KR101954120B1 - Composition for cryopreservation of stem cells having improved cryopreservation effect and method using the same - Google Patents

Abstract

The present invention relates to a composition for cryopreservation of a stem cell having an improved cryopreservation effect, comprising a recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) (FBS) as an active ingredient, and to a composition for cryopreserving stem cells. When the composition for cryopreservation according to the present invention is used for cryopreservation of stem cells, the number of cells before and after freezing is constant despite the cryopreservation, the survival rate and proliferation after thawing are excellent, the expression of cell surface markers is maintained, Cellularity is maintained. Therefore, the present invention can be used as a cryopreservative of stem cells, and it is characterized by its excellent clinical applicability because it does not contain any animal or heterogeneous material at all.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for cryopreserving stem cells,

The present invention relates to a composition for cryopreservation of stem cells improved in cryopreservation effect, and more particularly, to a composition for cryopreservation of stem cells, which comprises a xeno-free and an animal-derived component The present invention relates to a composition for cryopreserving stem cells having an improved cryopreservation effect while maintaining animal component-free, and a method of using the same.

Stem cells are cells that can multiply indefinitely while maintaining undifferentiated state and can differentiate into specific functions and forms when given a certain environment and conditions. Immunity and pluripotency of these stem cells provide a good in vitro model for the study of human development. Drug testing and toxicity testing of homogeneous human tissues or cells from stem cells can facilitate the development of new drugs. Further, it is possible to obtain a large amount of cells or tissues capable of replacing the damaged tissue, so that it can be used for the treatment of intractable diseases.

A cell therapy agent is a drug manufactured by physical, chemical, or biological manipulation, such as living proliferation or selection of a living individual, allogeneic or xenogeneic cell, and the stem cell treatment agent is a stem cell- Medicines to be administered are classified into bone marrow-derived, cord blood-derived, peripheral blood-derived, and fat-derived medicines according to their origins.

These stem cell and (stem) cell therapy agents are sensitive to environment because living cells are used as raw materials, and short shelf life is short. Therefore, to preserve the characteristics of these stem cells and to preserve them for a long period of time, it is essential to preserve the cells. In general, cryopreservation is used in the cryopreservation of cells since the cells are damaged by the crystallization of intracellular ice.

Presently, the cryopreservation agents used for stem cells include heterologous such as fetal bovine serum (FBS), human serum, or animal-derived components. Therefore, infection or immunological reaction of virus, And there is a problem that it is not suitable for preservation of a cell therapeutic agent or the like used for therapeutic purposes.

Korean Patent Laid-Open No. 10-2012-0117209, which is a domestic patent related to the cryopreservation of stem cells, discloses that sucrose, ethylene glycol, and polyvinylpyrrolidone in a PBS (phosphate buffered saline) Discloses a stem cell supernatant cryopreservation solution.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop cryopreservation agents that are excellent in the cryopreservation effect of stem cells without using animals and other materials derived from animal such as serum of animals as a culture additive. As a result, in the case of a composition comprising the recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) of the present invention as an active ingredient, The present inventors have found that the cryopreserving effect on stem cells is superior to the preservative, and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for producing recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) as an active ingredient, wherein fetal bovine serum (FBS) The present invention provides a composition for cryopreserving stem cells.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising recombinant human serum albumin, trehalose, and methylcellulose as an active ingredient, and containing fetal bovine serum (FBS) as an active ingredient The present invention provides a composition for cryopreserving stem cells.

As used herein, the term " recombinant human serum albumin " means a recombinant human serum albumin ("rHSA") synthesized in plant cells, insects, animal cells or bacteria by genetic engineering of albumin, And thus there is no need for unnecessary immunological reaction or contamination.

According to one embodiment of the present invention, the recombinant human serum albumin of the present invention may have a concentration of 0.5-3 (w / v)% based on the total volume of the composition for cryopreservation, more specifically 0.5-2, 0.5-1.5, 1-1.5 (w / v)%, most specifically 1 (w / v)%.

As used herein, the term " dimethyl sulfoxide (DMSO) " is a cell membrane permeable freezing inhibitor which serves to prevent formation of ice crystals and rupture of the cell membrane.

As used herein, the term " cryopreservation " means that when a subculture is continued during the cultivation of a proliferative cell, a certain proportion of mutants may be naturally produced and turned into a cell population that is completely different from the original cell population. Means to preserve cells at a low temperature of 0 ° C or lower in order to stably preserve the cell line and prevent loss due to contamination by minimizing the mutation and preventing aging and transformation. During cryopreservation, cellular organs and functions caused by low-temperature exposure of cells are disturbed, resulting in mechanical and physical damage due to the formation of ice crystals. A cryoprotectant is necessary to prevent cell damage and damage due to cryopreservation.

