Method for preserving cells

Abstract

The present invention relates to a method for preserving free cells, such as blood cells, by freezing in the presence of a non-toxic cryoprotectant, i.e. one that can be administered to a living human or animal organism. The present invention thus relates to methods, compositions and kits for freezing and storing non-adherent cells, such as red blood cells, for extended periods of time while preventing the lesions that can occur during the storage thereof and, in the case of red blood cells, while preserving deformability and increasing survival.

Description

[0001] Cell preservation method

[0002] The present invention relates to a method for preserving free cells, such as blood cells, by freezing in the presence of a non-toxic cryoprotective agent, i.e. compatible with administration to a living human or animal organism.

[0003] The present invention thus relates to methods, compositions and kits for freezing and storing non-adherent cells, such as red blood cells, for extended periods of time while preventing damage that may occur during their storage, and, in the case of red blood cells, maintaining their deformability and increasing their survival.

[0004] In this context, and as an example, blood transfusion is widely used for medical purposes, particularly for cardiovascular surgery, severe burns, transplant surgery, massive trauma, pregnancy-related complications, and therapy of solid and hematological malignancies.

[0005] Typically, blood for transfusion is stored for up to 42 days at 4°C. However, blood stored at this temperature for more than 21 days is considered old blood, in which depletion of metabolites, loss of cell membrane integrity, immune mediators, and hemolysis may be observed.

[0006] The use of anticoagulants and suitable storage media has made it possible to limit the damage caused to red blood cells during storage and to increase the safety and clinical efficacy of allogeneic or autologous blood transfusions.

[0007] An alternative to this preservation method is freezing; freezing is of particular interest, particularly for military use and the storage of blood cells for the treatment of rare diseases. There are two methods generally used for the cryopreservation of red blood cells:

[0008] - the slow cooling technique (approximately -1°C / min) in a medium with a high glycerol content (approximately 40%) and allows storage at -80°C; and

[0009] - the rapid cooling technique (above -100°C / min) in a low glycerol medium (approximately 20%) and allows storage at -196°C. However, excessive amounts of glycerol lead to osmotic imbalance problems, which can cause hemolysis. To avoid this, clinical staff must perform complex and lengthy deglycerolization operations after thawing, including the preparation of different concentrations of washing solution, multiple centrifugations, and repeated removal of used supernatant fluid.

[0010] Glycerol, like ethylene glycol, DMSO or DMF, used as a cryoprotectant protects red blood cells by limiting the formation of ice crystals in the intracellular space, decreasing the exposure of cells to excessive electrolyte concentrations during freezing and preventing excessive contraction of the cytoplasmic volume.

[0011] There are other extracellular cryoprotectants that can be used for red blood cell cryopreservation, including, for example, hydroxyethyl starch, polyvinylpyrrolidone (PVP), and dextran, which have the main advantage of not crossing the cell membrane. Although these non-permeable cryoprotectants are biodegradable and tolerated by the human body, it is still necessary to remove them after thawing.

[0012] Finally, cryoprotectants such as trehalose, whose cryoprotective action in living systems is known, do not spontaneously cross the cell membrane but have a predominant action in the intracellular space, are often used in the presence of adjuvants allowing its transport such as hydroxyapatite particles or in modified forms such as acetylated trehalose.

[0013] However, it remains necessary to optimize the cryopreservation of cells; this is what the Applicant has achieved by developing a method of cryopreservation of cells which does not require toxic cryopreservation agents which must be eliminated after thawing and also has the advantage of being able to be implemented with mechanical freezers.

