Injectable preserving medium for preserving cells from placental blood, from bone marrow and from peripheral blood

Abstract

The present invention relates to a solution for the preservation of cells from placental blood, from bone marrow and from peripheral blood.

Claims

1. An injectable solution for preserving cells, characterized in that it comprises a physiological saline solution of between 5 and 7 g/l sodium chloride and potassium chloride, between 0.05 and 2.4 g/l magnesium sulfate, citric acid, between 0.25 and 4.15 g/l of sodium bicarbonate, between 0.001 and 0.006 g/l of vitamin E, between 0.0002 and 0.0008 g/l of vitamin A, between 0.001 and 0.1 g/l of vitamin C, between 0.3 and 0.7 g/l of glutamine, between 5 and 20 g/l of human albumin and between 0.2 and 2.7 g/L of lactate, wherein the solution with the cells is injectable to a subject without the need for washing the cells and the solution does not comprise HEPES, sodium pyruvate or phosphate buffer.

2. The injectable solution according to claim 1, further comprising one or more elements selected from additional amino acids other than glutamine, glucose and mannitol.

3. The injectable solution according to claim 1, characterized in that it further comprises additional amino acids other than glutamine, glucose and mannitol.

4. A kit comprising the injectable preserving solution according to claim 1 and a sterile container for receiving cells from placental blood, from bone marrow and from peripheral blood.

5. A sterile container for receiving cells from placental blood, from bone marrow and from peripheral blood comprising the injectable preserving solution according to claim 1.

6. The container according to claim 5, characterized in that it further comprises cells from placental blood, from bone marrow and from peripheral blood.

7. The container according to claim 6, characterized in that the cells comprise CD34+cells.

8. A method for preserving cells from placental blood, from bone marrow and from peripheral blood, comprising the contacting of cells from placental blood, from bone marrow and from peripheral blood with the injectable solution according to claim 1.

9. The method according to claim 8, characterized in that the cells comprise CD34+cells.

10. The injectable solution according to claim 1, said solution further comprising cells from placental blood, bone marrow or peripheral blood.

11. The injectable solution according to claim 10, said cells being CD34+.

12. The injectable solution according to claim 1, further comprising between 1 and 25 g/L mannitol.

13. The injectable solution according to claim 1, characterized in that it comprises less than 10% by weight of components other than sodium chloride, potassium chloride, magnesium sulfate, citric acid, sodium bicarbonate, vitamin E, vitamin A, glutamine, human albumin, lactate, vitamin C, amino acids other than glutamine, glucose and mannitol.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Preservation yield of amplified CD34.sup.+ cells from placental blood, stored 48 h at 4° C. (n=12). For each condition, on the left, the TNC yield (total viable nucleated cells) and on the right the CFU (colony forming unit) allow evaluation of clonogenic functionality. The tested conditions were the following: D10 (end of culture), 4% HSA (medium comprising 4% human serum albumin), HP02 (reference medium by Macopharma), Formula 12 components (such as described in the examples in Table 2), without HSA (Formula 12 medium without human serum albumin), without Vits C,E,A (Cernevit®) (Formula 12 medium without Cernevit®), Vit C alone (Formula 12 medium without Cernevit® and with vitamin C), Vit E alone (Formula 12 medium without Cernevit® and with vitamin E), and Vit A alone (Formula 12 medium without Cernevit® and with vitamin A). Apart from D10, the other measurements are taken after 48 h storage of the cells at 4° C. The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes with de 2.5 ml of cell suspension for maximum avoidance of the presence of air).

(2) FIG. 2. Preservation of placental blood units after collection, CFU (n=9). D1, D3 and D7 respectively correspond to preservation for 1, 3 or 7 days at 4° C. PVC corresponds to storage in a PVC pouch, PVC NaCl comprises the addition of 0.9% sodium chloride, PVC SEC comprises the addition of SEC medium such as described in the examples in Table 2.

