METHOD FOR FREEZING AND STORING NEONATAL STROMAL CELLS

Abstract

The present invention relates to a method for freezing and preserving a composition comprising a population of neonatal stromal cells (NSCs) and a cryoprotector, characterised in that it comprises a step of freezing the composition at a temperature of between −70° C. and −140° C., then a step of preserving the composition at between −10° C. and −40° C. The present invention also relates to a composition comprising a population of NSCs and a cryoprotector, characterised in that it is preserved at between −10° C. and −40° C., said NSCs being in particular placental NSCs.

Claims

1. Method for freezing and preserving neonatal stromal cells (NSCs) comprising 1) preparing a composition comprising a population of neonatal stromal cells (NSCs) and a cryoprotector; 2) freezing said composition at a temperature of between −70° C. and −140° C.; then 3) storing said composition at a temperature of between −10° C. and −40° C.

2. The method according to claim 1, wherein the population of NSCs is a population of placental NSCs.

3. The method according to claim 1, wherein step 2 is conducted for at least 2 h.

4. The method according to claim 1, wherein step 3 is conducted for 1 day to 5 months.

5. The method according to claim 1, wherein step 3 is conducted for at least 21 days.

6. The method according to claim 1, wherein the cryoprotector is selected from glycerol, dimethyl sulfoxide (DMSO), propylene glycol, proteoglycans, trehalose, polyethylene glycol (PEG), polyacrylic acid, poly-L-lysine, ethylene glycol or a combination of a plurality of these cryoprotectors.

7. The method according to claim 1, wherein the composition comprises DMSO as the cryoprotector.

8. The method according to claim 1, wherein said composition comprises a population of NSCs of phenotype MHC-IL/CD90H.

9. Pharmaceutical composition comprising a population of NSCs and a cryoprotector, wherein the pharmaceutical composition has been preserved at between −10° C. and −40° C.

10. Pharmaceutical composition comprising a population of NSCs according to claim 9, wherein said NSCs are placental NSCs.

11. Pharmaceutical composition comprising a population of NSCs according to claim 9, wherein said population of NSCs is a population of NSCs of phenotype MHC-IL/CD90H.

12. Pharmaceutical composition comprising a population of NSCs according to claim 9, wherein said population of NSCs is free from NSC of phenotype MHC-IH/CD90L.

13. Pharmaceutical composition according to claim 9, wherein the cryoprotector is DMSO.

14. The method of claim 1, wherein the step of freezing is performed at a temperature of between −70° C. and −100° C.

15. The method of claim 14, wherein the step of freezing is performed at a temperature of −80° C.

16. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition has been preserved at a temperature between −10° C. and −35° C.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition has been preserved at a temperature between −15° C. and −30° C.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition has been preserved at a temperature between −17° C. and −20° C.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition has been preserved at a temperature of −18° C.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0209] FIG. 1 shows the procedure for analysing the cell viability by flow cytometry. A first analysis window (FSC-H/SSC-H) is traced so as to take into account only the cells (left-hand graph). The viability of these cells is analysed by measuring the fluorescence of propidium iodide (IP) on channel FL-2 of the cytometer.

[0210] FIG. 2 is a graph showing the cell viability evaluated with Trypan Blue (BT) and with propidium iodide (IP) after preservation of the NSCs at −80° C. for 15 days (J15), 1 month (1 M), 2 months (2 M), 3 months (3 M) and 5 months (5 M).

[0211] FIG. 3 is a graph showing the percentage of positive cells for expressing the CD29, CD44 and CD90 markers, measured in flow cytometry, after various preservation times for the NSCs at −18° C. (from 15 days to 3 months).

[0212] FIG. 4 is a graph showing the proliferative activity of the NSCs through the number of doublings (DN) of the cell population in 7 days, after preservation of the cell population at −80° C. or at −18° C. for 15 days, 1 month, 2 months, 3 months or 5 months.

[0213] FIG. 5 is a graph showing the proliferative activity of the NSCs through the doubling time (DT) of the cell population during 7 days, after preservation of the cell population at −80° C. or at −18° C. for 15 days, 1 month, 2 months, 3 months or 5 months.

[0214] FIG. 6 is a graph showing the proliferative activity of the NSCs after 24 h, 48 h and 5 days in culture measured by means of the CCK8 colorimetric kit, after preservation of the NSCs at −80° C. or at −18° C. for 15 days, 1 month, 2 months or 3 months.