Examples of commonly used cryopreservatives include glycerol, sucrose, glucose, methanol, lactose and the like, in addition to dimethylsulfoxide (DMSO) described above. Dimethylsulfoxide and glycerol are most commonly used. The frozen medium generally contains a culture medium, fetal bovine serum (FBS), and a cryoprotectant. The composition is usually prepared by adding 10-15 (v / v)% cryoprotectant (DMSO, glycerol) 25 (v / v)%. It is ideal to use an electronic automatic cell freezer for freezing for the cryopreservation. However, if there is no such equipment, a freezing vial is placed in a freezing container containing isopropyl alcohol and stored at 70 ° C to 80 ° C for one night. Store in a liquid nitrogen container at 196 ° C.

According to an embodiment of the present invention, the dimethylsulfoxide of the present invention may have a concentration of 1-10 (v / v)% based on the total volume of the composition for cryopreservation, more specifically 3-7, 5 -7 (v / v)%, most specifically 5 (v / v)%. The dimethylsulfoxide is known to exhibit cytotoxicity at concentrations above 10 (v / v)%.

The composition for cryopreservation of the present invention is characterized by xeno-free and animal component-free. In the present invention, the term 'non-heterozygous' means that a component derived from a heterologous animal such as fetal bovine serum is not included. In the present invention, the term 'non-animal derived component' means not only a heterozygous animal but also a component derived from a homologous animal do.

According to one embodiment of the present invention, a novel composition of a cryoprotectant composition comprising recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) of the present invention (Including CPA2 with 5% DMSO and 5% rHSA, CPA3 with 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4 with 5% DMSO and 1% rHSA, 0.05% methylcellulose) There was no significant difference in terms of cytotoxicity (Example 2 and Fig. 2) as compared with the cryopreservation agent (CPA1; containing 5% DMSO and 20% FBS ).

According to another aspect of the present invention, the composition for cryopreservation of the present invention may further comprise trehalose as an active ingredient.

As used herein, the term " trehalose " is a non-reducing saccharide having a crystal structure of this type, which is a kind of saccharides widely present in nature such as microorganisms, plants, and insects. It is widely used as a cryoprotectant, and it is known that it is a non-penetrating cryoprotectant which is not toxic and stabilizes the cell membrane when dehydration occurs. In general, the concentration of trehalose used as a cryoprotectant is 0.1-1.0 M.

According to one embodiment of the present invention, the trehalose may be in a concentration of 1-30 mM in the cryopreservation composition of the present invention, more specifically 1-20, 5-20, 7-15, 7-12 , 10-12 mM, most particularly 10 mM, which is extremely small compared to the concentration of trehalose commonly used.

According to another aspect of the present invention, the composition for cryopreservation of the present invention may further comprise methylcellulose as an active ingredient.

The methylcellulose is a high molecular weight polymer known as a non-permeable cryoprotectant that does not penetrate into cells, and plays a role in preventing cell membrane damage by promoting the osmotic equilibrium inside and outside the cell by promoting the outflow of water before cell freezing . Merten et al. (1995, Biologicals 23: 185189.) reported that in the experiments using Vero cells and BHK-21 cells, cells frozen in standard freezing medium (containing 10% Fetal Calf Serum) and serum-free medium (containing methylcellulose) There was no difference.

According to one embodiment of the present invention, the methylcellulose of the present invention may have a concentration of 0.001-0.5 (w / v)% based on the total volume of the composition for cryopreservation, more specifically 0.01-0.3, 0.01 -0.2, 0.01-0.1 (w / v)%, more specifically 0.01-0.05 (w / v)%, most specifically 0.05 (w / v)%.

According to one embodiment of the present invention, there is provided a novel recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) One composition of the cryopreservative composition (CPA2, including 5% DMSO and 5% rHSA, CPA3, 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4, 5% DMSO and 1% rHSA, There was no significant difference in the total cell count before and after freezing compared with the cryopreservation agent (CPA1; containing 5% DMSO and 20% FBS) containing animal-derived components (Example 3 and Fig. 3 A).

Also, according to another embodiment of the present invention, there is provided a novel recombinant human serum albumin, trehalose, methylcellulose, and dimethylsulfoxide (DMSO) (CPA2 with 5% DMSO and 5% rHSA, CPA3 with 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4 with 5% DMSO and 1% rHSA, 0.05% methylcellulose) (Example 3 and Fig. 3B) , both in terms of the survival rate of the cells before and after freezing, as compared with the cryopreservation agent (CPA1 containing 5% DMSO and 20% FBS) containing the derived components .

In particular, the composition for cryopreservation (CPA2; 5% DMSO and 5% DMSO) containing the recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) Including 5% rHSA, CPA3, 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4, 5% DMSO and 1% rHSA, 0.05% methylcellulose) (CPA1, including 5% DMSO and 20% FBS). Furthermore, the composition for cryopreservation (CPA3 containing 5% DMSO and 1% rHSA, 10 mM trehalose) containing recombinant human serum albumin, dimethylsulfoxide and trehalose as the active ingredient showed the best cell survival rate.