(3) FIG. 3. Preservation yield of amplified CD34.sup.+ cells from placental blood, stored 72 h at 4° C. (n=11). For each condition, on the left, the TNC yield (total viable nucleated cells) and on the right the CFU yield (colony forming units) allowing evaluation of clonogenic functionality. The tested conditions were the following: D10 (end of culture); M8 NaCl (medium comprising human serum albumin, amino acids, potassium chloride, glucose, glutamine, sodium bicarbonate, ACD A and 0.9% NaCl) with or without the addition of Cernevit®; M8 RL (medium comprising human serum albumin, amino acids, potassium chloride, glucose, glutamine, sodium bicarbonate, ACD A, and Ringer Lactate instead of 0.9% NaCl) with or without the addition of Cernevit®; M8 NaCl without HSA (M8 NaCl devoid of human albumin serum) with or without the addition of Cernevit®; M8 NaCl without AA (M8 NaCl devoid of amino acids) with or without the addition of Cernevit®; M8 NaCl without KCl (M8 NaCl devoid of KCl) with or without the addition of Cernevit®; M8 NaCl without GG (M8 NaCl devoid of glucose and glutamine) with or without the addition of Cernevit®; M8 NaCl without AAGG (M8 NaCl devoid of amino acids, glucose and glutamine) with or without the addition of Cernevit®; SEC 4 NaCl (components of Formula 12 medium without Ringer Lactate); SEC 4 RL (components of Formula 12 medium without 0.9% NaCl); HP02 (reference medium by Macopharma). The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air).

(4) FIG. 4. Preservation yield of amplified CD34.sup.+ cells from placental blood, stored 72 h at 4° C. (n=9). For each condition, on the left, the TNC yield (total viable nucleated cells) and on the right the CFU yield (colony forming units) allowing evaluation of clonogenic functionality. The tested conditions were the following: D10 (end of culture); HP02 (reference medium by Macopharma); SEC (SEC medium such as described in the examples in Table 2); without Mg and Mann (SEC medium but without magnesium and mannitol); without Magnesium (SEC medium but without magnesium); without Mannitol (SEC medium but without mannitol). The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air).

(5) FIG. 5. Preservation yield of amplified CD34.sup.+ cells from placental blood, stored for 48 h at 4° C. (n=9). For each condition, on the left, the TNC yield (total viable nucleated cells) and on the right the CFU yield (colony forming units) allowing evaluation of clonogenic functionality. The tested conditions were the following: D10 (end of culture); HP02 (reference medium by Macopharma); standard SEC (0.5% HSA) (SEC medium such as described in the examples in Table 2); SEC Mannitol (depleted of NaCl) (components of Formula 12 medium without 0.9% NaCl replaced by an identical volume of 4.55% mannitol [300 mos]); SEC Glucose (depleted of NaCl) (components of Formula 12 medium without 0.9% NaCl replaced by an identical volume of 5% glucose solution); SEC 3% HSA (SEC medium such as described in the examples in Table 2 but with 3% human serum albumin); SEC PVC pouch (the container here is a 20 ml gas-permeable sampling pouch in PVC). The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air) except with the PVC pouch.

(6) FIG. 6. Preservation yield of amplified CD34+ cells from placental blood, stored for 48 h at 4° C. (n=8) for a study on a range of CERNEVIT® volumes (and hence a molarity range of vitamin E) added to the «basic» SEC medium composed of 11 other components. The tested conditions were the following: Human serum albumin (4% HSA); HP02 (reference medium by Macopharma); «basic» SEC without CERNEVIT®; then «basic» SEC supplemented with decreasing volumes of CERNEVIT®, namely: 10 ml/l per 45 μM of vitamin E; 5 ml/l per 22.5 μM of vitamin E; 2 ml/l per 9 μM of vitamin E; 1 ml/1 per 4.5 μM of vitamin E; 0.5 ml/l per 2.25 μM of vitamin E; 0.2 ml/1 per 0.9 μM of vitamin E; 0.1 ml/l per 0.45 μM of vitamin E 0.05 ml/l per 0.225 μM of vitamin E; 0.025 ml/l per 0.112 μM of vitamin E. The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air).

