Frozen formulation of cells

Abstract

The present invention relates to a frozen cell formulation that can significantly improve the cell viability of cryopreserved cells, preferably stem cells. According to the present invention, the cell viability can be improved when thawing cells after freezing, thereby preventing a decrease in therapeutic efficiency that may occur during the storage and transportation of cell therapy agents.

Description

[Technical Field]

[0001] The present invention relates to a frozen preparation of cells, preferably stem cells, and more particularly to a frozen preparation of stem cells that contains glutathione as an active ingredient and can significantly improve the survival rate of cells, such as cryopreserved stem cells.

[0002] [Background technology]

[0003] Cryopreservation is commonly used to preserve cells, including stem cells. Because cells have different physiological characteristics depending on their origin, tissue source, and internal microenvironment, cryopreservation methods must be tailored to each cell type. Stem cells, in particular, require optimized cryopreservation conditions to maintain their inherent specific division and differentiation capabilities. Since they can be used as cell therapy immediately after storage, a low-toxicity preservation solution is essential. Developing effective cryopreservation techniques for stem cells is also an important step toward establishing stem cell banks. Cryopreservation typically involves storing samples at ultralow temperatures between -80°C and -196°C, maintaining cellular function and enabling long-distance transport and long-term storage.

[0004] Cryoprotective agents reduce physical and chemical cell damage during the freezing and thawing process and improve cell viability after thawing. A cryopreservative agent (CPA) consisting of 10% (v / v) dimethyl sulfoxide (DMSO) and 90% (v / v) FBS is commonly used for cell cryopreservation. This formulation has been widely used for many types of stem cells. However, the use of DMSO and FBS has many drawbacks. For example, serum (e.g., FBS) in the cryopreservative agent can induce infection and immune responses (Kocaoemer et al., Stem Cells., 25:1270-1278, 2007). Cryopreservatives are often a heterogeneous mixture of components, derived from animals, including humans. The ratio of these components can vary with each production run, creating numerous problems for both experimental and industrial use. Therefore, it is preferable to minimize serum use or to use serum-free media. Therefore, there is a need for the use of well-defined cryopreservatives, including substances approved for clinical application of cryopreserved cells and biological materials not of animal origin.

[0005] Together, dehydration, oxygen free radical formation, and apoptosis are important causes of decreased cell viability during or after freezing. Disaccharides such as sucrose and trehalose have been widely used as non-toxic natural cryopreservatives, additives for freeze-drying, and cell membrane stabilizers during dehydration. Peroxide radicals cause oxidative damage to cells, including lipid peroxidation, protein oxidation, and DNA damage.

[0006] Therefore, in the present invention, we have made extensive efforts to develop a freezing formulation for cells, such as stem cells, that can minimize the decrease in cell viability caused by peroxide radicals during cryopreservation of cells, preferably stem cells. As a result, we have found that when cells, such as stem cells, are cryopreserved using a freezing formulation containing glutathione, the cell viability after thawing is significantly increased, which led to the completion of the present invention.

[0007]

[0008] Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a frozen formulation of cells, preferably stem cells, that can significantly improve cell viability upon thawing after cryopreservation. [Means for solving the problem]

[0010] To achieve the above object, the present invention provides a frozen formulation of cells, preferably stem cells, containing glutathione and stem cells.

[0011] [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of screening antioxidants for frozen formulations of stem cells.

[0013] [Figure 2] FIG. 1 shows the results of optimizing glutathione concentration for frozen formulations of stem cells.

[0014] [Figure 3] This figure shows the results of measuring cell viability by freezing stem cells at a low concentration (5 x 105 cells / 500 μl) using the frozen formulation (4) of the present invention for one week, thawing them, and culturing them for 4 and 8 hours.

[0015] [Figure 4] This figure shows the results of measuring cell viability by freezing stem cells at a high concentration (5 x 106 cells / 100 μl) using the frozen formulation (4) of the present invention for one week, thawing them, and culturing them for 4 and 8 hours.

[0016] [Figure 5] This figure shows the results of measuring cell viability by thawing stem cells after freezing them for one month using the frozen formulation of the present invention (EN001 suspension) and culturing them for 4 and 8 hours.

[0017] [Figure 6] FIG. 1 shows the results of examining the cell aggregation phenomenon (A) and cell viability depending on the culture time after thawing stem cells frozen for 18 months using the frozen formulation (EN001 suspension) of the present invention.

[0018] [Figure 7] FIG. 1 shows the results of confirming the stability of umbilical cord-derived mesenchymal stem cells (WJ-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose-derived mesenchymal stem cells (AD-MSCs), placenta-derived mesenchymal stem cells (PL-MSCs), fibroblasts, and the brain cancer cell line H4, which were frozen and stored using the frozen dosage form of the present invention, and then thawed at 4°C and 37°C, respectively.

