Composition for increasing stability after thawing of frozen stem cell formulations

Abstract

The present invention relates to a composition (frozen formulation) used for cryopreservation of stem cells, and a cryopreservation method of stem cells using the composition. According to the cryopreservation solution of stem cells and the cryopreservation method using same of the present invention, the stem cells can be preserved for a long period of time, exhibit a high survival rate after being thawed, and can be directly applied to a living body without additional treatment after thawing.

Description

Composition for increasing the stability of frozen stem cell formulations after thawing

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

[0002]

[0003] The current problem or limitation of frozen stem cell formulations is their short shelf life of 3-10 days, which hinders distribution. To overcome this, the urgent task is to develop a formulation that extends the stability period of pure, highly effective stem cell therapeutics, ensuring long-term storage (specifically, more than a year or even several years) without safety issues. This formulation maintains product stability and efficacy, enabling immediate administration to patients upon thawing without additional processing. However, frozen stem cell formulations must meet safety, stability, and efficacy requirements.

[0004] Although several cryopreservation formulations have been developed for stem cell storage, only one has been commercialized as a cryopreservation formulation for direct human administration: Osiris' Prochymal, a stem cell therapy indicated for graft-versus-host disease (GVHD). However, Prochymal's excipients contain 10% DMSO.

[0005] Efforts have been made to prepare a composition for cell cryopreservation using recombinant human serum albumin, etc. (Korean Patent Publication No. 10-1407355), and there was an attempt to use human serum instead of fetal bovine serum (Transplantation. 2000 Dec 27;70(12):1780-7.), but this has resulted in problems such as altering the original characteristics of stem cells, such as reducing or rather promoting the proliferation or differentiation ability of stem cells.

[0006] Currently, dimethyl sulfoxide (DMSO) is the most widely used cell cryopreservative. While DMSO is essential for cell freezing, concentrations exceeding 5% can potentially be harmful to tissues. In this regard, Prochymal also presents safety concerns. Therefore, there is a need to reduce the DMSO content, and the development of a stem cell cryopreservation formulation that maintains stability and efficacy even with reduced DMSO concentration is essential.

[0007] Accordingly, the inventors of the present invention have made great efforts to develop a stem cell cryopreservation formulation that maintains the stability and efficacy of stem cells even at low DMSO concentrations, and as a result, they have confirmed that when adding glucose to a stem cell therapeutic cryopreservation formulation (freeze formulation), it is possible to manufacture a stem cell therapeutic cryopreservation formulation that is safe for the human body and has high product stability while maintaining efficacy even when the DMSO content is lowered, thereby completing the present invention.

[0008]

[0009] Summary of the invention

[0010] The purpose of the present invention is to provide a composition for stem cell cryopreservation having high stem cell storage stability and effectiveness even at low DMSO levels.

[0011] Another object of the present invention is to provide a method for cryopreserving stem cells using the above-described stem cell cryopreservation formulation.

[0012] To achieve the above purpose, the present invention provides a composition for cryopreservation of stem cells containing glucose, DMSO and human serum as active ingredients.

[0013] The present invention also provides a method for cryopreservation of stem cells, which comprises the step of storing and freezing stem cells in a cryopreservation formulation of the stem cells.

[0014]

[0015] Figure 1 shows the results of microscopic examination of the survival rate of stem cells in the Ad-MSC Fresh group and the Ad-MSC cryopreservation group by staining with trypan blue.

[0016] Figure 2 shows the FACS results of the surface antigen of the Ad-MSC Fresh group stem cells.

[0017] Figure 3 shows the FACS results of the surface antigen of the Ad-MSC cryopreservation group stem cells.

[0018] Figure 4 shows the results of Oil-red O staining after differentiation of stem cells from the Ad-MSC Fresh group and the Ad-MSC frozen group into adipocytes.

[0019] Figure 5 shows the results of Alizarin red S staining after differentiation of stem cells from the Ad-MSC Fresh group and the Ad-MSC frozen group into osteocytes.

[0020] Figure 6 shows the results of inducing chondrogenic differentiation of stem cells in the Ad-MSC Fresh group and the Ad-MSC frozen preparation group.

[0021] Figure 7 shows the results of confirming the adhesion ability of stem cells from the Ad-MSC Fresh group and the Ad-MSC frozen preparation group after culturing them for 8 hours.

[0022] Figure 8 shows the results of the sterility test (direct method) of the Ad-MSC Fresh group (A) and the Ad-MSC frozen formulation group (B).

[0023]

[0024] Detailed description of the invention and preferred embodiments

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise defined herein. Generally, the nomenclature used herein is well known and commonly used in the art.