[0215] FIG. 7 is a graph showing the inhibiting activity of the NSCs on the proliferation of lymphocytes (PBMC proliferation ratio), after freezing of the NSCs at −80° C. and preservation at −18° C. for 15 days, 1 month, 2 months or 3 months. The results are compared with the lymphocyte control stimulated with concanavalin A (Con A).

[0216] FIG. 8 is a graph showing the inhibiting activity of the NSCs on the proliferation of lymphocytes (PBMC proliferation ratio), after freezing of the NSCs at −80° C. and preservation at −18° C. for 15 days, 1 month, 2 months or 3 months. The results are compared with the inhibiting activity of the NSCs frozen at −80° C. and preserved at −80° C. (−80° C.) for 2 months.

[0217] FIG. 9 is a graph showing the cell viability of the NSCs measured by Trypan Blue counting, after a method according to the invention (freezing of the NSCs at −80° C., then preservation at −18° C. for 1 week or 3 months) of after a method comprising freezing of the NSCs at −18° C. and then preservation at −18° C. for 1 week or 3 months.

[0218] FIG. 10 is a graph showing the inhibiting activity of the NSCs on the proliferation of lymphocytes (PBMC proliferation ratio), after freezing at −18° C. and preservation at −18° C. of the cell population for 3 months, for co-cultures of ratio 1:10 and 1:20.

[0219] FIG. 11 is a graph showing the inhibiting activity of the NSCs on the proliferation of lymphocytes (PBMC proliferation ratio), after freezing of the NSCs at −18° C. and preservation at −18° C. for 3 months, versus freezing of the NSCs at −80° C. and preservation at −18° C. for 3 months, for co-cultures of ratio 1:10.

[0220] FIG. 12 shows examples representing various cytometric profiles and the associated characteristics thereof. NSCs are isolated in canine placenta and amplified over several passages (2 to 5). Several samples of NSC are analysed in flow cytometry to evaluate the expression of CD90 (FL2-PE) and of MHC-I (FL4-APC). According to the CD90/CMH-I phenotype profile, the NSCs can be classified in several categories depending on their biological characteristics and their composition. Here the cells do not meet the criteria for expression of CD90/CMH-I, as described in the invention.

[0221] FIG. 13 shows examples representing various cytometric profiles and the associated characteristics thereof. NSCs are isolated in canine placenta and amplified over several passages (2 to 5). Several samples of NSC are analysed in flow cytometry to evaluate the expression of CD90 (FL2-PE) and of MHC-I (FL4-APC). According to the CD90/CMH-I phenotype profile, the NSCs can be classified in several categories depending on their biological characteristics and their composition. Here the cells meet the criteria for expression of CD90/CMH-I, as described in the invention.

[0222] FIG. 14 shows the viability of the thawing of a population of cells frozen at −80° C. and then preserved at −20° C. over two months (on the right), according to its composition in terms of phenotype MHC-I.sup.L/CD90.sup.H cells (on the left).

EXAMPLES

[0223] l. Isolation of the NSCs from Canine Placenta

[0224] The canine extra-embryonic annexes (placenta, umbilical cord) are aseptically sampled during caesareans practiced in gestating dogs at term. As soon as the new-born puppy is removed from the amniotic sac and placed in safety, the extra-embryonic tissue is immediately transferred to a transport box containing a buffered saline solution with Dulbecco's phosphate (D-PBS) to be transported to the laboratory. The treatment of the extra-embryonic tissue is carried out at most within 48 h following sampling. All of the treatment steps of the tissue are carried out in a controlled environment, under a biosafety cabinet (BSC).

[0225] The tissue is transferred to a 100 cm.sup.2 Petri dish and the residual amniotic membrane is mechanically removed by dissection. The placenta is placed embryonic face against the plastic surface of the box and the uteroverdin present on the face of maternal origin is separated from the placenta by scraping the tissue. The placenta is rinsed 3 to 5 times in successive baths of D-PBS. The blood vessels and the umbilical cord are then mechanically removed from the placenta. The placental tissue is dissected into fragments of about 10-20 mm.sup.2 then subjected to an enzymatic digestion by incubating the tissue fragments in a solution composed of DMEM (Dulbecco's modified Eagle medium) containing 0.5-4 mg/ml of type I collagenase, and more specifically a concentration of 1 mg/ml of type I collagenase. The enzymatic digestion takes place at 37° C. for 1 h but a digestion comprised between 30 min and 16 h can be carried out by decreasing the incubation temperature (ambient temperature (18-22° C. or 4° C.). At the end of the digestion, the enzymatic activity is stopped by dilution, by adding DMEM containing at least 10% foetal bovine serum (FBS) in a quantity equivalent to the solution of enzymatic digestion. The solution is then filtered over a 70-100 μm screen. The recovered cells are centrifuged at 800 g for 10 min. The cell residue containing the neonatal stromal cells is rinsed with DMEM and again centrifuged at 800 g for 10 min. The cell residue is taken in culture medium constituted of DMEM, 10% FBS, 2 mM glutamine and from 0 to 20 ng/ml of fibroblast growth factor (FGF). The cells are counted and seeded in culture dishes at a density comprised between 10.sup.4 and 2.10.sup.4 cells/cm.sup.2. The cells are then cultivated in the culture medium described hereinabove in a controlled atmosphere at 37° C. and containing 5% CO.sub.2. The medium is changed after 48 h then every 2-3 days. The cells are passed when the confluence reaches 70-80%.