5% DMSO and 5% rHSA, CPA3, 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4, 5% DMSO and 1% rHSA , 0.05% methylcellulose) exhibited excellent cryopreservation effect which can replace the cryoprotectant composition containing animal-derived components such as conventional fetal bovine serum.

In addition, a novel composition of the cryoprotectant composition (including recombinant human serum albumin, trehalose, methylcellulose, and dimethylsulfoxide (DMSO) of the present invention, which does not contain FBS CPA2 contains 5% DMSO and 5% rHSA, CPA3 contains 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4 contains 5% DMSO and 1% rHSA and 0.05% methylcellulose) As a result of the surface antigen analysis of the stem cells before and after freezing compared with the preservative (CPA1, including 5% DMSO and 20% FBS), the expression rates were lower than those of the negative markers (CD34 and CD45) , CD105) exhibited an expression rate higher than the reference level, confirming that stem cell characteristics were maintained even after cryopreservation and thawing of the stem cells (Example 3 and Fig. 4) .

According to yet another embodiment of the present invention there is provided a novel composition comprising recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) but not FBS (CPA2 with 5% DMSO and 5% rHSA, CPA3 with 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4 with 5% DMSO and 1% rHSA, 0.05% methylcellulose) (CPA1, including 5% DMSO and 20% FBS), which showed no significant difference in the ability of cells to recover before and after freezing ( Example 4 and Figures 5 and 6) .

Specifically, a composition for cryopreservation of a novel composition containing recombinant human albumin and dimethylsulfoxide as an active ingredient in comparison with a conventional composition for cryopreservation (CPA1 containing 5% DMSO and 20% FBS) containing an animal serum component (Including CPA2 with 5% DMSO and 5% rHSA, CPA3 with 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4 with 5% DMSO and 1% rHSA, 0.05% methylcellulose) Test), total cell count, and cell survival rate.

As a result, the cryoprotectant compositions of the novel composition of the present invention (CPA2, including 5% DMSO and 5% rHSA, CPA3, 5% DMSO and 1% rHSA, 10 mM trehalose, CPA4, 5% DMSO and 1% % methylcellulose) showed excellent cryopreservation effect to replace the cryoprotectant composition containing animal-derived components such as conventional fetal calf serum.

In one embodiment of the present invention, the stem cells to which the composition for cryopreservation of stem cells of the present invention can be applied include embryonic stem cells, adult stem cells / Tissue-specific stem cells, A mesenchymal stem cell, an induced pluripotent stem cell, and the like.

The embryonic stem cell (ESC) of the present invention refers to a multipotent stem cell derived from an inner cell population of a blastocyst. After the oocyte and sperm are combined to become fertilized eggs, embryos that start with one cell become blastocysts composed of several cells through cell division. Inside the blastocyst, there is a mass of cells called myocytes. These cells are called embryonic stem cells. They are called pluripotent cells because they can differentiate into cells of all the tissues in blood, bone, skin, liver, There is an ethical problem because embryos can only be obtained by destroying them.

The adult stem cell / Tissue-specific stem cell of the present invention is an undifferentiated cell in which only a small amount exists in each tissue of the body, and is a cell that replaces dead cells or damaged tissue. A representative example is pluripotent stem cells. Unlike embryonic stem cells, adult adult cells are not ethically problematic because they do not destroy embryos, but they have few stem cells, are difficult to cultivate, and can only differentiate into specific cells.

The mesenchymal stem cell (MSC) of the present invention grows adherently in the form of fibroblasts in vitro and can form a cell community from a single cell, and can differentiate into bone, fat, chondrocyte It says.

The inducible pluripotent stem cell (iPSC) or degenerated stem cell of the present invention may be used to express four specific genes (Sox2, c-Myc, and the like) that cause the differentiation into somatic cells such as skin cells of non- The stem cells of the present invention can be used to express stem cells, such as embryonic stem cells, which are derived from the differentiation-inducing proteins derived from cells transfected with Klf4, Oct-4, Induced pluripotent stem cells do not have the ethical problems of conventional embryonic stem cells, and they convert the somatic cells of the patients into stem cells.

According to still another aspect of the present invention, there is provided a method for cryopreserving a stem cell according to the present invention comprising mixing a stem cell-cryoprotecting composition of the present invention with a stem cell to prepare a stem cell mixture, Freeze storage method.

The method for cryopreserving stem cells according to the present invention is a method for using the above composition for cryopreserving stem cells as it is. Therefore, in order to avoid the complexity of the present invention, a description within the overlapping range with the above-mentioned composition will be omitted.

The features and advantages of the present invention are summarized as follows:

The present invention relates to a pharmaceutical composition containing recombinant human serum albumin, trehalose, methylcellulose and dimethylsulfoxide (DMSO) as an active ingredient, but not containing fetal bovine serum (FBS) The present invention provides a composition for cryopreserving stem cells.