[0026] DMSO (dimethyl sulfoxide), currently the most commonly used cryopreservative, is essential for freezing cells. However, if the concentration exceeds 5%, it can be harmful to tissues, necessitating a reduction in its content. To achieve this, the inventors discovered glucose. Inspired by hibernating animals, glucose is a crucial factor in their survival during freezing. Glucose travels through the bloodstream to vital organs and muscles, entering the cells. This series of mechanisms prevents cells from freezing and, even after thawing, allows them to resume their pre-hibernation state without damage. This means synthesizing glucose and using it as a cryoprotectant. Furthermore, glucose is safe for the human body.

[0027] In the present invention, a cryopreservation composition (freeze-type) for stem cell therapy was developed that maintains efficacy while maintaining safety for the human body and product stability by including glucose in the composition to reduce DMSO levels. Of particular importance is maintaining cryostability, but also maintaining post-thawing stability and viability. The inventors focused on this aspect in developing the formulation.

[0028] The term "cryopreservation" in the present invention refers to the long-term stabilization of cells or tissues through freezing. Cells typically undergo mutations at a rate of approximately one in ten thousand in culture. If cells are passaged over a long period of time, they may transform into a population of cells distinct from the original. In severe cases, specific cell functions may be lost during passage. Furthermore, mycoplasma infection may occur during passage. Due to these problems, cells or tissues are frozen and preserved before their unique characteristics are lost, enabling them to be extracted and used when needed. In particular, stem cells, for therapeutic purposes, require immediate access to healthy stem cells when needed. Therefore, a method for effectively cryopreserving stem cells is particularly necessary.

[0029]

[0030] Accordingly, the present invention relates, from one aspect, to a composition for cryopreservation of stem cells (cryopreservation formulation) containing glucose, DMSO and human serum as active ingredients.

[0031] In the cryopreservation formulation 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%.

[0032] In the cryopreservation formulation 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%.

[0033] In the cryopreservation formulation of the present invention, the human serum may be contained at a concentration of 91 to 97% (v / v).

[0034] The frozen formulation of the stem cell treatment agent according to the present invention is not limited to local injection (intra-articular, intramuscular, subcutaneous injection) and can also be administered intravenously.

[0035] From another perspective, the present invention relates to a method for cryopreserving stem cells, comprising the step of storing and freezing stem cells in a composition for cryopreserving stem cells.

[0036] In the present invention, the stem cells may be mesenchymal stem cells, embryonic stem cells, or induced pluripotent stem cells, but are not limited thereto.

[0037] The frozen storage period of stem cells using the cryopreservation composition of the present invention can be used without limitation, but 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.

[0038] In the present invention, it was confirmed that in the case of a cryopreservation formulation containing 2% glucose, the survival rate of stem cells after thawing was similar to or higher than that of the control group even when the concentration of DMSO, which is harmful to cells, was reduced.

[0039] In addition, in one embodiment of the present invention, it was confirmed that the adipose-derived mesenchymal stem cells (Ad-MSC) were stored using a cryopreservation formulation containing glucose to reduce the DMSO content (human serum 95% + Glucose 2% + DMSO 3%), and then the thawed stem cells exhibited a high survival rate, expressed stem cell surface antigens (see FIGS. 2 and 3), and maintained differentiation potential into adipocytes, osteocytes, and chondrocytes (see FIGS. 4, 5, and 6).

[0040] In another aspect of the present invention, it was confirmed that the adhesion ability of adipose-derived mesenchymal stem cells (Ad-MSC) was well maintained after storage using the cryopreservation formulation of the present invention (human serum 95% + Glucose 2% + DMSO 3%) and then thawed (see Fig. 7).

[0041] In another embodiment of the present invention, it was confirmed that the adipose-derived mesenchymal stem cells (Ad-MSC) were stored using the cryopreservation formulation of the present invention (human serum 95% + Glucose 2% + DMSO 3%), and then the thawed stem cells maintained a sterile state, were mycoplasma negative, and had an endotoxin content lower than the allowable level for injectable therapeutic agents (see Example 5).

[0042]

[0043] [Example]

[0044] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0045]

[0046] Example 1: Preparation and Validation of the Efficacy / Stability of a Glucose-Containing Cryopreservative

[0047] After culturing stem cells and harvesting them, 1x107 cells were divided into groups, suspended in 1 mL of the solution for each group, and then placed in cryovials for freezing. The composition of the cryopreservation solution for each group is as follows.

[0048]

[0049] The purpose of developing the cryopreservation solution of the present invention is to maintain the survival rate by minimizing the DMSO content as much as possible, and therefore 5% DMSO, a concentration known to be safe for the body, was used as a control.

