of HbAm.Figure 4 represents the percentage of detected lactate dehydrogenase (LDH) released into the preservative surfactant by porcine LLC PK1 renal tubular cells (axis of orders) after 24 h at 4°C depending on the haemoglobin concentration of Arenicola marina (0,The dose of the test chemical should be measured at a concentration of 0.39 mg/ ml to 1.25 mg/ ml (abscissa axis) in an organ preservative (ViaSpan®, Bristol-Myers-Squibb). The black columns represent the results obtained for the different concentrations of HbAm.

The experimental part Example 1: Stability of haemoglobin in Arenicola marina in various organ conservation environments.

These studies were carried out in the laboratory to assess the stability of HbAm in different organ preservation media (colloidal solutions commonly used in organ transplantation and provided by Prof. T. Hauet's laboratory).

Kinetic analyses were carried out over 48 h from the chromatogram area under the curve and the percentage at 414 nm of the various subunits (corresponding to the absorbance of heme). where kd is the dissociation constant of HbAm.

These studies have shown that in these different organ conservation environments, HbAm is stable, non-oxidised and functional for at least 48 hours, which is fully compatible with the lifetime of the organs awaiting transplantation (Figure 1).

The dissociation constants and half-lives obtained for the different solutions indicate that HbAm is stable in these different organ conservation environments tested over 48 hours (dissociation < 3%, Table I). - What? Tableau I : Constantes de dissociation et temps de demi-vie de HbAm dans les différents milieux testés.

	HbAm
	0.0001	0.0002	0.0011	0.0004	0.0006
	∞	∞	625	1154	1705

Example 2: Function of the haemoglobin of Arenicola marina in different organ conservation media.

The measurement of P50 was carried out using the hemox technique (A. Toulmond et al., Biol. Bull. 179 366-373 (1990)) at 4° and over 48 h. The n50 was measured on the oxygen saturation curves of a respiratory pigment obtained by the hemox technique.

The results obtained in Figure 2 show that HbAm is functional in the different organ conservation environments tested as indicated by the observed P50 and n50 values (UW, IGL1, Celsior, Scot Maco, BPMS).

The affinity of HbAm for O2 is strong, ranging from 1-3 to ∼0.2 in SCOT Maco. The affinity is slightly higher in different organ conservation settings and particularly in SCOT Maco and increases slightly over 48 hours.

Cooperation is constant ∼1.5 between different conservation environments and over time.

Example 3: Efficacy test of haemoglobin from Arenicola marina at a concentration of 1,25 mg/ml to 20 mg/ml in an in vitro cold store model of kidney cells 1) Cold storage of the renal cell line - Biological materials

The experiments were carried out on the porcine renal tubular cell line LLC-PK1 (CL-101, Lot 1928865) (ATCC, LGC-Promochem, Molsheim, France) provided free of charge by the Inserm E0324 Laboratory 'Ischemia-reperfusion in renal transplantation' in Poitiers, led by Prof. G. Mauco.

The LLC-PK1 line is an untransformed cell line, established from epithelial cells from porcine proximal convoluted kidney tubes.

- Cell culture of LLC-PK1

The cells of LLC-PK1 are cultured in M199 medium (Ref 31150, Gibco-BrL, Invitrogen Life Technology) supplemented with 3% foetal calf serum (F7524, Lot 085K3397, Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (P4333, Sigma-Aldrich) and 2 mM L-glutamine (25030, Gibco-BrL). The cells are cultured at 37°C in a humid atmosphere containing 95% air and 5% CO2.

The test chemical is used to determine the concentration of the active substance in the test chemical.

In cold storage experiments, the cells are seeded in a 6 well culture plate (140675, Nunc) at a concentration of 2 x 105 cells/mL in 2 mL of culture medium per well. After 48 h of culture, the overlying material is removed and the cell mat is washed twice with a saline phosphate swab (PBS, 70011, GIBCO-BrL). The cells are then stored for 24 h at 4°C in the presence of 1.2 mL of a commercial preservative solution, UW solution (ViaSpan®, Bristol-Myers-Squibb), pre-added with Hb at concentrations of 0, 1.25, 2.5, 5, 10 or 20 mg/ml.

2) Detection of the release of lactate dehydrogenase (LDH)

The impact of the addition of HbAm preserved in Hemorgan Storage Buffer on the viability of LLC-PK1 cells preserved for 24 h at 4°C in UW solution was investigated.

The release of this enzyme into the extracellular environment is a reflection of the permeability of the plasma membrane of the cells and consequently cell death.

- How to operate

After 24 h of storage, the supernatant is removed, and the cell mat is then rinsed 3 times with 2 mL of PBS and the adhering cells are lysed in 1.2 mL of PBS containing 0.1% Triton®X-100 (X100, Sigma-Aldrich).