When the composition for cryopreservation according to the present invention is used for the cryopreservation of stem cells, the total number of cells before and after freezing, despite the cryopreservation is uniform, the cell survival rate after thawing and the cell proliferation during culture after thawing are excellent, Expression of specific cell surface markers is maintained. Therefore, the composition for cryopreservation according to the present invention can be used as a cryopreservation agent for stem cells, and in particular, it is characterized in that it has excellent clinical applicability because it does not contain any kind of heterologous or animal-derived material.

Brief Description of Drawings Fig. 1 is a graph showing physical and chemical properties (A.sub.2 concentration, B, pH) of the composition for cryopreservation according to the present invention (CPA1 is 5% DMSO + 20% FBS, CPA2 is 5% DMSO + , 5% DMSO + 1% rHSA + 10 mM trehalose for CPA3, 5% DMSO + 1% rHSA + 0.05% methylcellulose for CPA4).
FIG. 2 is a graph showing the cytotoxicity of bone marrow-derived mesenchymal stem cells according to the composition of the composition for cryopreservation according to the present invention compared with fresh cells in which the cells are not frozen.
FIG. 3 shows cell survival rates of bone marrow-derived mesenchymal stem cells after freezing and thawing according to the composition of the composition for cryopreservation according to the present invention, compared with fresh cells.
FIG. 4 is a view showing cell surface markers of bone marrow-derived mesenchymal stem cells after freezing and thawing according to the composition of the composition for cryopreservation according to the present invention, in comparison with fresh cells not frozen.
FIG. 5 is a graph showing the cell proliferation potency of bone marrow-derived mesenchymal stem cells after freezing and thawing according to the composition of the cryopreservation composition of the present invention using a CCK-6 assay.
FIG. 6 is a graph showing cell proliferation of bone marrow-derived mesenchymal stem cells after freezing and thawing according to the composition of the composition for cryopreservation according to the present invention by analysis of total cell number.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, the percentages used to denote the concentration of a particular substance, unless otherwise noted, are as follows: solid / solid is (weight / weight), solid / liquid is (weight / volume) The liquid is (vol / vol)%.

(5% DMSO + 5% rHSA), CPA3 (5% DMSO + 1% rHSA + 10 mM trehalose), CP4 (5% DMSO + 1% rHSA + 0.05% methylcellulose) was selected. In this example, to determine the optimal cryopreservation agent among the three cryopreservatives, frozen storage stability and cell characteristics were tested.

Experimental Method

Cell culture

1. Thawing of cryopreserved cells

Vials containing 1x10 ⁶ cells of frozen cells (P1) were thawed in a constant-temperature water bath at 37 ° C, and 2x10 ⁵ cells of cells were inoculated in a T175 flask containing the cell culture medium The culture medium was changed for 7 days and the medium was changed on the 4th day. When the cell culture conditions are culture conditions CO ₂ 7%, 37 ℃ was, CSBM-A06-A06 CSBM culture medium is basal medium (basal medium) L-alanyl- L-glutamine (200 mM), Penicillin- streptomycin a (10,000 U / mL) and 10% FBS (fetal bovine serum).

The cells used in the present invention are human bone marrow-derived mesenchymal stem cells.

2. Subculture

When the cell culture flask reached 80-90% confluency, cells were desorbed using 0.125% Trypsin-EDTA. The cells were inoculated with 2 x 10 ⁵ desensitized cells in a T175 flask containing the cell culture medium, and then cultured for 7 days. The culture medium was changed on the 4th day after culturing. When the cell culture incubated environmental conditions were CO ₂ 7%, 37 ℃, the cell culture medium is CSBM-A06 basal medium (basal medium) L-alanyl- L-glutamine (200 mM), Penicillin-streptomycin (10,000 U / mL in ) And 10% fetal bovine serum (FBS).

3. Cell harvesting

When the cell culture flask reached 80-90% confluency, the cells were desorbed using 0.125% trypsin-EDTA and suspended in the CSBM-A06 basal medium. Cell culture medium was prepared by adding L-alanyl-L-glutamine (200 mM), Penicillin-streptomycin (10,000 U / mL) and 10% fetal bovine serum (FBS) to a basal medium of CSBM-A06.

Cell freezing (cell cryopreservation)

1. Manufacture of semi-finished products (frozen cells)

1) Cells were harvested after 80-90% confluent cells in a cell culture flask incubated for 7 days in total were desorbed by rypsin / EDTA treatment.

2) The harvested cells were suspended in a cell culture medium, and the suspension was dispensed into tubes according to the number of cells to be frozen per one kind of the cryopreservative (CPA) composition, and centrifuged to remove the supernatant.

3) A cryopreservative (CPA) suspension (cell concentration: 1x10 ⁶ cells / 800 uL) was prepared by inserting each cryopreservative composition (stored at 2-8 캜) into a cell pellet-containing tube and then suspended.