[0014] The present invention thus relates to an original method for preserving non-adherent cells by directional freezing comprising the steps of: a) suspending said non-adherent cells, at a concentration which may be between 1x10 6 and 5xl0 9 cells / mL, preferably the cell concentration is between 1x10 8 and 5xl0 9cells / mL, in a cryopreservation medium composed of an isotonic aqueous solution, preferably saline, for example an aqueous solution at 0.9% by weight of NaCl, and at least one non-toxic cryoprotective agent, preferably at a concentration of between 4 and 50% by weight / volume, preferably this cryoprotective agent is a natural protein, preferably natural proteins with a molecular weight of between 10 and 80 kDa, and may be chosen from albumins, alpha and beta globulins and fibrinogen; optionally, the mixture may also comprise a cryoprotective additive such as carboxymethyl cellulose, sucrose, trehalose or glucose; b) optionally, conditioning the mixture obtained in step a); c) directional freezing of the mixture obtained in step a); d) storage at a temperature between -79°C and -197°C, preferably at approximately -80°C.

[0015] This method allows the preservation of any so-called non-adherent or free cell, i.e. circulating cells or temporarily suspended adherent cells, i.e. detached from their physiological support / environment; in particular, these cells are chosen from:

[0016] • Blood cells (red blood cells, white blood cells, platelets);

[0017] • Hepatocytes;

[0018] • Neuronal cells;

[0019] • Rare cells (linked to particular pathologies) whose conservation could be of future interest;

[0020] • Immunomodulatory cells (Tregs, etc.);

[0021] • Stem cells, iPSCs.

[0022] Preferably, these are blood cells and even more preferably red blood cells.

[0023] Prior to implementing step a), it may be necessary to separate the cells from their medium; this is particularly the case for blood cells present in a blood sample which should be separated from the plasma; this is carried out using conventional techniques known to those skilled in the art, such as centrifugation for example. The cryoprotective agent is non-toxic, that is to say it can be administered to a human or animal patient; it is therefore not essential to purify the medium containing the cells after they have been thawed; the cryoprotective agent is a natural protein which may be chosen from:

[0024] • Human or bovine albumin, ovalbumin;

[0025] • Alpha-1 glycoprotein, fibrinogen, ...

[0026] Preferably, when the cells preserved by the method according to the invention are intended to be administered to a human, the cryoprotective agent is human serum albumin (also known as HSA for human serum albumin).

[0027] Preferably, serum albumin is present in the cryopreservation medium at a concentration of between 4 and 50% weight / volume, more preferably between 20 and 50% weight / volume.

[0028] According to one embodiment, the cryopreservation medium consists of an isotonic aqueous solution and at least one of the aforementioned cryoprotective agents.

[0029] According to another embodiment, the cryopreservation medium consists of an isotonic aqueous solution, at least one of the aforementioned cryoprotective agents and at least one cryoprotective additive chosen from sucrose, glucose, trehalose and carboxymethyl cellulose.

[0030] According to this embodiment, carboxymethyl cellulose can be used at a concentration of between 0.1 and 1% w / v; trehalose at a concentration of between 0.5 and 5% w / v and sucrose at a concentration of between 0.5 and 5% w / v; glucose has a concentration of between 0.5 and 5% w / v.

[0031] Directional freezing is a freezing technique that consists of linearly moving a sample from a thermalized support at a temperature higher than the sample's melting temperature (called a hot block) to a thermalized support at a temperature lower than the sample's melting temperature (called a cold block). The distance between the two supports as well as the temperatures of each of the supports can be chosen independently to achieve a controlled thermal gradient. The temperature range of the hot block is between 0.5 and 40°C and the temperature range of the cold block is between -60 and -100°C). The speed at which the sample, placed in the freezing chamber, is moved between the hot block and the cold block corresponds to the growth rate of the ice front and is controlled by a dedicated motor that operates between 10 and 200 pm / s.As an example, directional freezing can be implemented according to the method described in Q / n et al., 2020.

[0032] The storage temperature depends on the device used for storing frozen cells; for example, it could be around -80°C with a variation of + / -3°C in a mechanical freezer, around -150°C with a variation of + / -3°C in a freezer capable of producing lower temperatures, around -196°C if storage is carried out in liquid nitrogen and between -135°C and -190°C if storage is carried out in nitrogen vapor.

