How to preserve 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 preserving 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 enhancing viability.

Description

[Technical field]

[0001] 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, ie, one that can be administered to a living human or animal organism.

[0002] Thus, the present invention 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 storage and, in the case of red blood cells, maintaining their deformability and increasing their viability. [Background technology]

[0003] In this regard, for example, blood transfusions are widely used for medical purposes, particularly in cardiovascular surgery, severe burns, transplantation surgery, major trauma, pregnancy-related complications, and the treatment of solid and hematological malignancies.

[0004] Generally, blood for transfusion is stored at 4° C. for up to 42 days. However, blood stored at this temperature for more than 21 days is considered old blood, where depletion of metabolic products, loss of cell membrane integrity, immune mediators, and hemolysis can be observed.

[0005] The use of anticoagulants and appropriate storage media has made it possible to limit the damage caused to red blood cells during storage and increase the safety and clinical efficiency of allogeneic or autologous transfusions.

[0006] An alternative to this method of preservation is freezing. Freezing is of particular interest for the preservation of blood cells, especially for military use and for treating rare diseases. There are two methods commonly used for the cryopreservation of red blood cells: - a slow cooling technique (approximately -1°C / min) in a medium with a high glycerol content (approximately 40%), which allows storage at -80°C; and - Rapid cooling technique (>-100°C / min) in medium with low glycerol content (approximately 20%), allowing storage at -196°C.

[0007] However, excessive amounts of glycerol can lead to osmotic imbalance problems and cause hemolysis. To avoid this, clinical staff must perform a complex and time-consuming deglycerolization procedure after thawing, which includes preparation of different concentrations of washing solutions, multiple centrifugations, and repeated removal of spent supernatants.

[0008] Glycerol, like ethylene glycol, DMSO or DMF, used as cryoprotectants protects red blood cells by limiting the formation of ice crystals in the intracellular space, reducing the exposure of cells to excessive electrolyte concentrations during freezing, and preventing excessive shrinkage of the cytoplasmic volume.

[0009] There are other extracellular cryoprotectants that can be used for the cryopreservation of red blood cells, including, for example, hydroxyethyl starch, polyvinylpyrrolidone (PVP), and dextran, which have the main advantage of not crossing the cell membrane. These non-permeating cryoprotectants are biodegradable and tolerated by the human organism, but it is still necessary to remove them after thawing.

[0010] Finally, cryoprotectants such as trehalose, known for their cryoprotective effect in biological systems, do not spontaneously cross cell membranes but rather have a predominant effect in the intracellular space and are often used in the presence of adjuvants that enable their transport, such as hydroxyapatite particles, or in modified forms, such as acetylated trehalose.

[0011] However, there remains a need to optimize the cryopreservation of cells, which the applicant has achieved by developing a method for cryopreserving cells that does not require the removal of toxic cryopreservatives after thawing and also has the advantage that it can be performed in a mechanical freezer. Summary of the Invention [Means for solving the problem]

[0012] Thus, the present invention provides a unique method for preserving non-adherent cells by directional freezing, comprising: a) The non-adherent cells are cryopreserved in a cryopreservation medium consisting of an isotonic aqueous solution, preferably physiological saline, e.g., 0.9% by weight NaCl in water, and at least one non-toxic cryoprotectant, at a concentration of 1×10 6 ~5×10 9 Concentration may be 1×10 cells / mL, preferably 1×10 8 ~5×10 9 suspending the cells at a cell concentration of 100000000 cells / mL, preferably at a concentration of 4-50% weight / volume, preferably the cryoprotectant is a natural protein, preferably a natural protein having a molecular weight of 10-80 kDa, and may be selected from albumin, alpha and beta globulins, and fibrinogen; optionally the mixture may also contain a cryoprotectant additive such as carboxymethylcellulose, sucrose, trehalose or glucose; b) optionally conditioning the mixture obtained in step a); c) directional freezing of the mixture obtained in step a); d) storing at a temperature between −79° C. and −197° C., preferably at approximately −80° C.; The present invention relates to a method comprising the steps of:

[0013] This method makes it possible to preserve any so-called non-adherent or free cells, i.e. circulating or adherent cells, temporarily in suspension, i.e. detached from their physiological support / environment, and in particular these cells are chosen from: ·Blood cells (red blood cells, white blood cells, platelets); ·Hepatocytes; ·Nerve cells; Rare cells whose preservation may be of future interest (associated with certain pathologies); ·Immune regulatory cells (Treg etc.); ·Stem cells, iPSCs.