[0226] II. Cell Passage and Amplification

[0227] At sub-confluence, the cells undergo a cell passage and optionally an amplification procedure. The NSCs are rinsed with D-PBS and treated with 0.05% trypsin-EDTA for 2-5 min at 37° C. This makes it possible to detach the cells and to form a population of isolated cells. The cells are then taken with amplification medium constituted of DMEM, 10% FBS, 2 mM glutamine and from 0 to 20 ng/ml of fibroblast growth factor (FGF) and centrifuged between 300-500 g for 5 to 10 min. The NSCs are taken in amplification medium, and counted via manual counting (Trypan Blue and Malassez cells) or using a cytometer. They are then seeded for 1,500 to 5,000 cells/cm.sup.2 and cultivated on a plastic cell culture support in an amplification medium and in a controlled atmosphere at 37° C. and containing 5% CO.sub.2. During the amplification process the cells can undergo between 0 and 15 cell passages.

[0228] III. Optional Cryopreservation of the NSCs—Formation of a Masterbank

[0229] At the end of the first or second cell passage (P1-P2), the cells can be cryopreserved in seed units. To do this, after counting, the NSCs are centrifuged between 300-500 g for 5 to 10 min and the cell residue is taken in the freezing medium comprised either of DMEM medium enriched with 5-20% FBS and 5-10% (vol:vol) DMSO or in a commercial cryopreservation medium, containing or not a fraction of DMSO. The cell concentration is comprised between 1.10.sup.6 and 15.10.sup.6 cells per ml of freezing medium. The freezing of the cells is carried out in controlled falling temperature conditions, using for example a CoolCell® Cell Freezing Container (BioCision) and by following the freezing procedure as described by the manufacturer. The cells are then transferred for negative cold storage at temperatures comprised between −70° C. and −196° C.

[0230] The cell units can be used to generate cell units for therapeutic use. The seed units are thawed at 37° C. for 3-6 min and amplified in vitro. The cells are seeded in the culture medium at the density of 1,500-3,000 cells/cm2. The cells are amplified by successive passage in vitro. When a significant number of cells are produced (for example >150.10.sup.6cells), the cells are frozen according to the protocol described hereinabove. The cells are distributed into hermetically sealed bottles with seals at a rate of 1.10.sup.6-15.10.sup.6 cells/ml in a freezing medium free from product of animal origin (such as for example the cryopreservation medium Recovery™ Cell culture freezing medium (Thermo Fisher), or Cryostem™ freezing medium (Biological Industries). The bottles are lowered in temperature according to a controlled falling temperature protocol, at a rate of −1° C./min to −80° C. The bottles are then transferred at −80° C. for storage. Once obtained, the population of NSCs is characterised on the one hand by its structural characteristics (presence/absence of markers) and on the other hand by its functional characteristics (proliferation, differentiation etc.).

[0231] IV. Preparation of the Pharmaceutical Composition and Freezing of the Unit Doses

[0232] At the end of the amplification of the NSCs corresponding to a phase of production of the unit doses for therapeutic purposes, the cells can be frozen in the form of a pharmaceutical composition and packaged in unit doses. To do this, after counting, the NSCs are centrifuged at between 300 and 500 g for 5 to 10 min and the cell remainder is taken up in freezing medium free from any product of animal origin composed of Cryostor® CS5 medium (stem cell technology) or any other commercial or preformulated medium containing 5% DMSO. The cells are distributed in hermetically sealed cappable bottles at the rate of 1×10.sup.6-15×10.sup.6 cellules/ml. The cells are frozen in a controlled temperature drop condition, using for example a programmable cryogenic freezer of the FREEZAL type (biofluid) and programmed for a temperature drop of 1° C./min for an initial temperature of between 10° C. and 4° C. and for a final temperature of −80° C. The cells are then transferred for negative cold storage at temperatures of between −70° C. and −80° C. or directly stored for final storage at a temperature of between −10° C. and −40° C.