(7) FIG. 7. Preservation yield of the clonogenicity of mononuclear cells (MNCs) and CD34+ cells (after immunomagnetic selection) of peripheral blood mobilised after 48 h and storage time of 120 h at 4° C. (n=1). The MNC cells, after thawing and removal of the cryoprotector, were divided into 2 aliquots one of which was resuspended in SEC medium (components of described Formula 12) and the other treated by immunomagnetic selection to obtain CD34+ cells of purity higher than 80% which were placed in suspension in the same SEC medium (components of described Formula 12). The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of suspension for maximum avoidance of the presence of air).

(8) FIG. 8. Preservation yield of the clonogenicity (CFU) of amplified CD34+ cells after storage in SEC medium at +4° C. for 48 h followed by primary grafting in NSG immunodeficient mice. The operations of this study consisted in different steps: graft production: placing in culture of CD34+ cells from placental blood (selected via immunomagnetic sorting) in a medium containing a cocktail of adapted cytokines for a period of 12 days. After culture, the amplified cells were harvested and divided into two parts: one part was grafted onto a batch of immunodeficient mice, each mouse receiving a quantity of amplified cells produced by 1000 CD34+ cells on start of culture; the other part was placed in SEC preserving medium (PVC pouch) at +4° C. for 48 h then injected into a second batch of immunodeficient mice under the same quantitative conditions. 8 weeks after grafting, the mice were sacrificed, the human clonogenic progenitors were analysed in the bone marrow of the mice: these progenitors are generated by the stem cells (key to graph: 1 circle corresponds to a grafted, analysed mouse (CFU/femur); the dotted line corresponds to the mean; the solid line corresponds to the median).

(9) FIG. 9. Preservation yield of the clonogenicity of CD34+ cells (cells of interest) from bone marrow (n=5). The CD34+ cells had been banked in LN2 several years ago (from 3 to 6 years). After thawing, the CD34+ cells were resuspended in SEC medium and stored at 4° C. After a storage time of 72 h at +4° C., clonogenic assays were performed. The assays were performed in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air).

(10) FIG. 10. Preservation yield of the viability and clonogenic potential of CD34+ cells from placental blood. The placental blood CD34+ cells were thawed and divided into 3 aliquots: suspending of the CD34+ cells in SEC medium, in HP02 medium and in 4% human albumin serum, followed by storage at 4° C. After a storage time of 72 h at +4° C., the analyses (counting of total viable nucleated cells and clonogenic functional tests) were carried out. The assays were conducted in gas-impermeable containers (2.5 ml capacity polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air).

EXAMPLES

(11) After finding a certain number of active ingredients in the Codex, the inventors selected injectable pharmaceutical preparations that they associated together to obtain a basic formulation that was subsequently assayed in comparison with the HP02 reference medium (Macopharma). The preservation results obtained after a storage time of 48 h and 72 h at +4° C. were practically identical for both media.

(12) The inventors therefore tested the advantage of each product and set out to determine the optimal effective concentration. Some components were excluded from the initial formulation either on account of no global action (SeNa), or because of possible recently recognized renal toxicity (6% Hydroxy-Ethyl-Starch).

(13) The inventors therefore evidenced the importance of vitamin E (Alpha-Tocopherol) and to a lesser extent that of vitamins A (Retinoic Acid) and C (Ascorbic Acid), of K+, Mg++ ions, and of Lactate and human albumin on the maintaining of viability and above all on the functionality (clonogenicity) of amplified CD34+ cells.

(14) Finally, the components of 12 pharmaceutical preparations were included in the formulation of the cell preserving biological medium. The preserving medium was prepared under sterile conditions in an EVA plastic pouch (Ethylene vinyl acetate) impervious to gases to prevent entry of atmospheric gases, in particular of dioxygen (O2), and especially so that the carbon dioxide (CO2) dissolved during pH adjustment (action of citric acid on Na bicarbonate) remained in the medium (promoting cell preservation at low temperature); thereby the pH of the medium therefore remains at the desired value for a period of several years. These pouches are used in the pharmaceutical industry to package injectable preparations, and by suppliers of biological reagents for the preservation inter alia of culture media.

