KR102569522B1 - Composition for cryopreservation of cell and method for cryopreservation of cell using the same - Google Patents

Abstract

It relates to a cell cryopreservation composition and a cell cryopreservation method using the same. According to this, genetic or generational variations can be minimized in long-term preservation of cells, and cell viability is excellent during freezing and thawing.

Description

Cell cryopreservation composition and cell cryopreservation method using the same {Composition for cryopreservation of cell and method for cryopreservation of cell using the same}

It relates to a cell cryopreservation composition and a cell cryopreservation method using the same.

When cells are cultured for preservation, it is known that their characteristics are highly likely to be changed due to genetic or generational variations, and unwanted contamination may occur due to environmental factors. Conventionally, ultra-freezing preservation methods have been used to preserve cells while minimizing such genetic or generational changes and preventing contamination by environmental factors. The ultra-low temperature cryopreservation method for cells requires a temperature condition as low as -195°C and a cryopreservative to prevent freezing/thawing damage of the cells. Conventionally, 15% glycerol has been used as a general cryopreservative, but cell damage may often occur due to an incomplete buffering function during the freezing/thawing process. Conventionally, dimethyl sulfoxide (DMSO) is also used as a cryopreservative, but it is expensive and can be toxic to cells when used in excess. Therefore, there has been a need for a cryopreservative agent capable of minimizing damage caused by cell freezing/thawing while having excellent cell preservation effect.

One aspect provides a composition for cryopreservation of cells.

Another aspect provides a cell cryopreservation method comprising the step of adding cells to a preservation solution containing the composition.

One aspect provides a composition for cryopreservation of cells.

The composition may include glycols, glucose, ascorbic acid, glutamine, tetramethylchromancarboxylic acid or tocopherol. The composition may include glycols, glucose, ascorbic acid, glutamine, and tetramethylchromancarboxylic acid or tocopherol. The cell cryopreservation composition functions as a cryoprotectant.

The glycols may have a glycol having 1 to 6 carbon atoms or a glycol repeating unit having 1 to 6 carbon atoms. The glycols may be ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, hexylene glycol, or a combination thereof.

The glycols may be included in about 0.14% to about 70% in the composition. The ethylene glycol may be included in about 0.4 to 20% in the composition. The polyethylene glycol may be included in about 1 to 50% of the composition. Here, % means v/v%.

The glucose is a type of sugar, which provides an energy source necessary for cell division, survival, or differentiation, and may be contained in an amount of about 0.0025M to about 1.0M, or about 0.005M to about 0.5M in the composition. The glucose may be, for example, D-(+)-glucose (Dextrose).

The ascorbic acid (Ascorbic Acid) has an antioxidant function, and may be included in about 5uM to about 2000uM, or about 10uM to about 1000uM in the composition.

The glutamine maintains the volume and moisture of the cells, and may be contained in an amount of about 0.1 mM to about 40 mM or about 0.2 mM to about 20 mM in the composition. The glutamine may be, for example, L-glutamine.

The tetramethylchromancarboxylic acid (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, trolox, trolox) and tocopherol each have an antioxidant function, and the tetramethylchromancarboxylic acid or Tocopherol may be contained in about 5uM to about 2000uM or about 10uM to about 1000uM in the composition.

The composition may include phosphate buffered saline (PBS).

The composition may not include dimethyl sulfoxide (DMSO). Since the composition does not contain DMSO, there is no DMSO toxicity that occurs at room temperature, and cells can be preserved without damaging cells even when large-scale batches are produced.

The composition may not contain animal-derived serum. The composition can prevent the formation of extracellular ice crystals, which is the role of serum proteins during cryopreservation, without including serum. In addition, cell contamination by serum proteins can be prevented during cryopreservation.

The cells may be undifferentiated cells, differentiated cells, or differentiated cells. The cells may be stem cells. The term "stem cell" refers to a cell having potency and self-renewal ability. Stem cells are classified into pluripotency, multipotency, or unipotency according to their differentiation ability. The stem cells include embryonic stem cells (ESC) (inner cells of embryos before implantation), adult stem cells (undifferentiated cells present in each tissue and organ), and dedifferentiated stem cells (induced It may be at least one selected from the group consisting of pluripotent stem cells: iPSCs (cells dedifferentiated by inserting genes and/or proteins into somatic cells, or induced pluripotent stem cells).

The stem cells may be mesenchymal stem cells. The term "mesenchymal stem cell (MSC)" is an adult stem cell, which has multipotency and self-renewal ability, has excellent proliferation ability and is genetically stable. The mesenchymal stem cells are cells that assist in making fat, cartilage, bone, bone marrow epilepsy, muscle, nerve, etc., and can differentiate into various cells, such as fat cells, chondrocytes, skin cells, and bone cells. .

The type and origin of the stem cells are not limited as long as they have the ability to differentiate and self-renew. The stem cells may be derived from, for example, mammals, humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice or rabbits. The stem cells may be derived from separated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid. The term "isolated" means present in an environment different from that of a naturally occurring cell or tissue. Separation of umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid and obtaining stem cells therefrom may be performed by conventional anatomical methods and known methods.

In addition, the cells are CHO cells, HEK cells, MCF-7 cells, MDCK cells, Vero cells, bone marrow cells, peripheral blood cells, primary cells, hepatocytes, and tissue-derived cells, such as liver, heart, lung, and small intestine. , It may be a cell derived from the large intestine, kidney, brain, or pancreas.