[0014] Preferably, these are blood cells, even more preferably red blood cells.

[0015] Before carrying out step a), it may be necessary to separate the cells from their medium. This applies in particular to blood cells present in the blood sample, which are to be separated from the plasma. This is carried out using conventional techniques known to those skilled in the art, such as, for example, centrifugation.

[0016] The cryoprotectant is non-toxic, i.e. it can be administered to a human or animal patient. Therefore, it is not necessary to purify the medium containing the cells after they have been thawed. The cryoprotectant is a natural protein that may be selected from: Human or bovine albumin, ovalbumin; Alpha-1 glycoprotein, fibrinogen,...

[0017] Preferably, if the cells preserved by the method according to the invention are intended to be administered to humans, the cryoprotectant is human serum albumin (also called HSA).

[0018] Preferably, serum albumin is present in the cryopreservation medium at a concentration of 4 to 50% weight / volume, more preferably 20 to 50% weight / volume.

[0019] According to one embodiment, the cryopreservation medium consists of an isotonic aqueous solution and at least one of the cryoprotectants mentioned above.

[0020] According to another embodiment, the cryopreservation medium consists of an isotonic aqueous solution, at least one of the cryoprotectants mentioned above, and at least one cryoprotectant additive selected from sucrose, glucose, trehalose and carboxymethylcellulose.

[0021] According to this embodiment, carboxymethylcellulose may be used at a concentration of 0.1-1% w / v; trehalose may be used at a concentration of 0.5-5% w / v, sucrose may be used at a concentration of 0.5-5% w / v; and glucose has a concentration of 0.5-5% w / v.

[0022] Directional freezing is a freezing technique that consists of moving a sample linearly from a thermalized support (called a hot block) at a temperature higher than the melting temperature of the sample to a thermalized support (called a cold block) at a temperature lower than the melting temperature of the sample. The distance between the two supports and the temperature of each support can be independently selected to achieve a controlled thermal gradient. The temperature range of the hot block is 0.5-40 °C, and that of the cold block is -60-100 °C. The speed at which the sample is placed in the freezing chamber and moved between the hot and cold blocks corresponds to the growth rate of the ice surface and is controlled by a dedicated motor operating at 10-200 μm / s. For example, directional freezing can be performed according to the method described in Qin et al., 2020.

[0023] The storage temperature depends on the equipment used to store the frozen cells. For example, this can range from -80°C with a + / -3°C variation for mechanical freezers, to -150°C with a + / -3°C variation for freezers that can generate lower temperatures, to -196°C if storage is in liquid nitrogen, and between -135°C and -190°C if storage is in nitrogen vapor.

[0024] The present invention also relates to a method for thawing suspension cells preserved according to the preservation method of the present invention.

[0025] This thawing method is therefore preferably carried out after the preservation method according to the invention, which is i. contacting the freezing chamber containing the frozen cells with a surface heated to a temperature of 35-45° C. for 10-40 seconds until the ice crystals are completely melted; ii. centrifuging; iii. suspending the cells from the centrifugation supernatant in an isotonic aqueous solution containing 1-10% w / v proline; Includes.

[0026] The present invention further relates to a composition comprising or consisting of non-adherent cells and an isotonic aqueous solution, preferably saline, containing at least one non-toxic cryoprotectant selected from albumin, alpha and beta globulins, and fibrinogen at a concentration of 4-50% weight / volume. Optionally, the composition may also contain a cryoprotectant additive, such as sucrose, trehalose, glucose or carboxymethylcellulose. In this embodiment, carboxymethylcellulose may be used at a concentration of 0.1-1% w / v; trehalose may be used at a concentration of 0.5-5% w / v, sucrose may be used at a concentration of 0.5-5% w / v, and glucose may be used at a concentration of 0.5-5% w / v.

[0027] Preferably, the cryoprotectant is human serum albumin.

[0028] The present invention further relates to a kit for storing non-adherent cells, comprising at least one sealing device for storing a sterile biological fluid, flexible at room temperature and capable of withstanding storage temperatures up to -196°C without damage, and an isotonic aqueous solution, preferably saline, comprising at least one non-toxic cryoprotectant selected from albumin, alpha and beta globulins, and fibrinogen in a concentration of 4-50% by weight, and optionally cryoprotective additives such as sucrose, trehalose, glucose and carboxymethylcellulose, preferably carboxymethylcellulose may be used in a concentration of 0.1-1% w / v, trehalose may be used in a concentration of 0.5-5% w / v, sucrose may be used in a concentration of 0.5-5% w / v, and glucose may be used in a concentration of 0.5-5% w / v. [Brief description of the drawings]