[0033] The present invention also relates to a method of thawing suspended cells preserved according to the preservation method of the invention.

[0034] This thawing method is thus preferably implemented after the preservation method according to the invention; it comprises the steps of: i. bringing the freezing chamber containing frozen cells into contact with a surface heated to a temperature between 35 and 45°C for between 10 and 40s, until the ice crystals have completely melted; ii. centrifugation; iii. suspending the cells from the centrifugation supernatant in an isotonic aqueous solution comprising between 1 and 10% by weight / volume of proline.

[0035] The present invention also relates to a composition comprising, or consisting of, non-adherent cells and an isotonic aqueous solution, preferably saline, comprising at least one non-toxic cryoprotective agent at a concentration of between 4 and 50% w / v and chosen from albumins, alpha and beta globulins and fibrinogen. Optionally, the composition may also comprise a cryoprotective additive such as sucrose, trehalose, glucose or carboxymethyl cellulose; in this embodiment, carboxymethyl cellulose may be used at a concentration of between 0.1 and 1% w / v; trehalose at a concentration of between 0.5 and 5% w / v, sucrose at a concentration of between 0.5 and 5% w / v and glucose at a concentration of between 0.5 and 5% w / v.

[0036] Preferably, the cryoprotective agent is human serum albumin.The present invention also relates to a kit for preserving non-adherent cells comprising at least one sealed device for storing sterile biological fluids, flexible at room temperature and capable of withstanding storage temperatures down to -196°C without damage and an isotonic aqueous solution, preferably saline, comprising at least one non-toxic cryoprotective agent at a concentration of between 4 and 50% by weight and chosen from albumins, alpha and beta globulins and fibrinogen and, optionally, a cryoprotective additive such as sucrose, trehalose, glucose and carboxymethyl cellulose, then, preferably, carboxymethyl cellulose can be used at a concentration of between 0.1 and 1% w / v, trehalose at a concentration of between 0.5 and 5% w / v, sucrose at a concentration of between 0.5 and 5% w / v and glucose at a concentration of between 0.5 and 5% p / v.

[0037] Figure 1: Red blood cell (RBC) recovery after directional freezing with varying BSA volume fractions (4>BSA) (0.04, 0.14, and 0.28) at varying translational speeds. Upper panel: Image of centrifuged samples after the freeze-thaw process. The red color is due to hemoglobin released from lysed cells. Neg, represents the negative control obtained without being frozen. Lower panel: Cell recovery after freezing and thawing.

[0038] Figure 2: Cell recovery after directional freezing of RBCs (5xl0 9 cells. mL 1 ) at 100 pm.s -1 . ns, does not indicate any significant difference.

[0039] Figure 3: SEM and flow cytometry of RBCs after freezing and thawing. RBCs were stained with Calcein-AM for flow cytometry. Negative and positive controls indicate unstained and calcein-stained cells without undergoing freezing. Varying BSA volume fractions and ice front velocities were applied to the experimental groups. Scale bar, 2 μm.

[0040] Figure 4: (A) Cell size (FSC-A) of calcein-stained RBCs. (B) Comparison of cell size between positive control (fresh cell) and cryopreserved cells. Cells frozen with a 4>BSA of 0.14 showed significantly smaller size than fresh cells. ***, indicates P<0.01. Figure 5: Cell recovery (A) and flow cytometry (B) of RBCs after 106 days of frozen storage.

[0041] Figure 6: (A) Cell size (FSC-A) of Calcein-stained RBCs after 106 days of storage in a -80°C freezer. (B) Comparison of cell size between the positive control (fresh cell) and cryopreserved cells. ***, indicates a significant difference at P<0.01.

[0042] Figure 7: Resuspension efficiency of thawed cells followed by freezing at 100 pm.s 1 . Positive control, hemolysis absorbance in deionized H2O with the same amount of cells compared to that in the presence of BSA.

