Composition to improve the stability of stem cell cryopreservatives after thawing

Abstract

The present invention relates to a composition (freezing agent form) used for cryopreservation of stem cells and a method for cryopreserving stem cells using the composition. The cryopreservation solution for stem cells and the cryopreservation method using the present invention enable long-term preservation of stem cells, have a high survival rate after thawing, and have the excellent effect of being immediately applicable to living organisms without any further processing after thawing.

Description

Technical Field

[0001] The present invention relates to a composition (cryoprotectant form) used for cryopreservation of stem cells and a method for cryopreserving stem cells using the composition.

Background Art

[0002] Currently used cryoprotectant forms for stem cells have a short expiration period of within 3 to 10 days, so they have problems or limitations in distribution. To overcome this, it is urgent to increase the stability period of pure and highly effective stem cell therapeutics, have no stability problems even after long-term storage (specifically, for one year or more or up to several years), maintain the stability and effectiveness of the product, and develop a dosage form that can be directly administered to the human body without additional treatment immediately after thawing when needed by patients. However, the cryoprotectant form of stem cells must satisfy safety, stability, and effectiveness.

[0003] A number of cryopreservation compositions have been developed for the purpose of preserving stem cells. However, as a product commercialized as a cryo-injection dosage form directly administered to humans, there is only "PROCHYMAL", a stem cell therapeutic agent of Osiris that treats graft-versus-host disease (GVHD). However, 10% DMSO is added to the excipient of "PROCHYMAL".

[0004] Efforts have been made to produce a composition for cryopreserving cells using recombinant human serum albumin, etc. (Korean Patent No. 10-1407355), and there have been attempts to use human serum instead of fetal bovine serum (Transplantation. 2000 Dec 27;70(12):1780-7.). However, this has caused problems such as changes in the original characteristics of stem cells because it reduces or rather promotes the proliferation ability or differentiation ability of stem cells.

[0005] Currently, DMSO (dimethyl sulfoxide) is widely used as a cryopreservation agent for cells. While DMSO is essential for freezing cells, concentrations exceeding 5% may be harmful to tissues. In this respect, "Prochimal" also has safety concerns. Therefore, it is necessary to reduce the DMSO content, and there is a need to develop a stem cell cryopreservation formulation that maintains the stability and efficacy of the formulation even with a reduced DMSO content.

[0006] Therefore, the inventors made diligent efforts to develop a stem cell cryopreservation formulation that maintains the stability and efficacy of stem cells even at low DMSO concentrations. As a result, they confirmed that by adding glucose to the cryopreservation formulation (freezing formulation) of a stem cell therapeutic agent, it is possible to manufacture a stem cell therapeutic agent cryopreservation formulation that maintains efficacy even when the DMSO content is reduced, is safe for the human body, and enhances product stability. This led to the completion of the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a stem cell cryopreservation composition that has high storage stability and efficacy of stem cells even at low DMSO content.

[0008] Another object of the present invention is to provide a method for cryopreserving stem cells using the aforementioned cryopreservation formulation for stem cells. [Means for solving the problem]

[0009] To achieve the above objective, the present invention provides a composition for cryopreservation of stem cells containing glucose, DMSO, and human serum as active ingredients.

[0010] Furthermore, the present invention provides a method for cryopreserving stem cells, which includes the step of storing and freezing the stem cells in the form of a cryopreservation agent for stem cells. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the results of examining the viability of stem cells in the Ad-MSC Fresh group and the Ad-MSC cryopreserved form group under a microscope after staining with trypan blue. [Figure 2] This figure shows the FACS results for surface antigens of stem cells in the Ad-MSC Fresh group. [Figure 3] This figure shows the FACS results for stem cell surface antigens in the Ad-MSC cryopreservation group. [Figure 4] This figure shows the results of staining with Oil-red O after differentiating stem cells into adipocytes from the Ad-MSC Fresh group and the Ad-MSC cryopreserved form group. [Figure 5] This figure shows the results of staining with Alizarin red S after differentiating stem cells into osteocytes from the Ad-MSC Fresh group and the Ad-MSC cryopreserved form group. [Figure 6] This figure shows the results of inducing chondrogenic differentiation of stem cells in the Ad-MSC Fresh group and the Ad-MSC cryopreserved form group. [Figure 7] This figure shows the results of confirming the adhesion ability of stem cells from the Ad-MSC Fresh group and the Ad-MSC frozen formulation group after 8 hours of adhesion and culture. [Figure 8] This figure shows the results of sterility testing (direct method) for the Ad-MSC Fresh group (A) and the Ad-MSC frozen dosage form group (B). [Modes for carrying out the invention]