4) The cryopreservative suspension was gently dispensed into each of the two cryovials labeled with the name of the cryoprotectant composition by 800 uL, and then the freezing vial cap was closed.

2. Storage using CRF (Controlled Rate Freezer) equipment

The semifinished product (frozen cells) prepared in 1 above was placed in the CRF equipment set as the CRF freezing protocol condition, and the freezing process was performed according to the freezing program. After freezing, they were stored in LN ₂ tank for 3 days (<-150 ° C).

Thawing of cryopreserved cells

1) After 3 days of freezing, the frozen cells were taken out from the LN ₂ tank and rapidly thawed in a water bath at 37 ° C. The thawed cell suspension was slowly transferred to a tube containing the cell culture medium, and further, the cell culture medium was added to suspend the cells, centrifuge the cell suspension, and remove the supernatant.

3) The cell pellet on the bottom of the tube was suspended in a cell culture medium, and the cell viability and cell viability were confirmed to confirm the survival rate of the frozen cells according to the cryoprotectant composition (Example 4).

4) In order to confirm the cell yield among the test items for the recovery of frozen cells, 2 × 10 ⁵ cells were inoculated into a T175 flask containing cell culture medium based on the number of viable cells measured above, and then cultured for 7 days. In addition, the cell proliferation rate test using CCK-8 cell proliferation assay was also carried out in 96-well cells in an amount of 5.0 × 10 ³ cells / well, based on the number of viable cells, and CCK-8 cell proliferation Assay (Example 5).

5) The culture conditions for the cell culture were 7% CO ₂ and 37 ° C. Cell culture medium was prepared by adding L-alanyl-L-glutamine (200 mM), Penicillin-streptomycin U / mL) and 10% FBS.

Total cell count test

1) Cells of 80-90% confluency in cell culture flasks cultured for a total of 7 days were treated with Trypsin / EDTA and harvested.

2) The trypan blue dye exclusion method was used to stain the harvested cells and the total cell number was confirmed using a hemocyte counter.

3) The number of cells was measured by mixing the cell suspension with the same amount of 0.4% trypan blue solution, and then using a hemocyte counter. The number of cells was measured five times repeatedly.

4) The total cell count can be expressed by the expression "(number of living cells + death cell average) X 10 ⁴ / mL x (1 / number of measurement compartments) x cell suspension (mL) x dilution multiple" The population doubling level (PDL) is an index representing the growth rate of a cell and can be expressed as a population doubling (PD) = log (final harvested cells / number of cells inoculated per second) / log 2.

Cell viablilty test

It is a test to determine the cell death state by observing the cell survival rate change during the subculture.

1) Cells of 80-90% confluency in a cell culture flask incubated for a total of 7 days were treated with Trypsin / EDTA and harvested.

2) The cell suspension was mixed with the same amount of 0.4% trypan blue solution and allowed to react for 2 minutes. The number of cells was measured using a hemocyte counter and repeated 5 times.

3) Cell viability is calculated as the ratio of the numbers of stained cells to untained cells on the trypan blue. Cell viability was calculated from the median values except the upper and lower values of the 5 measurements.

4) The method of calculating the cell survival rate can be expressed by the expression "cell survival rate (%) = number of unstained cells / number of whole cells x 100%".

The CCK-8 cell proliferation assay (CCK-8 cell proliferation assay)

Cell Counting Kit-8 (Dojindo Molecular Technologies, US).

On the third day of storage, the frozen cells were removed from the LN ₂ tank and thawed rapidly in a 37 ° C water bath. The cell suspension was transferred to an empty tube and cultured in a cell culture medium (CSBM-A06 + 10 % FBS).

3) based on the of the cell harvesting viable cells 5.0 × 10 ³ cells / in density of the well per sample conditions in a 96-well plate was inoculated by 100 ul to three times 37 ℃, was 7 days incubation at 7% CO ₂ conditions , And the medium was changed on the fourth day of culture. Cell numbers were measured at 0, 4, and 7 days intervals.

4) 10 μL of CCK-8 reagent was added to each well, followed by reaction at 37 ° C. for 1 hour. Absorbance was measured at 450 nm using an ELISA reader (Bio-rad). The slope value was calculated by measuring the O.D values of the 0th, 4th, and 7th day of each cryopreservative (CPA) composition.

Immunophenotype analysis of cell surface antigen

1) To examine the surface antigen expression of cells using fluorescence activated cell sorting (FACS).

2) Cells confluency over 70-80% in cell culture flasks were harvested by trypsin-EDTA. The harvested cells were washed with FACS buffer (DPBS supplemented with 2% FBS), and a cell suspension at a concentration of 1 × 10 ⁵ cells / 100 μl was added to the test tube. An antibody that binds to 5-20 μL of the cell surface antigen was added to the test tube, For 30 minutes. The stained cells were then suspended and analyzed with 500 uL of FACS buffer.