[0029] [Figure 1]Figure 1 shows the recovery of red blood cells (RBCs) after directional freezing with different BSA volume fractions (φBSA) (0.04, 0.14, and 0.28) at different translational speeds. Top panel: Images 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 freezing. Bottom panel: Cell recovery after freezing and thawing. [Diagram 2] FIG. 13 shows that cell recovery after directional freezing of RBCs (5x109 cells.mL-1) at 100 μm.s-1.ns shows no significant difference. [Diagram 3] SEM and flow cytometry of RBCs after freezing and thawing. RBCs were stained with calcein-AM for flow cytometry. Negative and positive controls show unstained and calcein-stained cells that had not undergone freezing. Various BSA volume fractions and ice surface speeds were applied in the experimental groups. Scale bar, 2 μm. [Figure 4] (A) Cell size (FSC-A) of calcein stained red blood cells. (B) Comparison of cell size between positive control (fresh cells) and cryopreserved cells. Frozen cells with φBSA of 0.14 showed significantly smaller size than fresh cells. *** indicates P<0.01. [Diagram 5] FIG. 1 shows cell recovery (A) and flow cytometry (B) of RBCs after 106 days of freezer storage. [Figure 6] (A) Cell size (FSC-A) of calcein stained RBCs after 106 days of freezer storage at -80°C. (B) Comparison of cell size between positive control (fresh cells) and cryopreserved cells. *** indicates significant difference at P<0.01. [Figure 7] Figure 14. Efficiency of resuspension of thawed cells followed by freezing at 100 μm.s-1. Absorbance of hemolysis in deionized H2O with the same amount of cells compared to the positive control, in the presence of BSA. [Figure 8] FIG. 1 shows a post-freeze cell suspension test, the histogram showing the measured values ​​of hemoglobin in the supernatant after suspension. [Figure 9] FIG. 1 shows a post-freeze cell suspension test, the histogram showing the measured values ​​of hemoglobin in the supernatant after suspension. [Figure 10] FIG. 1 shows a post-freeze cell suspension test, the histogram showing the measured values ​​of hemoglobin in the supernatant after suspension. [Figure 11] FIG. 1 shows a post-freeze cell suspension test, the histogram showing the measured values ​​of hemoglobin in the supernatant after suspension. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0030] Freezing Protocol a. Centrifuge 5 mL of sheep blood (1:1) in Alsever's solution (saline containing 2.05% dextrose, 0.8% sodium citrate, 0.055% citric acid, and 0.42% sodium chloride) at 600 g for 10 min at 4°C; b. Remove the supernatant and add 0.9% NaCl solution to return to the initial volume (5 mL); c. Centrifuge the resulting suspension at 600 g for 10 minutes at 4° C.; d. Remove the supernatant and add 0.9% NaCl solution to return to the initial volume (5 mL); e. Repeat steps c and d once more; f. Calculate red blood cell concentration using a hemocytometer; g.10 8 or 1.5*10 9 Transfer the cells into 2 mL of BSA solution (4-50 wt%) required to obtain a concentration of; h. The cell suspension was incubated at 50° C. in a 500 μL CoverWell™ incubation chamber (Grace Bio-Labs) 22 mm x 40 mm x 0.5 mm. -1 The specimen is frozen at a temperature gradient of 10, 50 and 100 μm.s (passing from a thermal block at +10 °C to a thermal block at -90 °C; gap between blocks 2 mm). The translation speeds of the specimen are 10, 50 and 100 μm.s -1 It is.

[0031] i. After freezing, keep the chamber containing the frozen cell suspension at -80 °C (for 1 day to 106 days).

[0032] Decompression Protocol a. Thaw the chamber containing the cells by placing it in contact with a 45°C surface for 10-15 seconds. b. Transfer 200 μL of the contents of each chamber into an Eppendorf and centrifuge under the following conditions: 1. For BSA concentrations of 20% or less: 3000 rpm, 20°C, 10 min. 2. For BSA concentrations above 20%, 6000 rpm, 20°C, 20 min. 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 and four more times for SEM characterization. e. The freeze-thaw efficiency is estimated from the measurement of hemoglobin concentration in the supernatant obtained in step b.1 or b.2 by UV-Vis absorbance at 541 nm.