[0043] Figures 8, 9, 10 and 11: Post-freezing cell suspension tests, histograms show the measurement of hemoglobin in the supernatant after suspension.

[0044] Freezing protocol a. 5 mL of sheep blood in Alsevers solution (saline solution comprising 2.05% dextrose, 0.8% sodium citrate, 0.055% citric acid, and 0.42% sodium chloride) (1:1) is centrifuged at 600g, 4°C for 10 mins; b. The supernatant is removed and 0.9% NaCl solution is added to restore the initial volume (5 mL); c. The resulting suspension is centrifuged at 600g, 4°C for 10 mins; d. The supernatant is removed and 0.9% NaCl solution is added to restore the initial volume (5 mL); e. Steps c and d are repeated once more; f. The red blood cell concentration is calculated using a hemacytometer; g. The cells needed to obtain a concentration of 10 8 or 1.5*10 9are transferred into 2 mL of BSA solution (between 4 and 50% by weight); h. The cell suspensions are frozen in 500 pL CoverWell™ incubation chambers (Grace Bio-Labs) 22mm X 40mm X 0.5mm on a thermal gradient of 50°C.mm 1 (transition from the thermal block at +10°C to the thermal block at -90°C; gap between the blocks 2mm). The translation speeds of the samples are 10, 50 and 100 pm.s -1 . i. After freezing, the chambers containing the frozen cell suspensions are kept at -80°C (from 1 day to 106 days).

[0045] Thawing protocol a. The chambers containing the cells are thawed in contact with a surface at 45°C for 10-15s. b. 200 pL of the contents of each chamber are transferred to an Eppendorf, and centrifuged according to the following conditions:

[0046] 1. For BSA concentrations less than or equal to 20%: 3000 rpm, 20°C, 10 mins

[0047] 2. For BSA concentrations above 20%: 6000 rpm, 20°C, 20 mins c. The supernatant is removed and the cells are suspended in a solution of 1% proline, 0.9% NaCl. d. Steps b and c are repeated two more times for FACS characterization; four more times for SEM characterization. e. The freeze-thaw efficiency is estimated from the measurement by UV-Vis absorption at 541 nm of the hemoglobin concentration in the supernatant obtained in step b.1 or b.2.

[0048] Other formulations were tested for post-thaw cell resuspension (results correspond to the measurement of hemoglobin in the supernatant after resuspension):

[0049] • Proline (1, 2, 4, 8, 10%)

[0050] • Proline (1, 2, 4, 8, 10%) then BSA 4%

[0051] (see Figure 8)

[0052] • BSA 4% + proline (0.1, 0.5, 1, 2, 5%)

[0053] (see Figure 9)

[0054] • BSA + PBS

[0055] • PBS

[0056] • 0.9% NaCl

[0057] • BSA + glucose

[0058] • BSA + glucose + trimethylglycine + proline

[0059] • BSA + glucose + trimethylglycine + proline + glycine + glutamine

[0060] • BSA + trimethylglycine + proline + glycine + glutamine (see Figures 10 and 11 where Cl: bsa+glucose; C2: Cl+betaine+proline; C3: C2+glycine+glutamine; C4: C3-glucose)

[0061] 1% Proline in 0.9% NaCl produces similar results to other proline concentrations, consistently superior to other formulations.

[0062] Results

[0063] Increasing the BSA volume fraction (4>BSA) resulted in improved cell recovery which was further improved with increasing the translational freezing rate implemented (Figure 1).

[0064] For volume fractions of 0.04 to 0.28, cell recovery increased from ~18% to ~30% at 10 pm.s 1 and from ~38% to ~95% at 100 pm.s -1 . For all volume fractions, increasing the translation speed resulted in better cell recovery, especially for the value 4>BSA = 0.28 which allows the increase by a factor of three when the translation speed varies from 10 to 100 ms. After thawing and centrifugation, the unlysed red blood cells (RBCs) were pooled at the bottom of the tube and the hemoglobin from the broken cells was released into the supernatant, leading to a red color. For the negative control, the fresh blood sample showed almost no cell hemolysis, as shown by the colorless supernatant. Samples processed under other conditions showed a red color, except for a 4>BSA between 0.28 and 50 at 100 pm.s 4, confirming the positive role of highly concentrated BSA systems on RBC recovery.