[0233] V. Details and Samples Tested: Freezing/Storage Comparison −80° C./−18° C. versus −80° C./−80° C.

[0234] Three batches of CSN coming from canine placentas of various amplification phases are used in this study.

[0235] Pharmaceutical compositions as described in the invention, comprising 15×10.sup.6 NSC in 1.5 ml of Cryostor® CS5 medium (stem cell technology), a medium comprising 5% DMSO, were packaged in bottles and were cryopreserved at −80° C. at the rate of a temperature drop of −1° C./minute using the Coolcell device. Three bottles in each batch were stored at −80° C. for a period of between 15 days and 3 months (Table 1), and then transferred to the freezer at −18° C. (condition −80° C./−18° C.).

[0236] [Table 1]

TABLE-US-00001 TABLE 1 Cell Storage times −80 for passage on analysis of intermediate Thawing Thawing Batch freezing viability viability (TB) viability (IP) 1 P3  3 months 94.2% 89.6% 2 P5  2 months 90.3%   85% 3 P5 15 days 90.2% ND

[0237] In parallel, cells stored only at −80° C., i.e. without transfer to −18° C., were used as controls (condition “ctrl −80° C.”).

[0238] On the day of the analyses, the bottles are taken out of −18° C. or −80° C., transferred to the laboratory in a polystyrene container containing a −20° C. eutectic pack. The bottles are thawed to ambient temperature (18-25° C.) for approximately 5 minutes. The cells are sucked from the bottles by means of a syringe (18 G needle) and transferred into a sterile Eppendorf for the following analyses.

[0239] V.1. Viability Under Trypan Blue and Propidium Iodide

[0240] For each condition, an aliquot of the initial suspension (50 μl) is sampled for counting with Trypan Blue (TB) using a Luna electronic counter.

[0241] Another aliquot of the initial suspension (50 μl) is sampled for marking with flow cytometer with propidium iodide (IP) (Sigma Aldrich). The aliquot is kept in a refrigerator until analysis.

[0242] The PI analysis window of the cytometric analysis (BD accury c6) is traced in accordance with the following figure. A “cell” window is traced in FSC-H/SSC-H and is used for the PI analysis. The PI analysis is carried out on the FL-2 channel by an FL2-A/FSC-H analysis (FIG. 1).

[0243] The cell viability was evaluated by TB and PI for all the conditions tested (15 days: J15 n=2; 1 month: 1 M n=3; 2 months: 2 M n=2; 3 months: 3 M n=1; 5 months: 5 M n=2) and compared with the viability of the cells after thawing of the cell batches stored at −80° C. (n=3) (FIG. 2).

[0244] The results obtained after the storage time at −80° C. for intermediate viability analysis (Table 1) did not show any differences compared with the cells frozen and stored in accordance with the protocol of the present invention (freezing at −80° C. and then storage at −18° C.) (FIG. 2).

[0245] The results obtained by TB or PI tally, suggesting that the two analytical methods can be used for verifying the viability parameter. The data reveal a viability >80% for the samples stored up to 2 months at −18° C. in TB and PI (greater variability at 1 month in PI). Analysis at 3 months shows a viability of 72% (TB) and 79% in PI. The analyses after 5 months at −18° C. show a mean viability of 40% (TB) and 42% (PI). The results show that the NSCs can be stored for a period of at least 3 months at −18° C. without drastically affecting their viability on thawing.

[0246] V.2. Analysis of Phenotype Markers

[0247] Cytometric analysis aims to determine the presence of membrane markers on the surface of the NSCs by means of the use of a panel of antibodies specific to each marker, for example: CD29, CD44, CD90.

[0248] In order to study the impact of storage at −18° C. on the frozen cells, 5×10.sup.5 cells are sampled from each previously mentioned condition for cytometric analysis. Each sampling of each condition is separated into 4 samples transferred into 1.5 ml Eppendorfs in order to implement 4 markings. The cells are taken up in 1 ml of cytometric buffer composed of D-PBS and 0.5-1% (v/v) bovine serum albumin (BSA) or

[0249] 0.5-2% (v/v) foetal bovine serum. These 4 samples are centrifuged at 500 g for 5 min. A second washing is carried out under the same experimental conditions. After elimination of the supernatant, the cells of the 4 tubes are respectively taken up in 1 volume of 30-100 μl, in particular 50 μl, of cytometric buffer. In total, 4 markings are implemented per freezing and storage condition: isotypic marking (FITC and PE), CD29 marking, CD44 marking and CD90 marking in accordance with Table 2.