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by skilled experts in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0013] Currently, DMSO (dimethyl sulfoxide), the most widely used cryopreservative for cells, is essential for freezing cells, but its concentration needs to be reduced because it can be harmful to tissues if it exceeds 5%. The inventors of this invention discovered glucose for this purpose. The idea for glucose was inspired by hibernating animals, as glucose is a crucial factor in their survival in a frozen state. Glucose is transported through blood vessels to major organs and muscles and taken up into cells. This series of mechanisms prevents cells from freezing, allowing humans to perform activities as before hibernation without cell damage after thawing. This means synthesizing glucose and using it as a cryoprotective agent. Furthermore, glucose is a safe substance for the human body.

[0014] In this invention, a cryopreservation composition (freezing formulation) for stem cell therapeutic agents was developed that incorporates glucose and reduces the DMSO content, thereby maintaining efficacy while being safe for the human body and enhancing product stability. While it is necessary to maintain cryopreservation stability, it is particularly important to maintain stability and viability after thawing. The inventors focused on this point and developed the formulation.

[0015] In this invention, the term "cryopreservation" means stably maintaining cells or tissues over a long period of time through freezing. Generally, cell mutations occur at a rate of about 1 in 10,000 cells during culture, and if cell subculturing continues for a long period, the cell population may change to one different from the original cell population, and in some cases, specific functions of the cells may be lost due to subculturing. In addition, infection with mycoplasma, etc., may occur during subculturing. Due to these problems, cells and tissues are cryopreserved so that their unique characteristics are not lost and they can be retrieved and used as needed, or tissues are cryopreserved. In particular, in the case of stem cells, it is necessary to be able to immediately use healthy stem cells when needed in order to use them as therapeutic agents, so there is a particular need for a method to effectively cryopreserve stem cells.

[0016] Therefore, in one aspect, the present invention relates to a composition for cryopreserving stem cells (cryopreservation dosage form) containing glucose, DMSO, and human serum as active ingredients.

[0017] In the cryopreservation dosage form of the present invention, the glucose may be contained at a concentration of 1 to 4% (v / v), preferably at a concentration of 1.5% to 3%, and more preferably at a concentration of 1.5% to 2.5%.

[0018] In the cryopreservation dosage form of the present invention, the DMSO may be contained at a concentration of 2 to 5% (v / v), and preferably at a concentration of 3 to 4%.

[0019] In the cryopreservation dosage form of the present invention, the human serum can be characterized by being contained at a concentration of 91 to 97% (v / v).

[0020] The cryopreservation dosage form of the stem cell therapeutic agent according to the present invention is not limited to local injection (intra-articular, intramuscular, subcutaneous injection), and intravenous administration is also possible.

[0021] In another aspect, the present invention relates to a method for cryopreserving stem cells, which includes the step of cryopreserving stem cells in the composition for cryopreserving stem cells.

[0022] In the present invention, the stem cells may be, but are not limited to, mesenchymal stem cells, embryonic stem cells, or dedifferentiated stem cells.

[0023] The cryopreservation period of stem cells using the composition for cryopreserving stem cells of the present invention is preferably 1 day to 20 years, more preferably 3 days to 10 years, and even more preferably 6 months to 3 years, but is not limited thereto.

[0024] In the present invention, in the case of the cryopreservation dosage form containing 2% glucose, it was confirmed that even when reducing the concentration of DMSO harmful to cells, the survival rate of stem cells after thawing is similar to or higher than that of the control group.

[0025] Furthermore, in one embodiment of the present invention, after preserving adipose-derived mesenchymal stem cells (Ad-MSCs) using a cryopreservation formulation containing glucose and a reduced DMSO content (human serum 95% + glucose 2% + DMSO 3%), it was confirmed that the thawed stem cells showed a high survival rate, expressed stem cell surface antigens (see Figures 2 and 3), and maintained the ability to differentiate into adipocytes, osteocytes, and chondrocytes (see Figures 4, 5, and 6).