3) All of the above antibodies were obtained from BD (Becton Dickinson, USA). PE-CD54, PE-CD44, PE-CD106, PE-Cy ^TM 7-CD73, FITC-CD105 and PerCP-Cy ^TM 5.5-CD90 were selected as the antibodies that detect mesenchymal stem cell markers. APC-CD34 and FITC-CD45 were selected as the antibodies to detect stem cell markers. As the negative control antibodies, PE-IgG1 κ Isotype Control, APC-IgG1 κ Isotype Control, FITC Mouse IgG1 κ Isotype Control, PerCP-Cy ™ 5.5 Mouse IgG1 κ Isotype Control and PE-Cy ™ 7 Mouse IgG1 κ Isotype Control were selected .

Statistical processing method (T-test verification)

The test results were expressed as mean ± SD (standard deviation, SD), and the comparison between the control and experimental groups was made by Student's T-test. Statistical significance was defined as p value <0.05.

Example 1: Physicochemical characterization of cryoprotectant

In order to analyze physicochemical properties of the cryoprotectant composition of the present invention, pH (hydrogen ion concentration) and osmotic pressure concentration were measured.

CPA2 (5% DMSO + 5% rHSA), CPA3 (5% DMSO + 20% FBS), test group CPA2 (control group, PH (hydrogen ion concentration) and osmotic pressure were measured by physicochemical properties of CP4 (5% DMSO + 1% rHSA + 0.05% methylcellulose) and 1% rHSA + 10 mM trehalose.

The results are shown in Fig.

As shown in Figure 1, the osmotic pressures of the compositions were determined using CPA1 (control), CPA2 (5% DMSO + 5% rHSA), CPA3 (5% DMSO + 1% rHSA + 10 mM trehalose) rHSA + 0.05% methylcellulose) compositions were measured at 1113 ± 4.0, 1120 ± 0.6, 1050 ± 1.5, and 1124 ± 1.2 (mOsmol / kg), respectively. The pH (hydrogen ion concentration) measurement results showed 9.15 ± 0.02, 7.96 ± 0.04, 8.22 ± 0.03 and 8.31 ± 0.01, respectively ( FIG. 1 ).

Example  2: Cytotoxicity test of cryopreservation agent (Cell toxicity test)

In order to confirm the cytotoxicity of the cryopreservative composition of the present invention, the harvested cells were suspended at a cell concentration of 1 x 10 ⁶ cells / 0.8 mL using each of the cryopreservative compositions, and then cultured at low temperature (2-8 ° C) Cellularity, total cell number and cell viability were analyzed.

1) Total cell count

The harvested cells were suspended at a cell concentration of 1 × 10 ⁶ cells / 0.8 mL in each of the four cryoprotectant compositions, and then analyzed for cellularity, total cell number and cell viability at low temperature (2 to 8 ° C.) And the occurrence of cytotoxicity was confirmed.

Each of the bone marrow-derived MSCs in the four kinds of frozen preservative compositions (CPA1, CPA2, CPA3, CPA4 ) 1X10 6 cells / 0.8 mL, and the cell shape and total cell number of the cell suspension were measured. As a result of the measurement, there was no difference in the characteristics of the microscopic-assisted visual cell. CPA1 (97.6 ± 4.4x10 ⁴ cells), CPA2 (104.9 ± 2.5x10 ⁴ cells) and CPA3 (88.8 ± 2.6x10 ⁴ cells) were used as the cryopreservative agent suspension (98.2 ± 10.7) ), CPA4 (96.4 ± 2.7 × 10 ⁴ cells), and no significant difference was observed in the results compared with CPA1, a conventional frozen-thaw preservative composition derived from animal serum (FBS) 2A).

2) Cell survival analysis

The cell viability of the four cryopreservative cell suspensions before freezing was evaluated by comparing the control group (99.1 ± 0.7) not frozen with the cryoprotectant and the test group [CPA1 (98.1 ± 1.0), CPA2 (99.2 ± 0.5) CPA3 (98.2 ± 0.9), CPA4 (99.5 ± 0.6)] showed more than 98% cell survival rate and there was no significant difference between all test groups compared to the control group (FIG. 2B).

From this, it was confirmed that the cryopreservative composition (CPA) of the present invention was free from toxicity to cells.

Example 3: Semi-finished product (frozen cell) survival rate test (Cell viability test)

In order to verify the cytoprotective effect of the cryopreservative composition of the present invention, the cell characteristics, total cell number and cell viability of the cryo-thawed cells were determined. In addition, it was confirmed whether the mesenchymal stem cell characteristics were maintained by comparing the biological characteristics of the cryo-thawed cells with those of the cells that had not undergone freezing and thawing (comparison of the expression rate of cell surface antigens).