[0250] [Table 2]

TABLE-US-00002 TABLE 2 Mark Fc Reference Name of product Supplier TypeAc Clone Isotype FITC 400110 FITC mouse IgG1 k isotype Ozyme IgG1k MOPC-21 Ctrl_Fc Isotype PE 400114 PE mouse IgG1 k isotype Ozyme IgG1k MOPC-21 Isotype_primary — COL2002 Mouse COL2002/COLIS205C Vetmed IgG2a COLIS205C IgG2a Secondary APC 17-4010-82 Goat F(ab′) 2 anti-mouse IgG Thermofisher IgG Polyclonal secondary a pc CD29 PE 303003 PE anti-human CD29 Ozyme IgG1k TS2/16 CD44 APC 103012 APC rat anti-human CD44 Ozyme IgG2bk IM7 CD90 PE 12-5900-41 PE anti-canine CD90 Thermofisher IgG2bk YK IC337,217 CMH1 — DG-BOV2001 Primary anti-canine CMH-1 Vetmed IgG2a DG-H58A

[0251] The optimum concentration of antibodies used for the marking must be determined beforehand by those skilled in the art. The incubation required for the marking must also be determined by those skilled in the art and comprised between 15 min and 10 h at 4° C. in a dark place. In particular, the cells are incubated 20 min at 4° C. in a dark place.

[0252] Isotypic controls (here FITC, PE and APC for fluorescene isothiocyanate, phycoerythrin and allophycocyanin) adapted to each marking have to be used as a negative control. Following the incubation with the antibodies, the cells are washed with D-PBS, centrifuged 5-10 min at 500 g and taken in a volume from 100 to 250 μI of marking buffer for analysis with the flow cytometer (Accuri C6, BD Biosciences).

[0253] Thus the expression of the CD29, CD44 and CD90 markers was evaluated by flow cytometry. The mean expression of the markers of the cells stored at −80° C. at variable times was taken as control (CD29=91%±7%; CD44=99.4%±0.3%; CD90=87.8±9%) (n=6).

[0254] The samples were analysed for 15 days: J15 n=2; 1 month: 1M n=3; 2 months: 2M n=2; 3 months: 3M n=1. For the 3 markers studied, a positive signal was revealed whatever the condition tested (FIG. 3).

[0255] FIG. 3 representing the percentage positive cells for the CD29, CD44 and CD90 markers. The results show a very similar expression of CD44 for all the conditions (>99% positivity). With regard to CD29, >85% positivity is observed for all the conditions (except a sample stored at −18° C. for 15 days). The CD90 marker shows a greater variability with 5 samples preserved at −18° C. the positive signal of which is >95% and two samples with a positive signal of approximately 80%. This variability is also observed with samples preserved at −80° C. (87.8±9%) and the samples in the course of amplification. Because of this, preservation at −18° C. does not affect the expression of the CD29, CD44 or CD90 markers.

[0256] For all the experiments relating to the pharmaceutical composition described in the invention, the MHC-1.sup.L/CD90.sup.H profile is determined in advance during the cell amplification. The profile is determined by flow cytometry analysis and immunomarking of the MHC-I and CD90. The details of the analysis are presented in the invention (paragraphs [0065] to [0104]). The profile is determined in accordance with a cytometric analysis matrix presented in FIG. 13. The profile is termed MHC-I.sup.L/CD90.sup.H if the MHC-1.sup.L/CD90.sup.H sub-population represents more than 80% of the total population at P5.

[0257] V.3. Proliferative and Metabolic Activity After Culture of the Thawed Cells

[0258] After thawing of the pharmaceutical composition and counting, the cells are diluted in amplification medium (DMEM, 10% SVF, 2 mM glutamine and 0 to 20 ng/ml of FGF) to obtain a concentration of approximately 10.sup.6 cells/ml. A 75 cm.sup.2 culture dish is seeded with 0.225×10.sup.6 cells for proliferation analysis for 1 week in amplification medium. In parallel, a 96 well plate is seeded to the extent of 10,000 cells/well for kinetic monitoring of the metabolism of the cells using Cell Counting Kit-8 (CCK8-Sigma-Aldrich) analysed by spectrophotometry.