[0026] In another embodiment of the present invention, it was confirmed that the adhesion ability of adipose-derived mesenchymal stem cells (Ad-MSCs) was well maintained after preservation using the cryopreservation formulation of the present invention (human serum 95% + glucose 2% + DMSO 3%) and thawing of the stem cells (see Figure 7).

[0027] In yet another embodiment of the present invention, adipose-derived mesenchymal stem cells (Ad-MSCs) were preserved using the cryopreservation formulation of the present invention (human serum 95% + glucose 2% + DMSO 3%), and it was confirmed that the thawed stem cells remained sterile, were mycoplasma-negative, and had an endotoxin content lower than the standard permissible value for injectable therapeutic agents (see Example 5). [Examples]

[0028] The present invention will be described in more detail below with reference to examples. It will be obvious to those ordinary in the art that these examples are merely illustrative and that the scope of the present invention should not be construed as being limited by these examples.

[0029] Example 1: Production of glucose-containing cryopreservation solution and confirmation of its effectiveness / stability

[0030] After culturing stem cells and harvesting them, the cells were divided into groups of 1 × 10⁷ cells, suspended in 1 mL of the solution for each group, and then frozen in cryovials. The composition of the cryopreservation solutions for each group is as follows.

[0031] [Table 1] Experiment 1 group [Terminology: GLU: glucose, DMSO: methyl sulfoxide, HS: human serum]

[0032] The purpose of developing the cryopreservation solution of this invention is to minimize the DMSO content while maintaining survival rates. Therefore, a 5% DMSO concentration, known to be safe for living organisms, was used as the control group.

[0033] [Table 2] Experiment 2 group

[0034] In the experiment, the glucose (GLU) concentration was fixed at 2%, then the DMSO concentration was adjusted. After freezing, the samples were stored in a deep freezer for 3 to 10 days, then thawed. The survival rate was checked at different time intervals to select the optimal dose of DMSO and glucose. (After thawing, the samples were stored under refrigeration, and the survival rate was checked at different time intervals.) The results are as follows:

[0035] [Table 3] Results of Experiment 1 (Survival rate before freezing: 89%, 0-6 hours after thawing)

[0036] [Table 4] Results of Experiment 1 (7 to 120 hours after thawing, survival rate before freezing: 89%)

[0037] [Table 5] Results of Experiment 2 (Survival rate before freezing: 92.9% from 0 to 6 hours after thawing)

[0038] [Table 6] Results of Experiment 2 (7 to 48 hours after thawing, survival rate before freezing: 92.9%)

[0039] In the case of a cryopreservation formulation containing 2% glucose, it was confirmed that even when the concentration of DMSO was reduced, the survival rate of stem cells after thawing was similar to or higher than that of the control group.

[0040] Example 2: Confirmation of stem cell characteristics after cryopreservation in cryopreservation form.

[0041] After culturing and harvesting adipose-derived stem cells (Ad-MSCs), they were suspended in the cryo formulation according to the present invention (human serum 95% + glucose 2% + DMSO 3%), placed in a cryo container, and frozen in a deep freezer. After 9 days, they were thawed in a 37°C water bath, and the viability, characteristics, efficacy, activity, sterility (direct method), mycoplasma negative test (RT-PCR), and endotoxin test of the stem cells were performed. Through these experiments, the cytotoxicity, characteristics, efficacy, activity, and safety of the Ad-MSC cryo formulation were confirmed, and it was confirmed that when stem cells are frozen using the cryo formulation of the present invention, they have excellent safety and can be immediately applied as a cell therapy without any further treatment after thawing.

[0042] [Table 7] Experimental group and conditions

[0043] 2-1: Confirmation of stem cell survival rate

[0044] The survival rates of the Ad-MS Fresh group and the Ad-MSC frozen dosage form group were confirmed using trypan blue.

[0045] As a result, as shown in Figure 1 and Table 8, a survival rate of over 90% was confirmed in both groups. In conclusion, it was confirmed that the cryopreserved form was non-toxic to cells after thawing.

[0046] [Table 8] Survival rate of Ad-MSC Fresh & Ad-MSC frozen formulations

[0047] 2-3: Confirmation of Ad-MSC surface antigen

[0048] Ad-MS Fresh and Ad-MSC cryopreserved forms were reacted with the stem cell surface antigen CD marker, and then the surface antigen was confirmed using FACS.