(15) The inventors called this preserving medium SEC.

(16) Description of the Preserving Medium

(17) Injectable Products Used

(18) Product C1: Human albumin 20% solution (composition: human album 200 gr; caprylic acid qs 1 litre): it has a protective role for some molecules; during our assays it exhibited a very distinct role in maintaining the viability of total nucleated cells; it allows better stability of the pH of the formulated medium in containers that are not gas-impermeable during storage of amplified cells at +4° C. (See FIG. 1)

(19) Product C2: Amino acids (Vaminolact® (Fresenius Kabi) composition qs 100 ml: L-Alanine 630 mg; L-Arginine 410 mg; L-Aspartic Acid 410 mg; L-Cysteine/L Cystine 410 mg; L-Glutamic Acid 710 mg; Glycine 210 mg; L-Histidine 210 mg; L-Isoleucine 310 mg; L-Leucine 700 mg; L-Lysine 560 mg; L-Methionine 130 mg; L-Phenylalanine 270 mg L-Proline 560 mg; L Serine 380 mg L-Taurine 30 mg; L-Threonine 360 mg; L-Tryptophan 140 mg; L-Tyrosine 50 mg; L-Valine 360 mg.) Amino acids make a non-significant contribution to improvement in functionality of the amplified cells, but one that is of interest however especially as some amino acids may have a protective role on the cells. (See FIG. 3)

(20) Product C3: KCl (10% preparation in H.sub.2O): throughout our assays, it showed signification action on the preserving of the functionality of amplified cells. (See FIG. 3)

(21) Product C4: Glucose (30% preparation): throughout out assays, it showed non-significant action but one of interest. (See FIG. 3).

(22) Product C5: Glutamine amino acid (Dipeptiven Composition (Fresenius Kabi): Alanyl-Glutamine 20 g/100 ml i.e. 13.2 g of Glutamine/100 ml; molecule stable versus glutamine): throughout our assays, it exhibited action that was non-significant but of interest; it would seem to have protective action on mitochondria. (See FIG. 3)

(23) Product C6: MgSO4 (15% preparation in H.sub.2O); throughout our assays, it showed significant action on the preserving of the functionality of amplified cells; it would seem to have a role on the mitochondria. (See FIG. 4)

(24) Products C7: Anti-Oxidants

(25) Either C7.Cernevit® (Baxter) (Composition for 5 ml: Retinol [Vitamin A] 3500 IU in the form of retinol palmitate; Cholecalciferol [Vitamin D3] 220 IU; Alpha-tocopherol or [Vitamin E] 11.2 IU i.e. 10.2 mg; Ascorbic Acid [Vitamin C] 125 mg; Thiamine [Vitamin B1] 3.51 mg; Riboflavin [Vitamin B2] 4.14 mg); Pyridoxine [Vitamin B6] 4.53 mg; Cyanocobalamin [vitamin B12] 0.006 mg; Folic acid [Vitamin B9]; Pantothenic acid [Vitamin B5] 17.15 mg; Biotin [Vitamin B8] 0.069 mg; Nicotinamide [Vitamin PP] 46 mg): throughout our assays, it exhibited preserving action on the functionality of the cells stored at +4° C. Among the vitamins assayed separately, vitamin E exhibited the most important action; vitamins A and C have lesser action but vitamin C is described as having a protective role on vitamin E; the addition of Cernevit® to the culture supernatant showed an identical effect to that of the formulated medium in the preservation of amplified cells at +4° C. Finally, each vitamin exists in the form of an injectable medicinal product and vitamins E and C only could be used in the formulation.

(26) Or C7a.Vitamin E (composition for 2 ml: Alpha-tocopherol 100 mg).

(27) Or C7b.Vitamin C (composition for 5 ml: Ascorbic acid 1000 mg).

(28) Or C7c.Vitamin A (composition for 2 ml: Retinol 100000 IU).