[0259] The proliferative activity of the NSCs is evaluated by calculating the number of doublings (DN) of the cells during 7 days of culture and calculating the doubling time (DT). After 7 days of culture, the cells are detached from their culture substrate by means of trypsin/EDTA 0.5% for 2-3 minutes. Culture medium is added and the cells are centrifuged 5 min/300 g. The cell residue is returned to a defined volume of culture medium and the cells are counted by a Trypan Blue exclusion technique by means of an electronic counter. The number of doublings (DN) is calculated with each cell passage according to the following formula: DN=LOG (Nf/Ni)/LOG(2) (Nf: number of final cells and Ni: number of initial cells). The doubling time (DT) is calculated in accordance with the following formula: DT=culture time (h)/DN.

[0260] FIGS. 4 and 5 present the proliferative activity of the cells in culture for 7 days. FIG. 4 presents the number of doublings of NSCs cultivated for 7 days and FIG. 5 the mean doubling time of the cultures. The data show a mean number of cell doublings (DN)=4.6 when the cells have come from −80° C. (n=12). This number is similar for the two samples stored at −18° C./15 days (DN=4.6). After 1 month at −18° C., ND=3.6±1.2 (n=3); after 2 months at −18° C., ND=3.9 (n=2) and after 3 months at −18° C., ND=4.1 (n=1). A significant reduction in the number of doublings is observed after 5 months at −18° C.; ND=1.7 (n=2) (FIG. 4).

[0261] With regard to the doubling times (DT), these are similar to −80° C. and after 15 days at −18° C.; respectively 37.5 h±5 (n=12) and 38.7 h (n=2). At 1, 2 and 3 months at −18° C., respective doubling times are observed of 48.5 h±18 (n=3), 48.7 h (n=2), and 41 h (n=1). After 5 months at −18° C., the doubling times increase substantially DT=112 h (n=2) (FIG. 5).

[0262] V.4. Metabolic Activity

[0263] A second analytical method was used for evaluating the proliferation of the post-thawing cells in 24 h, 48 h and 5 day kinetics as well as for checking the metabolic increase in the cells during culture. The CCK8 colorimetric test was used and analysed in plate reading at 450 nm.

[0264] The analyses were done on cells preserved at −18° C. for 15 days (n=2); 1 month (n=2); 2 months (n=2); 3 months (n=2) and 4 samples cryopreserved at −80° C. used as controls.

[0265] FIG. 6 presents the analysis of the proliferative activity of the cells after 24 h, 48 h and 5 days in culture calculated by means of the CCK8 colorimetric kit. An increase in the OD corresponds to an increase in the metabolic activity of the cells or in the number of viable cells in the wells.

[0266] The results show that, at 24 h post-seeding, the optical densities (ODs) at 450 nm are equivalent for all the conditions (OD.sub.450=0.22). This is consistent with the viability results obtained on thawing.

[0267] At 48 h, an increase in OD is observed in the −80° C. condition (OD.sub.450=0.313±0.1) whereas the ODs are not modified in the −18° C. tests (OD.sub.450 (J15)=0.27; OD.sub.450 (1 M)=0.27; OD.sub.450 (2 M)=0.24; OD.sub.450 (3 M)=0.26).

[0268] V.5. In Vitro Immunosuppressive Activity of the NSCs

[0269] The immunomodulating activity of the NSCs coming from the pharmaceutical composition is studied through their immunosuppressive activity, in particular their ability to inhibit proliferation of the lymphocytes in vitro.

[0270] This inhibiting activity is evaluated by co-cultivating the NSCs of each storage condition with blood mononuclear cells (PBMCs) in the presence of a mitogenic agent.