[0050]

[0051] After fixing the experimental glucose (GLU) dose at 2% and adjusting the DMSO dose, the experimental freezing was stored in a deep freezer for 3 to 10 days, and then thawed to check the survival rate over time to select the optimal dose of DMSO and glucose. (After the behavior, the survival rate was checked over time by storing it in a refrigerated state.)

[0052] The results are shown below.

[0053]

[0054]

[0055]

[0056]

[0057] For the cryopreservation formulation containing 2% glucose, it was confirmed that the survival rate of stem cells after thawing was similar to or higher than that of the control group even when the DMSO concentration was reduced.

[0058]

[0059] Example 2: Confirmation of stem cell characteristics after cryopreservation in a frozen formulation

[0060] After culturing and recovering adipose-derived stem cells (Ad-MSC), they were suspended in a cryogenic formulation (human serum 95% + Glucose 2% + DMSO 3%) according to the present invention, placed in a cryo container, frozen in a deep freezer, and after 9 days, thawed in a 37℃ water bath, and then tested for stem cell viability, stem cell characteristics, efficacy, activity, sterility (direct method), mycoplasma negativity test (RT-PCR), and endotoxin. Through the above experiments, the cytotoxicity, stem cell characteristics, efficacy, activity, and safety of the Ad-MSC cryogenic formulation were confirmed, and it was confirmed that when stem cells are frozen using the cryogenic formulation of the present invention, it has an excellent and safe effect that can be applied as a cell therapy agent immediately after thawing without separate treatment.

[0061]

[0062] 2-1: Checking stem cell survival rate

[0063] The survival rates of the Ad-MS Fresh group and the Ad-MSC frozen group were confirmed using Trypan blue.

[0064] As a result, as shown in Figure 1 and Table 8, both groups demonstrated a survival rate of over 90%. In conclusion, it was confirmed that the frozen formulation was not toxic to cells after thawing.

[0065]

[0066] 2-3: Confirmation of Ad-MSC surface antigens

[0067] After reacting Ad-MS Fresh and Ad-MSC frozen formulations with stem cell surface antigen CD markers, surface antigens were identified using FACS.

[0068] As a result, as shown in Figures 2-3 and Table 9, both groups were able to confirm the characteristics of adipose-derived mesenchymal stem cell surface antigens (positive markers: CD73, CD90 / negative markers: CD31, CD34, CD45).

[0069]

[0070] 2-3: Confirmation of differentiation ability (fat, bone, and cartilage differentiation) of Ad-MSCs

[0071] To confirm the differentiation potential of Ad-MSC into adipocytes, stem cells from the Ad-MSC fresh group and Ad-MSC frozen group were seeded in a 12-well culture dish, and adipogenesis was induced using the StemPro™ Adipogenesis Differentiation Kit (Gibco, A1007001). Oil Red-O staining was then performed to confirm adipogenesis.

[0072] As a result, as shown in Fig. 4, it was confirmed that fat differentiation was induced in both groups.

[0073] To confirm the differentiation ability of Ad-MSC into osteocytes, stem cells from the Ad-MSC fresh group and the Ad-MSC frozen preparation group were seeded in a 12-well culture dish, and osteogenic differentiation was induced using the StemPro™ Osteogenesis Differentiation Kit (Gibco, A1007201). Then, Alizarin red staining was performed to confirm osteogenic differentiation.

[0074] As a result, as shown in Fig. 5, it was confirmed that bone differentiation was induced in both groups.

[0075] To confirm the differentiation potential of Ad-MSCs into chondrocytes, stem cells from the Ad-MSC fresh group and Ad-MSC frozen group were seeded in Falcon tubes, and TGF-β3 (Lonza, PT-4124) was added to Human Mesenchymal Stem Cell (hMSC) Chondrogenic Differentiation Medium BulletKit™ (lonza, PT-3003) to induce chondrogenic differentiation.

[0076] As a result, as shown in Fig. 6, it was confirmed that both groups were well induced to form round cartilage differentiation.

[0077]

[0078] Example 3: Confirmation of stem cell therapeutic efficacy after cryopreservation in a frozen formulation.

[0079] To determine changes in the efficacy of mesenchymal stem cells after cryopreservation in a frozen formulation, it was confirmed whether the content of cartilage regeneration factors was maintained.

[0080] First, cartilage differentiation was induced using a cartilage differentiation induction medium, and it was confirmed that cartilage differentiation was induced in a round shape. The content of human TSP-2, a cartilage regeneration factor, was confirmed using an Elsia assay (Human TSP-2 Elisa Kit (R&D Systems, DTSP20)).