The harvested cells were frozen (-80 ° C) using CRF equipment with cryovials prepared at a cell concentration of 1 × 10 ⁶ cells / 0.8 mL in the form of four cryopreservatives (CPA1, CPA2, CPA3 and CPA4) . Frozen cells were stored for 3 days in an LN ₂ tank (<-150 ° C.), and the viability of the cryo-thawed cells was determined. The bioequivalence analysis of the frozen cells with fresh cells To confirm whether mesenchymal stem cell characteristics were maintained.

1) Cellularity and total cell count

Cellularity and total cell number were determined after freezing and thawing.

Cells stored frozen with the above-mentioned four types of cryopreservation compositions (CPA1, CPA2, CPA3, CPA4) were thawed and the total number of cells was measured. As a result, the control group (98.2 ± 10.7), which was not frozen, and the cryopreservative cell suspension (110.2 ± 4.5 × 10 ⁴ cells), CPA 2 (102.2 ± 4.5 × 10 ⁴ cells), CPA 3 (100.3 ± 7.7 × 10 ⁴ cells) and CPA4 (110.0 ± 4.5 × 10 ⁴ cells) Also, it was confirmed that CPA1, which is an antifreeze preservative composition derived from animal serum (FBS), was not significantly different from CPA2, CPA3 and CPA4 of the present invention (FIG. 3A).

From the above results, it was confirmed that each test group can be uniformly obtained without loss of cells during the freezing-thawing process.

2) Cell survival analysis

Next, cell viability after freezing and thawing was analyzed.

The cell viability of cryo-thawed cells after freezing storage in the cell suspension of four kinds of cryopreservative agents was measured. As a result, it was found that the cells (99.1 ± 0.7%), which were not subjected to the freezing process, CPA1 (75.9 ± 1.7%), CPA2 (88.4 ± 1.6%), CPA3 (90.4 ± 1.6%) and CPA4 (81.3 ± 2.6%) showed good cell viability (FIG. In particular, the inventive cryopreservative composition (CPA2, CPA3, CPA4), which is a xeno-free composition, was compared with CPA1 (90.4 ± 1.6%, p <0.001) (88.4 ± 1.6%, p <0.001) and CPA4 (81.3 ± 2.6%, p <0.05), respectively (Fig. 4B). CPA1, CPA2, and CPA4 were compared with CPA3 consisting of non-heterologous and non-animal derived substances (rHSA + trehalose) and showed a significantly lower survival rate in the order of CPA1 (p <0.001) and CPA4 (p <0.05) , And there was no significant difference in CPA2 (FIG. 3B).

From these results, it was confirmed that CPA2 (rHSA) and CPA3 (rHSA + trehalose) composition of the present invention is a cryopreservative agent capable of maintaining the highest cell viability.

3) Cell surface antigen analysis

Finally, cell surface antigen markers were analyzed to determine whether the freezing and thawed mesenchymal stem cells retained their mesenchymal stem cell characteristics.

The bone marrow-derived mesenchymal stem cells, which were cryopreserved for 3 days with the four kinds of cryopreservative compositions (CPA1, CPA2, CPA3, CPA4) of the present invention, were thawed and cultured for 7 days. Then, they were thawed by flow cytometry The expression of cell surface antigen markers in stem cells was compared and analyzed. Specifically, we compared the comparability of control cells, fresh cells, and cryo-thawed cells after freezing and thawing, and recovery cells after freezing and thawing. Selection of cell surface antigen markers and setting of test criteria were carried out based on the quality test method of Neuroanat - Al. Negative marker and CD45 <4% and CD45 <2% were classified as positive marker and CD29> 80%, CD44> 80% and CD105> 80%, respectively. The results are shown in Fig.

As shown in FIG. 4, when the expression ratios of CD34 (hematopietic lineage marker) and CD45 (hematopietic lineage marker) as the voice markers were examined, the expression rate was less than 1% in all the test groups. It has also been confirmed that it meets the test (purity test) standard. In addition, the expression ratios of positive markers such as CD29 (integrin marker), CD44 (adhesion molecule marker), CD73 (5'-nucleotidase) and CD105 (endoglin; SH2) , And the expression rate was 80% or more.

As a result, a comparison of the fresh cell and the cryo-thawed cell (recovery cell) showed that the cell surface markers of the cells after thawing of the frozen cells in all the cryopreservatives (CPA1, CPA2, CPA3, CPA4) Of the respondents. Therefore, it was confirmed that the cryopreservative of the present invention retains the characteristics of mesenchymal stem cells even after cryopreservation and thawing of the stem cells.

Example 4: Recovery test of semi-finished product (frozen cell)

In order to examine the effect of the cryopreservation composition of the present invention on the cell-freezing preservation effect, the cells cultured in a single cryo-thawed cell were subjected to cell growth and CCK-8 assay to change cell proliferation rate Respectively. In order to compare the bioequivalence of the cell with the control cell (fresh cell, passage 4) of the same passage number as the recovery cell after freezing and thawing, total cell count and flow cytometry were used to analyze the cryopreserved cells And maintained the characteristics of mesenchymal stem cells.