[0271] To achieve this, the PBMCs are isolated from a blood sample taken from a donor dog or a donor horse over a Ficoll gradient. The PBMCs are then incubated with a fluorescent dye (CellTrace CFSE, Thermo Fisher) which makes it possible to measure cellular proliferation over several generations. 0.2.10.sup.6 PBMCs marked by Celltrace are added to the wells of a 96 well plate in which the NSCs were seeded the day before in a concentration (2.10.sup.4 NSC/wells); thus making it possible to obtain a ratio of NSC:PBMC equivalent to 1:10. The NSCs are treated with mitomycin (10 μg/ml for 1.5-2 h at 37° C.) and rinsed 3 times in culture medium before the addition of PBMCs. The proliferation medium of the lymphocytes is added (RPMI, 10% SVF, 2 mM glutamine, 10 mM hepes, 50 μM β-mercaptoethanol, 5 μg/ml concanavalin A). The PBMC/NSC co-cultures and the control cultures (PBMC without NSCs) are incubated for 4 days in an incubator at 37° C. Following the culture, the non-adherent cells are recovered from the wells, centrifuged and washed in D-PBS. The cells are then marked with an anti-CD3 antibody coupled with a FITC fluorochrome for 30 min at 4° C. The cells are then washed in D-PBS before flow cytometry analysis (Accuri C6, BD Biosciences). The cytometric analysis consists of evaluating the Celltrace signal within the viable CD3+ population. At the end of incubation, the T-lymphocytes specifically marked with CD3 are analysed by cytometer. For each test implemented, the PBMCs were isolated from 2 or 3 patients. A lymphocyte proliferation index is calculated for each experimental condition using the Modfit® analysis software. The lymphocyte proliferation index in the presence of NSCs is standardised with respect to the lymphocyte proliferation index under controlled condition, fixed at 1 (100% proliferation). This ratio is termed “PBMC proliferation ratio”. The statistical analyses were implemented by a t-test of comparison with the reference value (con A). The conditions stored at −18° C. were compared with the PNMCs stimulated with concavanalin A (con A) (FIG. 7) or with the results of samples stored at −80° C. (−80) (FIG. 8).

[0272] The data show a reduction in the lymphocyte proliferation in the presence of NSCs stored for 15 d, 1 M, 2 M and 3 M at −18° C. This difference is significant (p<0.05) with the control condition con A with the cells stored for 15 d, 1 M, 2 M and 3 M at −18° C. (FIG. 7).

[0273] The data obtained in this study are compared with the results observed when the PBMCs are co-cultivated with the NSCs stored at −80° C. (n=14), showing a proliferative activity=0.4±0.13 (FIG. 8). The results show a significant difference in lympho-inhibiting activity of the NSCs stored at −18° C. for 15 days, 1 month, 2 months and 3 months compared with the NSCs stored at −80° C. (t-test).

[0274] VI. Freezing/Storage Comparison: −80° C./−18° C. versus −18° C./−18° C.

[0275] In order to assess whether the cryopreservation mode of the NSCs with a 1.sup.st step at −80° C. is necessary for preserving the biological properties of the cells stored temporarily at −18° C., the following study is carried out by storing samples of NSCs in a cryopreservation medium directly in a freezer at −18° C.±5° C. without an initial temperature drop to −80° C.

[0276] Briefly, the NSCs are centrifuged at 300-500 g for 5 to 10 min and the cell residue is taken up in freezing medium composed either of DMEM medium enriched with 5-20% FBS and 5-10% (vol:vol) DMSO or in a commercial cryopreservation medium, containing or not a fraction of DMSO. The cell concentration is between 5×10.sup.6 and 10×10.sup.6 cells per ml of freezing medium. Some of the samples coming from 5 cell batches are frozen and preserved according to the description of the invention presented in part V.1 (−80/−18). Others are frozen and stored directly at −18° C. (−18/−18). The number of experimental samples per analysis time is presented in Table 3.

[0277] [Table 3]

TABLE-US-00003 TABLE 3 Number of doses −80/−18 −18/18 T0 3 2 1 week 2 2 2-3M 5 4

[0278] VI.1. Freezing/Storage Comparison: Viability Under Trypan Blue

[0279] The results in FIG. 9 show that the cell viability is only slightly affected by the two freezing conditions for storage of one week. A reduction of 9.42% is observed for −80/−18 and 2.20% for −18/−18 at 1 week in comparison with their respective viabilities on freezing. Nevertheless, after a storage of 2-3 months, a significant reduction in the viability can be observed for the −18/−18 case (reduction of 26.01% with respect to T0) while in the case −80/−18, a maintenance of viability is found (reduction of only 11.05% with respect to T0). The statistical analysis at 2-3 months indicates a significant difference in viability between the two methods of the order of 12.03% (comparison of the means per Student's T-test, p=0.041). Because of this, the freezing strategy described in the invention (−80/−18) favours storage of the NSCs at −18° C. for a period of 2-3 months in comparison with the −18/−18 method.

[0280] VI.2. Immunomodulator Effect of the NSCs According to the Freezing/Storage Method −18° C./−18° C.

[0281] The immunomodulating activity of the NSCs was studied through their immunosuppressive activity, in particular their ability to inhibit the proliferation of the lymphocytes in vitro as detailed in part V.5.