[0033] Other formulations were tested for post-thaw cell resuspension (results correspond to measurements of hemoglobin in the supernatant after resuspension): Proline (1, 2, 4, 8, 10%) Proline (1, 2, 4, 8, 10%), followed by 4% BSA (See Figure 8) 4% BSA + Proline (0.1, 0.5, 1, 2, 5%) (See Figure 9) ·BSA+PBS ·PBS 0.9% NaCl BSA + glucose ·BSA + glucose + trimethylglycine + proline ·BSA + Glucose + Trimethylglycine + Proline + Glycine + Glutamine ·BSA + Trimethylglycine + Proline + Glycine + Glutamine (See Figures 10 and 11, where C1: bsa + glucose; C2: C1 + betaine + proline; C3: C2 + glycine + glutamine; C4: C3 - glucose)

[0034] 1% proline in 0.9% NaCl produces results similar to other proline concentrations that are systematically higher than the other formulations.

[0035] result Increasing the BSA volume fraction (φBSA) improved cell recovery, which was further improved by increasing the translational freezing rate performed (Figure 1).

[0036] When the volume fraction is between 0.04 and 0.28, the cell recovery rate is 10 μm.s -1 It increases from about 18% to about 30% at 100 μm.s -1 For all volume fractions, increasing the translation speed resulted in better cell recovery, especially for a value of φ = 0.28, which was consistent with the increase in translation speed from 10 to 100 μm.s. -1 A three-fold increase in hemolysis was possible when the hemoglobin concentration in the 100 μm.s solution was changed to 100 μm. After thawing and centrifugation, unlysed red blood cells (RBCs) clustered at the bottom of the tube and hemoglobin from the disrupted cells was released into the supernatant, resulting in a red color. For the negative control, the fresh blood sample showed little cell hemolysis as indicated by a colorless supernatant. Samples treated under other conditions showed a 100 μm.s solution with 100 μm.s -1 All samples showed red color except for φBSA of 0.28 to 50, confirming the positive role of high-concentration BSA system on RBC recovery rate.

[0037] The cell density used in these experiments (10 8 cells mL -1 ) is the physiological density of the human body (approximately 5×10 9 cells mL -1 ) was 50 times lower than that of 100 μm.s. In the literature, cell density has a crucial positive effect on final cryosurvival. To account for this effect, -1 At 5×10 9 cells mL-1 Another test was performed at a cell density of 1000× (Figure 2). Frozen cells with a BSA volume fraction of 0.28 showed the highest recovery rate (approximately 80%), further confirming the beneficial effect of high BSA concentration on cell viability during directional freezing. 8 cells mL -1 A relatively lower viability was observed compared to samples containing , which may be due to increased mechanical stress between the cells.

[0038] Red blood cell quality Although the previous experiments made it possible to determine the amount of red blood cells whose membrane integrity was maintained, it is also useful to obtain additional information on the physiological state of the recovered cells. For this, observations by 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 that confers great flexibility and a high oxygen transport capacity. Figure 3A shows that the majority of fresh RBCs have a biconcave disc shape. In the presence of low BSA volume fractions (φBSA = 0.04 and 0.14), the diameter of the RBCs increased by 100 μm.s. -1 Cells frozen at RT showed a morphology similar to that of fresh samples (Figure 3B-C).

[0039] In the high volume fraction group, the majority of thawed cells have a disk shape without any concave features, and only a small portion of cells have a morphological change toward a slight sphere (Figure 3D). Varying the translational speed did not significantly change the shape of RBCs when φBSA = 0.28 (Figure 3E-F). Calcein-AM, a cell-permeable non-fluorescent dye that is converted to green fluorescent calcein by intracellular esterases in live cells, was used in the flow cytometry test. In Figure 3G, the negative control (unstained fresh cells) and the positive control (stained fresh cells) have clearly different fluorescence intensities, which makes it easier to determine the experimental groups. Regardless of the experimental conditions (BSA concentration and translational speed), more than 97% of the cells have high calcein intensity. This indicates that after resuspension, most of the recovered RBCs have esterase activity and are therefore viable. In other words, the slightly changed shape of RBCs does not affect their esterase activity. In flow cytometry analysis, "forward scatter gating" is generally regarded as an indicator of cell size [9]. For the experimental group, the thawed RBCs were slightly smaller in size, φBSA = 0.14 (100 μm s -1 All RBCs frozen in the presence of 0.1% DMSO showed median cell sizes similar to those of the positive control (Figure 4A-B). Considering both cell recovery rate and quality, more than 95% of RBCs were within 100 μm.s. -1 After being cryopreserved by directional freezing at φBSA=0.28 in , they have intact membranes with metabolic activity and similar cell size compared to the control.