[0049] As a result, as shown in Figures 2-3 and Table 9, the characteristic surface antigens of adipose-derived mesenchymal stem cells (positive markers: CD73, CD90 / negative markers: CD31, CD34, CD45) were confirmed in both groups.

[0050] [Table 9] FACS results for Ad-MSC Fresh & Ad-MSC frozen formulations *positive%

[0051] 2-3: Confirmation of the differentiation ability of Ad-MSCs (adipose, bone, and cartilage differentiation)

[0052] To confirm the differentiation ability of Ad-MSCs into adipocytes, stem cells from the Ad-MSC fresh group and the Ad-MSC frozen formulation group were seeded into 12-well culture dishes. After inducing adipogenesis differentiation using the StemPro™ Adipogenesis Differentiation Kit (Gibco, A1007001), the adipogenesis ability was confirmed by staining with Oil Red O.

[0053] As a result, as shown in Figure 4, we confirmed that adipogenic differentiation was induced in both groups.

[0054] To confirm the differentiation ability of Ad-MSCs into osteocytes, stem cells from the Ad-MSC fresh group and the Ad-MSC cryopreserved group were seeded into 12-well culture dishes. After inducing osteogenic differentiation using the StemPro™ Osteogenesis Differentiation Kit (Gibco, A1007201), the differentiation ability was confirmed by staining with alizarin red.

[0055] As a result, as shown in Figure 5, we confirmed that ossification was induced in both groups.

[0056] To confirm the differentiation ability of Ad-MSCs into chondrocytes, stem cells from the Ad-MSC fresh group and the Ad-MSC cryopreserved group were seeded into Falcon tubes, and chondrogenic differentiation was induced by adding TGF-β3 (Lonza, PT-4124) to hMSC (Human Mesenchymal Stem Cell) Chondrogenic Differentiation Medium Bullet Kit™ (Lonza, PT-3003).

[0057] As a result, as shown in Figure 6, we confirmed that round cartilage differentiation was successfully induced in both groups.

[0058] Example 3: Confirmation of the therapeutic efficacy of stem cells after cryopreservation in cryopreservation form.

[0059] After cryopreservation in cryogenic form, we checked whether the content of cartilage regeneration factors was maintained in order to confirm any changes in the efficacy of mesenchymal stem cells.

[0060] First, chondrogenic differentiation was induced using a chondrogenic culture medium. After confirming that round chondrogenic differentiation had been induced, the content of human TSP-2, a chondrogenic factor, was confirmed using the Elsia assay (Human TSP-2 Elisa Kit (R&D Systems, DTSP20)).

[0061] As a result, as shown in Table 10, we confirmed that the Ad-MSC cryopreserved form group expressed TSP-2 at a similar level to that expressed in the Ad-MSC fresh group, and in conclusion, we confirmed that the efficacy of stem cells is stably maintained even while stored in the Ad-MSC cryopreserved form.

[0062] [Table 10] Human TSP-2 concentration (pg / mL) generated by the Ad-MSC Fresh group and the Ad-MSC frozen dosage form group.

[0063] Example 4: Confirmation of stem cell adhesion ability after cryopreservation in cryopreservation form.

[0064] To confirm the activity of the Ad-MSC cryopreserved form, the adhesion ability was checked 8 hours after thawing and attaching stem cells.

[0065] Stem cells from the Ad-MSC fresh group and the Ad-MSC cryopreserved group were seeded into 12-well culture dishes. After 8 hours, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and the degree of adhesion was checked.

[0066] As a result, as shown in Figure 7, we were able to confirm that cells adhered well in both groups, and in conclusion, we were able to confirm that stem cell activity is maintained even after cryopreservation using the Ad-MSC cryopreservation formulation.

[0067] Example 5: Confirmation of stem cell stability after cryopreservation in cryopreservation form.

[0068] We aimed to confirm the stability of stem cells cryopreserved in the Ad-MSC cryopreservation form as a cell therapy that can be immediately applied to living organisms. To this end, we conducted sterility tests (direct method) and microplasma rejection tests to confirm that the cells were sterile, and confirmed that they were non-toxic when administered to humans using an endotoxin (colorimetric method) test.