[0081] As a result, as shown in Table 10, it was confirmed that the Ad-MSC cryopreservation group expressed TSP-2 in a similar amount to the content of cartilage regeneration factor (TSP-2) expressed in the Ad-MSC Fresh group, and thus, it was confirmed that the efficacy of stem cells was stably maintained even while preserved in the Ad-MSC cryopreservation group.

[0082]

[0083] Example 4: Confirmation of stem cell adhesion after cryopreservation in a frozen formulation

[0084] To confirm the activity of the Ad-MSC frozen formulation, stem cells were attached after thawing and the attachment ability was confirmed after 8 hours.

[0085] Stem cells from the Ad-MSC fresh group and Ad-MSC frozen group were seeded in 12-well culture dishes, and after 8 hours, the cells were fixed with 4% paraformaldehyde, and then crystal violet staining was performed to check the degree of attachment.

[0086] As a result, as shown in Fig. 7, it was confirmed that cells in both groups were well attached, and in conclusion, it was confirmed that the activity of stem cells was maintained even after cryopreservation using the Ad-MSC cryopreservation formulation.

[0087]

[0088] Example 5: Confirmation of the stability of stem cells after cryopreservation in a frozen formulation.

[0089] This study aimed to confirm the safety of cryopreserved stem cells from the Ad-MSC cryopreservation formulation as a cell therapy product that can be directly applied to living organisms. To this end, sterility was confirmed using a direct sterility test and a microplasma negative test. Furthermore, the absence of toxicity upon human administration was confirmed using an endotoxin (colorimetric) test.

[0090] 5-1: Sterility test (direct method)

[0091] For the sterility test, a sample is placed in a sterile medium and cultured in a BOD incubator. If no growth of microorganisms is observed with the naked eye on the final day (14th day) of the culture period, the medium is verified to be sterile (negative).

[0092] As a result of the test using stem cells from the Ad-MSC fresh group and the Ad-MSC frozen preparation group as samples, as shown in Fig. 8 and Table 11, both samples were confirmed to be negative, verifying the safety of the samples.

[0093]

[0094] 5-2: Mycoplasma negative test (real-time PCR)

[0095] Stem cells from the Ad-MSC fresh group and Ad-MSC frozen group were used as samples, and the DNA of the samples was prepared and tested for mycoplasma negativity using the MycoQSearch Mycoplasma qPCR Detection Kit (CellSafe, QDEP-100).

[0096] The criteria for the result judgment are that the Ct value of the HEX Channel, which is the PCR result of the internal DNA added to confirm that the test was performed properly, 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).

[0097] As a result of the mycoplasma negative test (real-time PCR) of the Ad-MSC Fresh group and the Ad-MSC frozen formulation group, as shown in Tables 12 and 13, the PC (Positive control) was positive, the NC (Negative control) was negative, and the sample was negative, confirming the safety of the sample in both the Ad-MSC fresh group and the Ad-MSC frozen formulation group.

[0098]

[0099]

[0100] 5-3: Endotoxin (colorimetric) test

[0101] Endotoxin refers to lipopolysaccharide (LPS), which constitutes 70% of the cell wall components of Gram-negative bacteria. It is a pyrogen that causes a feverish reaction when infecting human blood. Therefore, all drugs administered to the human body must not exceed the permissible endotoxin levels to be considered safe treatments.

[0102] Since the capacity of the Ad-MSC Fresh group and Ad-MSC frozen formulation group is 1 mL, if calculated using the endotoxin allowance calculation method below, the endotoxin allowance for intravenous and intramuscular injection is 349.6 EU / mL, and the endotoxin allowance for intrathecal injection is 13.9 EU / mL (see Table 14 of the United States Pharmacopeia (USP)).

[0103]

[0104] As a result of testing the endotoxin in the Ad-MSC Fresh group and the Ad-MSC frozen formulation group, the safety of the samples was proven as the endotoxin levels were confirmed to be much lower than the permissible level, as shown in Table 15.

[0105]

[0106] According to the composition for stem cell cryopreservation of the present invention and the cryopreservation method using the same, 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 able to be applied directly to a living body without separate treatment after thawing.

[0107]

[0108] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for cryopreservation of stem cells containing glucose, DMSO and human serum as active ingredients.

2. A composition for cryopreservation of stem cells, characterized in that the glucose is contained at a concentration of 1-4% (v / v) in the first paragraph.

3. A composition for cryopreservation of stem cells, characterized in that the DMSO is contained at a concentration of 2 to 5% (v / v) in the first paragraph.

4. A composition for cryopreservation of stem cells, characterized in that the composition contains human serum at a concentration of 91 to 97% (v / v) in the first paragraph.

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

6. A method for cryopreserving stem cells, characterized in that the stem cells in paragraph 5 are mesenchymal stem cells, embryonic stem cells, or induced pluripotent stem cells.

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