1) CCK-8 assay (cell proliferation assay)

Cryo-thawed cells were inoculated in a 96-well plate at a density of 5.0 × 10 ³ cells / well on a viable cell basis and cultured for 7 days. CCK-8 cells were cultured for 7 days at 0 day, 4 day, The cell proliferation ability was confirmed by assay. The slope values for different OD values on the 0th, 4th and 7th day of each CPA composition were CPA1 (0.086 ± 0.005), CPA2 (0.088 ± 0.008), CPA3 (0.091 ± 0.002), CPA4 (0.090 ± 0.007 ). As a result, it was confirmed that there was no significant difference between CPA1 as a frozen-preserving composition derived from animal serum (FBS) and CPA2, CPA3 and CPA4 as xeno-free cryopreservative compositions (FIG. Therefore, it was confirmed that there was no difference in cell proliferation between cryo-thawed cells after freezing and thawing according to the composition of each cryopreservative.

2) Total cell count

Cryo-thawed cells were inoculated into T175 flasks at a density of 2.0 × 10 ⁵ cells per viable cell count, cultured for 7 days, harvested at 7 days, Were measured. Non-frozen cells (fresh cell) in the control group (234.9 ± 4.8x10 ⁴ cells) and frozen cell suspension preservative in the test group ^{[CPA1 (258.4 ± 11.9x10 4 cells} ), CPA2 (260.3 ± 18.3 x10 4 cells), CPA3 (249.9 ± 6.0 x10 4 cells), CPA4 (258.9 ± 13.5 x10 4 (p <0.05). As a result of comparison between CPA1 and CPA2, CPA3 and CPA4, which are frozen-thaw preservative compositions derived from animal serum (FBS) , And there was no significant difference in the total number of cells (Fig. 6A).

From the above results, it was confirmed that there was no difference in the cell proliferation capacity between the cryo-thawed cells after freezing and thawing according to the composition of each cryopreservative.

3) Cell survival analysis

The cell viability of the cells cultured after the thawing was measured. As a result, the cell viability of the cells cultured after the thawing was measured and compared with the control group (99.2 ± 0.4%), which was not frozen, and the test group [CPA1 (97.6 ± 1.3%), CPA2 (98.8 ± 1.0%), CPA3 (98.3 ± 0.5%) and CPA4 (97.4 ± 0.8%) showed that CPA1 and CPA2 did not significantly differ in cell survival, And CPA4 significantly increased the cell survival rate (p < 0.05). Further, it was confirmed that the cell survival rates of CPA1, CPA2, CPA3 and CPA4, which are the frozen-thaw preservative compositions derived from animal serum (FBS), were not significantly different (Fig. 6B).

 From the above results, it was confirmed that there was no difference in the cell survival rate between the cryo-thawed cells after freezing and thawing according to the composition of each cryopreservative.

Furthermore, the population doubling level (PDL), which is an index indicating the cell growth rate, was analyzed based on the total cell counts measured above.

Compared with the control group (3.55), which is a fresh cell without the freezing process, and the test group [CPA1 (3.69), CPA2 (3.70), CPA3 (3.64), CPA4 , And it was confirmed that there was no difference in the level of cell multiplication between the cryo-thawed cells after freezing and thawing according to the composition of each cryopreservation agent and the control group, which is a fresh cell, .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

(BSA) containing 0.5-3 (w / v)% recombinant human serum albumin, 5-20 mM trehalose and dimethylsulfoxide (DMSO) as active ingredients, fetal bovine serum ) As an active ingredient for cryopreservation of stem cells.
delete The composition of claim 1, further comprising methylcellulose.
The composition of claim 1, wherein the concentration of said recombinant human serum albumin is 0.5-2 (w / v)%.
The composition of claim 1, wherein the concentration of dimethylsulfoxide is 1-10 (v / v)%.
6. The composition of claim 5, wherein the concentration of dimethyl sulfoxide is 3-7 (v / v)%.
delete delete 4. The composition of claim 3, wherein the concentration of methylcellulose is 0.001-0.5 (w / v)%.
The composition of claim 9, wherein the concentration of methylcellulose is 0.01-0.3 (w / v)%.
2. The composition of claim 1, wherein the composition is a xeno-free and an animal component-free.
The stem cell according to claim 1, wherein the stem cell is selected from the group consisting of an embryonic stem cell, an adult stem cell, a mesenchymal stem cell, an induced pluripotent stem cell, a pluripotent stem cell).
A method for producing a stem cell mixture comprising mixing a composition according to any one of claims 1, 3 to 6 and 9 to 12 with a stem cell to prepare a stem cell mixture and freezing the stem cell mixture, / RTI &gt;

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