(29) Vitamin E can be used alone or associated with vitamin C or A or a combination of both, with an effect close to or identical to the effect of Cernevit® in terms of preservation of amplified cells. Vitamin C would seem to protect vitamin E. (See FIG. 1, FIG. 3 and FIG. 6)

(30) Product C8: Mannitol (20% solution): this has an antioxidant role (hydroxyl anti-radical and throughout our assays it showed non-significant action. (See FIG. 4)

(31) Product C9: Ringer Lactate (composition: NaCl 6 gr; KCl 0.4 gr; CaCl.sub.22H.sub.2O 0.27 gr; Na lactate 5.16 gr qs 1 litre i.e. Na.sup.+ 131 mmol; K.sup.+5 mmol; Ca.sup.++; Lactate 29 mmol.) It has minor antioxidant action, a small buffer role (idem CO3.sup.− ion) is present in the culture supernatant; throughout our assays, it showed relatively significant action on preservation of the functionality of the amplified cells. (See FIG. 3)

(32) Product C10: NaCl (in the form of 0.9% physiological saline): it plays a solute role and allows the maintaining of physiological osmolarity. Throughout our assays, it proved to be of particular interest on clonogenic potential since, if it is replaced by an isotonic solute of glucose and mannitol, a major drop was observed in clonogenic functionality after storage for 48 h at +4° C. despite the maintained viability of the total nucleated cells. (See FIG. 5)

(33) Product C11: Sodium bicarbonate (1.4% NaHCO.sub.3 preparation). It allows the creation of a pseudo-buffer system with the citric acid of ACD A to adjust the pH of the medium to the desired value, and brings dissolved CO.sub.2 (protective action on the cells of interest at +4° C.).

(34) Product C12: ACD A (Citric acid Citrate Dextrose Solution A) (Dextrose monohydrate 24.5 g; Citric acid monohydrate 8 gr; sodium citrate dihydrate 22 gr qs 1 litre). It allows adjustment of pH after adding sodium bicarbonate; the citrate ion is described as having antioxidant action, and finally the citrate ion complexes part of the calcium ions introduced to the medium with the Ringer Lactate. ACD A is an anticoagulant conventionally used for transfusion procedure.

(35) The Table below gives the current formulation with the upper and lower values of the activities of interest of the components as evidenced throughout optimisation studies. The following components were not examined for optimisation and only the physiological values were taken into account: KCl (0.33 gr/l final by associating 10% KCL and the KCl contained in the Ringer Lactate), Glucose, NaCl, ACD A.

(36) The preferred pH of the preserving solution is 6.95±0.05.

(37) TABLE-US-00002 TABLE 2 weight in Gr of the molecule(s) of Volume in ml per 1 litre of medium interest per 1 litre of medium Components: injectable Current Upper Lower Current Upper Lower preserving preparations value value value value value value C1. 20% human albumin 75 200 25 15 40 5 C2. AA (Vaminolact) 30 50 20 1.959 3.265 1.306 C3. 10% KCl 2.74 2.74 2.74 0.274 0.33 0.33 C4. 30% Glucose 3.32 3.32 3.32 1 1 1 C5. Glutamine (Dipeptiven) 5.3 5.3 2.65 0.7 0.7 0.35 C6. 15% MgSO4 0.68 16 0.34 0.1 2.35 0.05 C7. Polyvit (Cernevit) 2 4 0.0125 Vit E: Vit E: Vit E: 0.00408 0.00816 0.0000255 Vit C: 0.05 Vit C: 0.1 Vit C: Vit A: Vit A: 0.0003125 0.00042 0.00084 Vit A: 0.0000026 C8. 20% Mannitol 25 100 25 5 20 5 C9. Ringer Lactate 140 513 46.7 Na Lact: Na Lact: Na Lact: 0.722 2.647 0.24 KCl: 0.056 KCl: 0.205 KCl: 0.0187 C10. 0.9% NaCl 676.46 Solute ND solute ND 6.09 ND ND C11. 1.4% NaHCO3 37 295 18.5 0.518 4.14 0.259 C12. ACD A qs pH 6.95 ± 0.05 about 2.5 ND ND ND ND ND

(38) Assays on the Action of the Components of the Preserving Medium

(39) 1. On Placental Blood CD34+ Cells Selected and then Amplified Ex Vivo.