[0069] 5-1: Sterility test (direct method) The sterility test involves placing the sample in a sterile culture medium, culturing it in a BOD incubator, and verifying that the medium is sterile if no microbial growth is observed with the naked eye on the final day of the culture period (14 days) (negative result).

[0070] Tests using stem cells from the Ad-MSC fresh group and the Ad-MSC cryopreserved group confirmed negative results in both samples, as shown in Figure 8 and Table 11, thus verifying the safety of the samples.

[0071] [Table 11] Sterility test results for the Ad-MSC Fresh group and the Ad-MSC cryopreservation group

[0072] 5-2: Mycoplasma negative test (real-time PCR)

[0073] Stem cells from the Ad-MSC fresh group and the Ad-MSC frozen formulation group were used as samples. The DNA from the samples was prepared, and a mycoplasma negative test was performed using the MycoQSearch Mycoplasma qPCR Detection Kit (CellSafe, QDEP-100).

[0074] The criteria for determining the results are that the Ct value of the HEX Channel, which is the PCR result of internal DNA inserted to confirm that the test was performed correctly, must be positive (Ct value < 40), and the Ct value of the FAM Channel, which is the PCR result of mycoplasma, must be negative (Ct value = undetermined).

[0075] As shown in Tables 12 and 13, the results of the mycoplasma negative test (Real-time PCR) for the Ad-MSC Fresh group and the Ad-MSC frozen dosage form group confirmed that the PC (Positive control) was positive, the NC (Negative control) was negative, and the samples were negative, thus confirming the safety of the samples in both the Ad-MSC Fresh group and the Ad-MSC frozen dosage form group.

[0076] [Table 12] Results of Mycoplasma Negative Tests by the Ad-MSC Fresh Group

[0077] [Table 13] Results of the Mycoplasma Negative Test for the Ad-MSC Freezing Formulation Group

[0078] 5-3: Endotoxin (colorimetric method) test

[0079] Endotoxins are lipid polysaccharides (LPS) that make up 70% of the cell wall components of Gram-negative bacteria. They are pyrogenic substances that cause a fever reaction when they infect the human bloodstream. Therefore, all pharmaceuticals administered to the human body must not exceed the acceptable limit for endotoxins in order to be safe for use as therapeutic drugs.

[0080] Since the volume of the Ad-MSC Fresh group and the Ad-MSC frozen dosage form group is 1 mL, the endotoxin tolerance for intravenous and intramuscular injection is calculated to be 349.6 EU / mL, and the endotoxin tolerance for intrathecal injection is 13.9 EU / mL, based on the endotoxin tolerance calculation method described below (see Table 14, United States Pharmacopeia (USP)).

[0081] [Table 14] Endotoxin tolerance levels (EU / mL) according to the United States Pharmacopeia (USP) standards.

[0082] Endotoxin testing was performed on the Ad-MSC Fresh group and the Ad-MSC frozen dosage form group, and as shown in Table 15, the results were confirmed to be significantly lower than the acceptable endotoxin levels, thus confirming the safety of the samples.

[0083] [Table 15] Endotoxin test results for the Ad-MSC Fresh group and the Ad-MSC cryopreservation group [Industrial applicability]

[0084] According to the stem cell cryopreservation composition and cryopreservation method using the present invention, stem cells can be preserved for a long period of time, have a high survival rate after thawing, and have the excellent effect of being immediately applicable to living organisms without any further processing after thawing.

[0085] Although specific parts of the present invention have been described in detail above, it is clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for cryopreserving stem cells, characterized by containing glucose, DMSO, and human serum as active ingredients.

2. The stem cell cryopreservation composition according to claim 1, characterized in that the glucose is contained at a concentration of 1 to 4% (v / v).

3. The stem cell cryopreservation composition according to claim 1, characterized in that the DMSO is contained at a concentration of 2-5% (v / v).

4. The stem cell cryopreservation composition according to claim 1, characterized in that it contains human serum at a concentration of 91-97% (v / v).

5. A method for cryopreserving stem cells, characterized by comprising the step of storing and freezing stem cells in a stem cell cryopreservation composition according to any one of claims 1 to 4.

6. The method for cryopreserving stem cells according to claim 5, characterized in that the stem cells are mesenchymal stem cells, embryonic stem cells, or reverse-differentiated stem cells.

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