[0019]

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0022] In the present invention, in order to develop a cryopreservation formulation for cells, such as stem cells, that can minimize the decrease in cell viability due to peroxide radicals during cryopreservation of cells, preferably stem cells, stem cells were frozen using a variety of antioxidants, and then thawed to confirm cell viability. It was confirmed that when stem cells are cryopreserved in a cryopreservation formulation containing glutathione, one of these antioxidants, cell viability after thawing is significantly increased.

[0023] Accordingly, the present invention relates to a frozen formulation of cells containing glutathione and cells, preferably stem cells.

[0024] The frozen dosage form of the present invention can be characterized by using Hartmann's solution as a base solution and further comprising human serum albumin and DMSO.

[0025] In the present invention, the glutathione may be contained at a concentration of 50 μM to 1000 μM, preferably 50 μM to 500 μM, more preferably 50 μM to 200 μM, and even more preferably 60 μM to 150 μM.

[0026] In the present invention, the human serum albumin may be contained at a concentration of 1% to 30%.

[0027] In the present invention, the DMSO may be contained at a concentration of 0.5% to 30%.

[0028] In the present invention, the cells, preferably stem cells, are 5×10 5 cell / mL ~ 1 × 10 7 The antibody can be characterized by being contained at a concentration of 100 cells / mL.

[0029] In the present invention, the Hartmann solution may be characterized by containing 0.1 g / L to 0.5 g / L of calcium chloride, 0.1 g / L to 0.8 g / L of potassium chloride, 2 g / L to 10 g / L of sodium chloride, and 1 g / L to 5 g / L of sodium lactate, and preferably contains 0.27 g / L of calcium chloride, 0.4 g / L of potassium chloride, 6 g / L of sodium chloride, and 3.22 g / L of sodium lactate.

[0030]

[0031] [Example]

[0032] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0033]

[0034] Example 1: Antioxidant screening for frozen formulations of stem cells

[0035] In the examples of the present invention, umbilical cord blood-derived mesenchymal stem cells were used to establish a frozen formulation for stem cells.

[0036] Umbilical cord mesenchymal stem cells (Wharton's Jelly-mesenchymal stem cells, WJ-MSCs) were isolated from umbilical cords separated by the method of Park et al. (Arch. Pharm. Res. 39:1171-1179, 2016) after obtaining umbilical cords and placentas through a collaborative research project with the Department of Obstetrics and Gynecology at Samsung Medical Center in accordance with the standards of the IRB approved by Samsung Medical Center (IRB# 2015-09-023-003). Specifically, the umbilical cords were cut into 3-4 cm lengths, the tissue was minced, and treated with collagenase solution (Gibco, USA) for 60-90 minutes to degrade the extracellular matrix. Then, 0.25% trypsin (Gibco, USA) was added and further decomposition was induced at 37°C for 30 minutes. Then, fetal bovine serum (FBS; Biowest, USA) was added, and the cells were harvested by centrifugation at 1000 g for 10 minutes. The cells were then cultured in MEM Alpha (Minimum Essential Medium; Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS (fetal bovine serum; Invitrogen-Gibco) and 50 μg / mL gentamicin (Invitrogen-Gibco) at 37°C in a 5% CO environment. Human umbilical cord-derived mesenchymal stem cells (WJ-MSCs) were used for the experiments.

[0037] A common stem cell cryopreservation formulation uses Ringer's solution as the base solution, DMSO as the cryopreservative, and human serum albumin (Table 1).

[0038] [Table 1]

[0039] In this example, to screen for antioxidants that can minimize the decrease in cell viability due to peroxide radicals during cryopreservation of stem cells, acetadote, fursultiamine hydrochloride, vitamin C, and glutathione were used as antioxidants. WJ-MSCs were cultured at 1 × 10 in a 96-well plate. 4 After seeding and culturing for 24 hours, the effect of antioxidants on the cell viability of WJ-MSCs was confirmed by CCK-8 assay.

[0040] As a result, as shown in Figure 1, it was confirmed that when glutathione was used as an antioxidant, the cell survival rate was highest and cell proliferation actually increased.

[0041]

[0042] Example 2: Confirmation of the optimal concentration of glutathione in frozen dosage forms

[0043] To determine the optimal concentration of glutathione in frozen formulations, WJ-MSCs were treated with hydrogen peroxide (HO) at concentrations of 0 μM, 10 μM, 50 μM, and 100 μM to induce stress due to reactive oxygen species. Then, glutathione was added at concentrations of 0 μM, 10 μM, 50 μM, and 100 μM, and cell recovery was confirmed using a CCK-8 assay.