The amount of LDH present in the cell mat is determined by a colorimetric dosage as instructed by the supplier (TOX7, Sigma-Aldrich). This dosage is based on the reduction of NAD by LDH during the transformation of pyruvate into lactate. Briefly, 25 μL of the dosage sample is deposited in a 96-well plate before the addition of 25 μL of a reaction mixture containing 1 substrate volume (L2402), 1 cofactor volume (L2527) and 1 dye volume (L2277). The mixture is then gently homogenized and incubated for 5 min at room temperature in the dark. The reaction is performed by adding 5 μL of HCl to 1 N. The absorption of the sample is measured by photobenzene (DOX) and absorbed directly into the plastic sample at a temperature of 690 nm (905 nm) and the difference is measured by the photobenzene (DOX) and Bioxene (DOX) is measured at 630 nm (905 nm) and the absorption is measured by the photobenzene (DOX) in the sample is measured at a distance of 690 nm (905 nm) from the plastic sample.

- Results

The results shall be expressed as a percentage of the amount of LDH detected in the carpet of cells stored at 4°C compared to the amount of LDH detected in the carpet before the cold storage period (T0 cells).

- Conclusion

The preservation of LLC-PK1 cells for 24 hours at 4°C in UW (Viaspan) solution induces a high cell death (74%±9).

In addition, the presence of the Hemorgan Storage Buffer in the UW preservative solution does not alter the viability of the renal cells to a great extent.

On the other hand, the presence of HbAm during preservation, at a concentration of 1.25 g/L of the molecule, fully protects the viability of the cell mat.

Example 4: Efficacy test of haemoglobin from Arenicola marina at a concentration of 0,039 mg/ml to 1,25 mg/ml in an in vitro cold store model of renal cells

The biological material, cell culture and cold storage are prepared and carried out in the same way as in example 3.

- Concentration in HbAm:

The cells are stored for 24 hours at 4°C in the presence of 1.2 mL of a commercial preservative solution, UW (ViaSpan®, Bristol-Myers-Squibb), pre-added with HbAm at concentrations of 0.039, 0.078, 0.156, 0.312, 0.625 or 1.25 mg/mL.

- Results

The results shall be expressed as a percentage of the amount of LDH detected in the carpet of cells stored at 4°C compared to the amount of LDH detected in the carpet before the cold storage period (T0 cells). Tableau III : Pourcentage de libération de LDH obtenu pour les différentes concentrations en HbAm (les résultats correspondent à la moyenne de trois essais).

HbAm

- Conclusion

Preservation of LLC-PK1 cells for 24 h at 4°C in UW solution (ViaSpan®, Bristol-Myers-Squibb) induces significant cell death (76%±7). LDH release from renal tubular cells is markedly decreased in the presence of HbAm and this is dose-dependent. Thus, as low as 0.625 mg/ml, HbAm protects renal cells (25%±12 versus 76%±7) and at a concentration of 1.25 g/L, HbAm completely protects renal cells from cell death induced by 24 h cold conservation (7%5 versus 76%±7).

Claims (10)

1. Composition comprising:

   - at least one globin and/or at least one globin protomer and/or at least one extracellular hemoglobin of an invertebrate animal selected from the phylum Annelida, at a concentration, relative to the final volume, of 0.625 mg/ml to 100 mg/ml, and

   - an organ storage medium, said medium being capable of protecting the organs from the deleterious effects of ischemia while satisfying the minimum metabolic needs of the organs, and said medium being an aqueous solution containing electrolytes and impermeants.
2. Composition according to claim 1, characterized in that the globin and/or at least the globin protomer and/or at least the extracellular hemoglobin is of Arenicola marina.
3. Composition according to claim 1 or 2, characterized in that said organ storage medium further contains colloids.
4. Composition according to any one of claims 1 to 3, characterized in that said concentration relative to the final volume is of 0.625 mg/ml to 20 mg/ml, particularly of 0.625 mg/ml to 5 mg/ml, more particularly of 1.25 mg/ml.
5. Composition according to any one of claims 1 to 4, characterized in that said electrolytes are chosen from potassium, sodium, magnesium, calcium, chlorine or sulfate.
6. Composition according to any one of claims 1 to 5, characterized in that said impermeants are chosen from mannitol, raffinose, saccharose, glucose, fructose, lactobionate or gluconate.
7. Composition according to any one of claims 3 to 6, characterized in that said colloids are chosen from albumin, hydroxyethyl starch, polyethylene glycol or dextran 40.
8. Process for storage of an organ or tissue, or of organ or tissue cells, comprising a step of static or dynamic perfusion storage of said organ in a composition according to any one of claims 1 to 7.
9. Process for storage of an organ or tissue, or of organ or tissue cells according to claim 8, comprising the following steps:

   - rinsing of said organ or said tissue previously removed, or of said organ or tissue cells previously removed, at a temperature of 4°C to 15°C, with a composition according to any one of claims 1 to 7;

   - static or dynamic perfusion storage of said organ or said tissue, or of said organ or tissue cells, at a temperature of 4°C to 15°C, for a defined time, depending on said organ or tissue, or of said organ or tissue cells, in a composition according to any one of claims 1 to 7.
10. Process for storage of an organ according to claim 8, comprising the following steps:

    - rinsing of said organ previously removed, at a temperature of 25°C to 37°C, with a composition according to any one of claims 1 to 7;

    - static or dynamic perfusion storage of said organ, at a temperature of 25°C to 37°C, for a defined time, depending on said organ, in a composition according to any one of claims 1 to 7.