[0040] As mentioned before, the limited life span of RBCs, even in the presence of additives (less than 49 days), results in a high demand for daily blood donors. Here, cryopreservation of RBCs was performed by directional freezing, and then the RBCs were stored in a -80°C freezer for 3 months (106 days). In Figure 5A, the cell recovery followed the same survival trend after 1 day of storage. Red blood cell survival could be improved even more at higher translational speeds with increasing BSA content. After 106 days, 100 μm.s -1Samples prepared at a volume fraction of 0.28 in PBS showed approximately 95% viability, comparable to samples thawed after 1 day of storage. Additional calcein staining (Figure 5B) showed that more than 98% of the cells had esterase activity, regardless of the freezing conditions. In all samples, the size of the thawed cells was similar to that of the positive control, except for the group with a volume fraction of 0.14, which had a smaller size (Figure 6). Thus, directional freezing in the presence of BSA (φBSA=0.28) allows storage in the freezer for twice as long as the allowable shelf life of red blood cells (49 days) without significantly affecting cell viability and viability.

[0041] In conventional cryopreservation of RBCs, the deglycerolization process negatively impacts the final quality of the cells. For clinical applications, the efficiency of the resuspension of freeze-thawed cells must be considered to obtain a highly concentrated cell suspension equivalent to physiological cell density. Figure 7 shows the effect of the deglycerolization of thawed cells (φBSA=0.28, 100 μm.s) compared to the positive control, i.e., complete hemolysis of RBCs. -1 ) after resuspension, no hemolysis occurred (absorbance at 541 nm less than 0.005). In other words, the cells can be cryopreserved in a relatively dilute state, but once necessary, these cells could be thawed, concentrated, and then mixed into a specific solution without time-consuming and harmful post-purification of the cells, or even subjected to simple centrifugation, which is a substantial advantage of the method of the present invention.

[0042] Further analysis of metabolic activity based on esterases showed that more than 95% of the stored RBCs were viable after freezing and thawing, regardless of the experimental conditions. With regard to RBC viability and quality, the 0.28 BSA volume fraction group showed good freezing survival, resulting in cell sizes similar to those of fresh cells. Comparable recovery and viability of RBCs thawed after 1 day and 106 days of storage allowed extending the generally accepted shelf life of RBCs from 49 to 106 days. Considering the physiological cell density, the low hemolysis rate during resuspension of thawed cells provides a simple tool to enrich RBCs for transfusion.

Claims

1. A composition comprising non-adherent cells and an isotonic aqueous solution comprising at least one non-toxic cryoprotectant selected from albumin, alpha and beta globulins, and fibrinogen at a concentration of 4-50 weight / volume (w / v) %.

2. The composition of claim 1 , wherein the cryoprotectant is human serum albumin.

3. 3. The composition according to claim 1 or claim 2, further comprising a cryoprotective additive selected from sucrose at a concentration of 0.5-5% w / v, trehalose at a concentration of 0.5-5% w / v, glucose at a concentration of 0.5-5% w / v and carboxymethylcellulose at a concentration of 0.1-1% w / v.

4. The 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. 1. A method for preserving non-adherent cells by freezing, comprising: a) suspending the non-adherent cells in a cryopreservation medium composed of an isotonic aqueous solution containing at least one non-toxic cryoprotectant selected from albumin, alpha and beta globulins, and fibrinogen at a concentration of 4-50% by weight; b) optionally conditioning the mixture obtained in step a); c) directional freezing the mixture obtained in step a); d) storing at a temperature between −79° C. and −197° C., preferably at approximately −80° C.; The method includes:

6. 6. The method for preserving non-adherent cells by freezing according to claim 5, characterized in that the cryoprotectant is human serum albumin.

7. 7. The method for preserving non-adherent cells by freezing according to claim 5 or 6, characterized in that the cryopreservation medium further comprises a cryoprotective additive selected from sucrose, glucose, trehalose and carboxymethylcellulose.

8. 1. A method for thawing non-adherent cells, comprising: i. contacting the frozen cells with a surface heated to a temperature of 35-45°C; ii. centrifuging; iii. suspending the cells from the centrifugation supernatant in saline containing 1-10% proline; The method includes:

9. 1. A kit for storing non-adherent cells, comprising at least one containment device for storing a sterile biological fluid, and an isotonic aqueous solution comprising at least one non-toxic cryoprotectant selected from albumin, alpha and beta globulins, and fibrinogen at a concentration of 4-50% by weight, and optionally a cryoprotectant additive such as sucrose, carboxymethylcellulose, trehalose, or glucose.

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