(40) The cells, after ex vivo expansion, are more fragile than the original CD34+ cells, and the maintaining thereof is most critical at +4° C. (Duchez et al, 2013, supra).

(41) Throughout all the studies leading to the formulation of the biological preserving medium, the assays were conducted on placental blood CD34+ cells derived from a bank with storage at −196° C. Each assay was performed on one 1 or more samples.

(42) The placental blood CD34+ cells were isolated with an immunomagnetic system, then frozen and banked at −196° C.

(43) After thawing, the placental blood CD34+ cells (20000/ml) were placed in culture in HP01 medium in the presence of SCF (Stem Cell Factor) 100 ng/ml, Flt3 (FMS-like tyrosine kinase-3) 100 ng/ml, G-CSF (Granulocyte Colony Stimulating Factor) 10 ng/ml, and thrombopoietin (TPO) 20 ng/ml at 37° C., 5% CO.sub.2 and 85% humidity. At mid-culture, the addition was made of HP01 culture medium with the cytokines (1/5 dilution) and culture continued up to 9 to 12 days (generally 10 days).

(44) After culture, 2.5 ml of cell suspension were transferred to 2.5 ml gas-impermeable polypropylene tubes and centrifuged at 430 g for 10 minutes at 18° C. The supernatant was fully removed (with about 50 μl of supernatant remaining on the cell residue). Then, 2.45 ml of preserving medium to be assayed (at ambient temperature) were added to the tube and the tube sealed with the stopper; the cells being replaced in suspension with successive upturning movements. The samples were left at ambient temperature for 1 to 3 hours. Finally, the tubes were placed flat at +4° C. for times generally of 48 h to 72 h. Assays were also conducted in gas-permeable PVC pouches for comparative studies and in gas-impermeable EVA pouches.

(45) Analyses were performed on the amplified cells: counting and viability of TNCs (Total Nucleated Cells), clonogenic assays, sometimes with counting and viability of CD34+ cells. After the period at +4° C. for the defined times (48 h and 72 h), the cell samples were assayed: counting and viability of TNCs and clonogenic assays (at times also counting and viability of CD34+ cells).

(46) The quantity of viable total nucleated cells remaining after storage at 4° C. was compared with the quantity of viable total nucleated cells at the end of culture, to obtain a recovery yield of viable cells. Similarly, the yield of CFCs (Colony Forming Cells) was calculated. The values obtained were used to evaluate the advantage of one or more compounds in the formulation and to identify the optimal concentration of these compounds. The values were compared with those obtained with the HP02 preserving medium (Duchez et al, 2013, supra).

(47) The results are given in FIG. 1.

(48) The placental blood CD34+ cells amplified ex vivo were grafted onto NSG immunodeficient mice to examine the preservation of clonogenicity of the amplified CD34+ cells after being stored in SEC medium at +4° C. for 48 h. This study involved different handling steps: production of the graft with the placing in culture of placental blood CD34+ cells (selected by immunomagnetic sorting) in a medium containing a cocktail of adapted cytokines for a period of 12 days. After culture, the amplified cells were harvested and divided into two portions: one portion was grafted onto a batch of immunodeficient mice, each mouse receiving a quantity of amplified cells produced by 1000 CD34+ cells at the beginning of the culture; the other portion was stored in SEC medium (PVC pouch) at +4° C. for 48 h and then injected into a second batch of immunodeficient mice under the same quantitative conditions. 8 weeks after grafting, the mice were sacrificed, and the human clonogenic progenitors were analysed in the bone marrow of the mice: these progenitors are generated by the stem cells (key to graph: 1 circle corresponds to a grafted, analysed mouse (CFU/femur); the dotted line corresponds to the mean; the solid line corresponds to the median). The results show that preservation in SEC medium for 48 h at 4° C. does not modify the number of clonogenic progenitors derived from the stem cells grafted onto the mice.