[0282] The NSCs frozen at −18 and stored at −18° C. for 3 months were thawed and cultivated with PBMCs marked with CFSE. The lymphocyte proliferation index in the presence of NSC is standardised with respect to the lymphocyte proliferation index under controlled condition, fixed at 1 (100%). This ratio is termed the PBMC proliferation ratio. The results in FIG. 10 showed that, for co-cultures with an NSC:PBMC ratio of 1:10 or 1:20, no immunomodulating effect could be observed, indicating a loss of this effect following freezing at −18° C. and storage at −18° C. These results were compared with the methods of freezing at −80° C. and storage at −18° C. described in the invention (FIG. 11). The comparison showed that the method described in the invention allowed preservation of the lympho-inhibiting activity of the NSCs compared with a direct freezing at −18° C. (comparisons of the means according to the two techniques by Student's T-test, p=0.0017). In other words, in FIG. 11, the lower the bar, the more the lymphocyte proliferation is inhibited.

[0283] VII. Effect of the Proportion of MHC-I.sup.L/CD90.sup.H Sub-Population on the Viability of the −80° C./−18° C. Product on Thawing

[0284] Several expression profiles of the MHC-I and CD90 markers were identified among the populations of NSC coming from canine placenta. Six MSC populations were analysed during the manufacturing method: 2 to 7 analyses for passages of between P3 and P6. These analyses were implemented in accordance with the description of the invention. The expression profiles were determined by flow cytometry analysis and immunomarking of the MHC-I and CD90. A cytometric analysis matrix is established in accordance with FIGS. 12 and 13.

[0285] The MHC-I.sup.L/CD90.sup.H sub-population of interest was quantified and expressed in terms of percentage of the population. A cytometric profile analysis by double marking is implemented in accordance with the description of the invention. Two profiles emerged during these experiments, a profile termed MHC-I.sup.L/CD90.sup.H, which has at P5 more than 80% of the MHC-I.sup.L/CD90.sup.H sub-population (n=3). This profile corresponds to that of the pharmaceutical composition described in the invention. This profile is illustrated by FIG. 13. As well as a so-called “mixed” profile wherein the presence of the MHC-I.sup.L/CD90.sup.H sub-population remained below 80% during the manufacturing method (n=3). This profile does not correspond to that of the pharmaceutical composition described in the invention. This profile is illustrated by FIG. 12. For each NSC population, the percentage mean of the MHC-I.sup.L/CD90.sup.H population is calculated between the P3 to P6 passages. These results are presented in FIG. 14 (left). The results confirm a predominance of the MHC-I.sup.L/CD90.sup.H population on these passages for the populations termed MHC-I.sup.L/CD90.sup.H (comparison of the means by Student's T-test, p=0.0022).

[0286] Freezing and storage of these NSC populations are implemented in accordance with the description of the invention (i.e. freezing at −80° C. and storage at −18° C.). These cell products are preserved for 2 months at a temperature of −18° C. They are then thawed and a Trypan Blue viability study is implemented. The data are presented in FIG. 14 (right). The result show that the cells termed MHC-I.sup.L/CD90.sup.H have viability on thawing superior to the NSCs termed mixed (comparison of the means by Student's T-test, p=0.0135).

LIST OF DOCUMENTS CITED

[0287] For information, the following documents are cited: [0288] Marquez-Curtis, L. A., Janowska-Wieczorek, A., McGann, L. E., and Elliott, J. A. W. (2015). Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects. Cryobiology 71, 181-197. [0289] Espina, M., Jülke, H., Brehm, W., Ribitsch, I., Winter, K., and Delling, U. (2016). Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses. PeerJ 4, e1773. [0290] Chinese patent application CN102487939 A, filed by Shangai Angelife Biotechnology Co Ltd, published on 13 Jun. 2012. Inventors: Yingbo, C., Jiawei, Z., Limin, Y., and Yiqiang, J. [0291] Freshney, R. Ian. (2015). Culture of animal cells: a manual of basic technique and specialized applications. John Wiley & Sons, (p 307-p 312) [0292] Mazur, P. (1984). Freezing of living cells: mechanisms and implications. Am J Physiol 247, C125-142 [0293] Gao, D., et al. (1997). Fundamental cryobiology of mammalian spermatozoa, p. 263-328. In A. M. J. Karow and J. K. Critser (ed.), Reproductive tissue banking. Academic Press, San Diego, Calif.