[0065] The cell density used in these experiments (10 8 cells mL -1 ) was 50 times lower than the physiological density (~5xl0 9 cells mL 1 ) of the human body. In the literature, cell density has a significant positive effect on final cryosurvival. To account for this effect, another test was performed with a cell density of 5xl0 9 cells mL 1 at 100 pm 4 (Figure 2). Cells frozen with a BSA volume fraction of 0.28 showed the highest recovery (~80%), further confirming the beneficial effects of highly concentrated BSA on cell survival during directional freezing. However, relatively lower survival compared to samples containing 10 8 cells mL 1was observed, which can be attributed to the increase in mechanical stress between cells.

[0066] Red Blood Cell Quality While the previous experiments determined the quantity of erythrocytes with preserved membrane integrity, it is also useful to obtain additional information on the physiological state of the recovered cells. To this end, scanning electron microscopy and flow cytometry were performed on the cells after freezing and thawing. It is known from the literature that healthy RBCs have a biconcave shape, which gives them great flexibility and a high oxygen transport capacity; Figure 3A shows that a majority of fresh RBCs have a biconcave disc shape. Cells frozen at 100 pm.s 1in the presence of a low volume fraction of BSA (4>BSA = 0.04 and 0.14) presented a shape similar to that of fresh samples (Figure 3B-C).

[0067] For the high volume fraction groups, a majority of thawed cells exhibit a disc shape in the absence of the concave feature, and a small portion of cells exhibit morphological alteration toward a slightly spherical shape (Figure 3D). Varying the translational speed did not significantly change the shape of RBCs when 4>BSA = 0.28 (Figure 3E-F). Calcein-AM, a cell-permeable non-fluorescent dye that is converted to green fluorescent calcein by intracellular esterases in living cells, was used for the flow cytometry assay. In Figure 3G, the negative control (unstained fresh cells) and positive control (stained fresh cells) have apparent distinct fluorescence intensity, which facilitates determination of the experimental group.Regardless of the experimental conditions (BSA concentration and translational speed), more than 97% of the cells showed high calcein intensity. This indicates that, after resuspension, most of the recovered RBCs have esterase activity and are therefore alive. In other words, the slightly altered shape of the RBCs does not affect their esterase activity. In flow cytometric analysis, “Forward scatter gating” is generally considered an index of cell size [9]. For the experimental groups, thawed RBCs showed a median cell size similar to that of the positive control, except those frozen in the presence of 4>BSA = 0.14 (at 100 pm.s. 1) which are slightly smaller in size (Figure 4A-B). Considering both cell recovery and quality, more than 95% of RBCs exhibit intact membranes with similar metabolic activities and cell size compared to the control after being cryopreserved via directional freezing with 4>BSA = 0.28 at 100 pm.s -1 .

[0068] As previously described, the limited lifespan of RBCs, even in the presence of additives (< 49 days), leads to a high demand for daily blood donors. Here, RBC cryopreservation was performed by directional freezing, the RBCs were then stored in a -80°C freezer for three months (106 days). In Figure 5A, cell recovery followed the same survival trend as that after one day of storage. RBC survival could be improved with an increase in BSA content and even more so with a higher translational speed. After 106 days, samples prepared with a volume fraction of 0.28 at 100 pm.s 1showed a survival of ~95% comparable to that of thawed samples after 1 day of storage. Additional calcein staining (Figure 5B) indicated that more than 98% of cells exhibited esterase activity, regardless of freezing conditions. In all samples, the size of thawed cells was similar to that of the positive control, except for the group with a volume fraction of 0.14 which showed a smaller size (Figure 6). Thus, directional freezing in the presence of BSA (4>BSA = 0.28) allows twice as long storage in the freezer without significantly affecting cell viability and vitality as the permitted storage time of red blood cells (49 days).