[0044] As a result, as shown in Figure 2, the highest cell viability was confirmed when glutathione was used at a concentration of 100 μM, and it was decided to use glutathione at a concentration of 100 μM for the frozen formulation.

[0045]

[0046] Example 3: Confirmation of viability of frozen stem cell formulations

[0047] To select the optimal Ringer's solution suitable for stem cell cryopreservation, we used commercially available cryopreservation formulations CS5 (Sigma, USA) and CS10 (Sigma, USA) as controls, and confirmed the short-term stability of DSL 1:2:3 solution (CJ Healthcare) and Hartmann solution (Innoen) as base solutions.

[0048] Low concentration (5×10 5 WJ-MSCs that had been cryopreserved for one week at a concentration of 1000 μl (cells / 500 μl) were thawed at 4°C and 37°C, respectively, and the cell viability was measured after leaving the cells for 4 and 8 hours.

[0049] As a result, as shown in Figure 3, the highest cell viability was observed when H+HSA+G+DMSO (Hartmann's solution+human serum albumin+glutathione+DMSO) was used as the frozen formulation.

[0050] In addition, high concentration (5 × 10 6 WJ-MSCs that had been cryopreserved for one week at a concentration of 100 μl (cells / 100 μl) were thawed at 4°C and 37°C, respectively, and the cell viability was measured after leaving the cells for 4 and 8 hours.

[0051] As a result, as shown in FIG. 4, even when a high concentration of stem cells was used, the frozen formulation under the condition of H+HSA+G+DMSO showed the highest cell viability.

[0052] Therefore, the frozen formulation of cells, preferably stem cells, according to the present invention was determined to be a formulation containing human serum albumin (HSA), glutathione, and DMSO in Hartmann solution (Innoen), and was named "EN001 suspending agent."

[0053]

[0054] Example 4: Stability confirmation of frozen stem cell formulations

[0055] The short-term stability was confirmed by comparing the commercially available frozen preparations CS5 (Sigma, USA) and CS10 (Sigma, USA) with a frozen formulation containing FBS and DMSO and the "EN001 suspension," a frozen formulation of stem cells of the present invention.

[0056] Cells were cryopreserved in each frozen formulation for one month, and then thawed at 4°C and 37°C, respectively, and the cell viability was measured after leaving the cells for 4 and 8 hours.

[0057] As a result, as shown in FIG. 5, the highest cell viability was observed when the frozen formulation according to the present invention was used.

[0058] In addition, the cells were frozen and stored for 18 months in "EN001 suspension," a frozen formulation of the stem cells of the present invention, and then thawed to check the ratio of single cells to clumped cells, as well as the cell viability.

[0059] As a result, as shown in Figure 6, when the frozen formulation of stem cells of the present invention, "EN001 suspension," was used, the incidence of cell aggregation was significantly low (A), and cell viability (B) was maintained at 80% or more.

[0060]

[0061] Example 5: Scalability of frozen stem cell formulations

[0062] Using the frozen formulation of the present invention, the stability of not only umbilical cord-derived mesenchymal stem cells (WJ-MSCs), but also umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose-derived mesenchymal stem cells (AD-MSCs), placenta-derived mesenchymal stem cells (PL-MSCs), fibroblasts, and the brain cancer cell line H4 was confirmed by freezing and storing them for one month and then thawing them at 4°C and 37°C, respectively.

[0063] As a result, as shown in FIG. 7, when thawed at 4° C., high cell viability was observed for all types of cells used.

[0064] [Industrial Applicability]

[0065] According to the present invention, the cell viability can be improved when thawing stem cells or the like after freezing, thereby preventing a decrease in therapeutic efficiency that may occur during the storage and transportation of cell therapy agents such as stem cells.

[0066]

[0067] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. A frozen formulation of cells containing glutathione and cells.

2. 10. The frozen formulation of claim 1 further comprising Hartmann's solution, human serum albumin, and DMSO.

3. The frozen formulation of cells according to claim 1, wherein the glutathione is contained at a concentration of 50 μM to 1000 μM.

4. The frozen cell formulation of claim 2, wherein the human serum albumin is contained at a concentration of 1% to 30%.

5. The frozen cell formulation of claim 2, wherein the DMSO is contained at a concentration of 0.5% to 30%.

6. The frozen formulation of cells according to claim 1, wherein the cells are stem cells.

7. The stem cells were 5×10 ５ cell / mL~1×10 ７ The frozen formulation of cells according to claim 6, characterized in that it is contained at a concentration of 1000 cells / mL.

8. 3. The frozen formulation of cells according to claim 2, wherein the Hartmann's solution contains 0.1 g / L to 0.5 g / L of calcium chloride, 0.1 g / L to 0.8 g / L of potassium chloride, 2 g / L to 10 g / L of sodium chloride, and 1 g / L to 5 g / L of sodium lactate.

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