(49) The results are given in FIG. 8.

(50) 2. On Placental Blood Cells after Collection

(51) The placental blood was collected in a plastic pouch (PVC poly-vinyl-chloride) containing 23 ml of CPD anticoagulant (Citrate phosphate dextrose). The placental blood unit was stored at +4° C. awaiting treatment for banking (cryopreservation at −196° C.). The protocol requires that banking is performed within 36 hours to prevent cell deteriorations related to the storage mode (+4° C.) and to absence of preserving medium. To allow collections at far distant sites to meet therapeutic needs (tissue compatibility, rare HLA phenotypes, populations in French overseas departments and territories, etc. . . . ), and requiring transport times longer than 36 h, a first study was conducted to improve preservation of the placental blood unit after collection. It comprised associating a gas-impermeable pouch and the addition of non-injectable medium (HP02 by Macopharma) to a unit of placental blood. This made it possible to obtain a significant improvement in the time before banking (up to 72 h) whilst maintaining viability and functionality compatible with efficient grafting (WO 2014/057220, Chevaleyre et al, 2014, Stem Cells Dev, 23, 1820-30).

(52) Since the HP02 medium is not injectable, the SEC medium (of the present invention) was used to preserve units of placental blood. After a storage time of 72 h at +4° C., the addition of the SEC medium allowed the maintaining of significantly better functionality compatible with banking (Rodriguez, Master 2 thesis, Université de Bordeaux, presented on 11 Jun. 2014). The results obtained are given in FIG. 2.

(53) 3. On Selected Placental Blood CD34+ Cells after Thawing

(54) The placental blood CD34+ cells frozen at −196° C. were thawed, washed and divided into 3 aliquots: suspension of CD34+ cells in SEC medium, in HP02 medium and in 4% human albumin, followed by storage at 4° C. After a storage time of 72 h at +4° C., analyses were performed (count of viable total nucleated cells and clonogenic functional assays). The assays were conducted in gas-impermeable containers (2.5 ml polypropylene tubes containing 2.5 ml of cell suspension for maximum avoidance of the presence of air). The results show very good preservation of the CD34+ cells, quantitatively (number of viable cells) and qualitatively (clonogenic functionality) after storage for 72 h at +4° C. in SEC medium compared with the HP02 medium. The results obtained are given in FIG. 10.

(55) 4. On Bone Marrow CD34+ Cells after Thawing (n=5)

(56) The medullar CD34+ cells banked (frozen at −196° C. for 3 to 6 years), were thawed, washed and resuspended in 2.5 ml of SEC medium in 2.5 ml gas-impermeable polypropylene tubes; they were stored at +4° C. for 72 h. Clonogenic assays performed after thawing and storage for 72 h at 4° C. showed good preservation of the functionality of these medullar cells of interest (preservation yield of functionality >than 70%). The results obtained are given in FIG. 9.

(57) 5. On Peripheral Blood Cells Mobilised after Thawing (n=1)

(58) The peripheral blood cells after Ficoll gradient centrifugation (MNCs) were frozen to −196° C. After thawing and washing, they were divided into 2 aliquots: the first was left as such and the second was subjected to immunomagnetic sorting to obtain a suspension of CD34+ cells. The mononuclear cells (MNCs) and CD34+ cells were suspended in SEC medium (2.5 ml) in gas-impermeable 2.5 ml propylene tubes and stored at +4° C. from 48 to 120 h. The clonogenic assays performed after thawing the MNCs and/or purification of the CD34+ cells and then after storage at 4° C. show good preservation of the functionality of these cells of interest: preservation yield of functionality from 85% (purified CD34+ cells) to 100% (MNCs) after 48 h at +4° C., and from 60% (purified CD34+ cells) to 80% (MNCs) after 120 h at 4° C. The results obtained are given in FIG. 7.