[0069] In conventional RBC cryopreservation, the deglycerolization process has detrimental effects on the final cell quality. For clinical applications, the efficiency of resuspension of frozen-thawed cells must be considered in order to obtain a highly concentrated cell suspension equivalent to physiological cell density. In Figure 7, no hemolysis (absorbance at 541 nm less than 0.005) occurred after resuspension of thawed cells (4)BSA = 0.28, at 100 pm.s 1) compared to the positive control, i.e., complete hemolysis of RBCs. In other words, cells can be cryopreserved in a relatively dilute state, but once needed, these cells could be thawed, concentrated and then mixed in specific solutions without time-consuming and harmful post-purification of cells, or also subjected to simple centrifugation, which represents a substantial advantage of the method of the invention. Further analysis of esterase-based metabolic activity indicated that more than 95% of the RBCs preserved after freezing and thawing are alive, regardless of the experimental conditions. Regarding the viability and quality of RBCs, the 0.28 BSA volume fraction group showed good cryosurvival and led to a cell size similar to that of fresh cells. The comparable recovery and viability of

[0070] RBCs thawed after 1 day and 106 days of storage extended the commonly accepted shelf life of RBCs from 49 days to 106 days. Given the physiological cell density, the low rate of hemolysis upon resuspension of thawed cells provides a simple tool for enriching RBCs for transfusion.

Claims

DEMANDS 1. Composition comprising non-adherent cells and an isotonic aqueous solution comprising at least one non-toxic cryoprotective agent at a concentration between 4 and 50% by weight / volume (w / v) and selected from albumins, alpha and beta globulins and fibrinogen.

2. Composition according to claim 1, characterized in that the cryoprotectant agent is human serum albumin.

3. Composition according to claim 1 or claim 2, characterized in that it further comprises a cryoprotectant additive selected from sucrose at a concentration of between 0.5 and 5% w / v, trehalose at a concentration of between 0.5 and 5% w / v, glucose at a concentration of between 0.5 and 5% w / v and carboxymethyl cellulose at a concentration of between 0.1 and 1% w / v.

4. Composition according to any one of claims 1 to 3, characterized in that the non-adherent cells are blood cells, preferably red blood cells.

5. Method for preserving non-adherent cells by freezing comprising the steps of: a) suspending said non-adherent cells in a cryopreservation medium composed of an isotonic aqueous solution with at least one non-toxic cryoprotectant at a concentration of between 4 and 50% by weight and selected from albumins, alpha and beta globulins, and fibrinogen; b) optionally, conditioning the mixture obtained in step a); c) directional freezing of the mixture obtained in step a); d) storage at a temperature between -79°C and -197°C, preferably at approximately - 6. Method of preserving non-adherent cells by freezing according to claim 5, characterized in that the cryoprotective agent is human serum albumin.

7. Method of preserving non-adherent cells by freezing according to claim 5 or claim 6, characterized in that the cryopreservation medium further comprises a cryoprotectant additive selected from sucrose, glucose, trehalose and carboxymethyl cellulose.

8. Method for thawing non-adherent cells comprising the steps of: i. bringing the frozen cells into contact with a surface heated to a temperature between 35 and 45°C; ii. centrifugation; iii. suspending the cells from the centrifugation supernatant in a saline solution comprising between 1 and 10% proline.

9. Non-adherent cell preservation kit comprising at least one sealed sterile biological fluid storage device and an isotonic aqueous solution comprising at least one non-toxic cryoprotectant at a concentration between 4 and 50% by weight and selected from albumins, alpha and beta globulins and fibrinogen and, optionally, a cryoprotectant additive such as sucrose, carboxymethyl cellulose, trehalose or glucose.

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