The composition may inhibit damage caused by freezing/thawing of cells. In the process of freezing for long-term preservation of cells and thawing for reuse, cells are damaged by temperature changes. When freezing proceeds, the water outside the cell freezes first, and as a result, the environment outside the cell forms a state of higher salt than the inside of the cell. In order to maintain the equilibrium state, the movement of water inside the cell occurs, resulting Plasmolysis takes place. In order to prevent this, cryopreservatives are used, and cryopreservatives are divided into two types: cell-protective cryoprotectants and cell-penetrating cryoprotectants. Cell-protective short-acting cryopreservatives act to inhibit the formation of extracellular ice crystals, and cell-penetrating cryopreservatives penetrate cell membranes to displace intracellular water and inhibit the formation of ice crystals during freezing. do. For cryopreservation of cells, cell penetrating cryopreservatives such as DMSO or glycerol have been conventionally used. However, cell penetrating cryopreservatives can be toxic to cells when the concentration is high. When the composition for cryopreservation of cells of the present invention is used as a cryopreservative, the viability of cells can be increased without detrimental effects in cell penetrating cryopreservatives.

Another aspect provides a cell cryopreservation method comprising the following steps.

The method includes adding cells to a preservation solution containing the composition; And it may include the step of cryopreserving the preservation solution to which the cells were added at -50 ° C to -300 ° C.

The term "preservation solution" of the present invention refers to various media generally used for cell culture. The medium is not particularly limited as long as it can be used for cell culture, and for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10 , F-12, DMEM/F12, MEM-α (Minimal Essential Medium-α), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A badge, AmnioMax complete badge, AminoMax II complete badge, EBM (Endothelial Basal Medium) medium, Chang's Medium, MesenCult-XF, DMEM/HG (Dulbecco's Modified Eagle's Medium high glucose) medium, and MCDB + DMEM / LG (MCDB + Dulbecco's Modified Eagle's Medium low glucose) medium selected from the group consisting of It may contain one or more.

The preservation may be performed in a liquid nitrogen tank or a deep freezer. The liquid nitrogen tank may be maintained at about -100 ° C to about -300 ° C, about -150 ° C to about -250 ° C, or about -170 ° C to about -220 ° C, and the cryogenic refrigerator is about -50 ° C to about -100°C, or about -60°C to about -90°C.

The preservation may last for 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 2 months, 3 months, 6 months, or 1 year or longer.

The cells may be undifferentiated cells, differentiated cells, or differentiated cells. The cells may be stem cells. The stem cells may be embryonic stem cells, mesenchymal stem cells, or dedifferentiated stem cells.

The cells are about 0.5 x 10 ⁴ cells to about 1x10 ⁸ , about 0.5 x 10 ⁵ cells to about 1x10 ⁷ cells, about 1.0 x 10 ⁵ cells to about 0.5x10 ⁷ cells, about 2.0 x 10 ⁵ cells, based on 1 ml of the composition. to about 2.5x10 ⁶ cells, or about 5.0 x 10 ⁵ cells to about 1.0x10 ⁶ cells.

According to the cell cryopreservation method comprising the step of adding cells to a preservation solution containing the composition, cells can be safely protected during cryopreservation, and cell viability can be high even after thawing.

Cell viability means an object that exhibits the cell's original physiological function, and for example, it can be confirmed by counting cells that are not stained blue by trypan blue staining and show green color. .

According to the cell cryopreservation composition and the cell cryopreservation method using the cell cryopreservation method, genetic or generational changes can be minimized in long-term preservation of cells, and cell viability is excellent during freezing and thawing.

1 is an image showing the shape, viability and proliferation rate of stem cells after freezing/thawing them using the cell cryopreservation composition of the present invention.
Figure 2 is a graph showing the survival rate and proliferation rate of stem cells after freezing/thawing them using the cell cryopreservation composition of the present invention.
3 is a graph showing the size of stem cells after freezing/thawing them using the cell cryopreservation composition of the present invention.

Hereinafter, the present invention will be described in more detail by examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of cell cryopreservation composition and cell freezing/thawing, cell viability, cell proliferation, and cell size analysis

1. Preparation of cell cryopreservation composition

Ethylene Glycol, Polyethylene Glycol, Glucose, Ascorbic Acid, Trolox or a-Tocopherol in Phosphate Buffered Saline (PBS) ), and after adding L-glutamine (L-glutamine), filtered through a filter with a diameter of 0.22um, to prepare a composition for cell cryopreservation.

2. After cell freezing/thawing using the composition of 1, cell viability, cells proliferation rate , and cell size analysis

(1) Cell freezing/thawing using the composition of 1

Informed consent was obtained in advance from healthy mothers who gave birth normally, and the umbilical cord was separated from the placenta collected at the time of normal placenta delivery. Each of the separated umbilical cords was washed 2 to 5 times with Ca/Mg-free Dulbecco's Phosphate-Buffered Saline (DPBS) to remove blood. Then, after removing the artery and vein from the umbilical cord, the umbilical cord was cut to a size of about 1 to 5 mm. Thereafter, the umbilical cord was attached to a culture container and cultured for 10 to 15 days, and after confirming that the cells protruded from the cultured tissue, 200 U/mL of collagenase I was added for 5 to 6 hours to obtain an umbilical cord-derived stem Cells were each isolated. The isolated umbilical cord-derived stem cells were cultured in an incubator under conditions of 37°C, 95% air and 5% CO ₂ .

The obtained stem cells were added in an amount of about 5.0 x 10 ⁵ cells or about 1.0 x 10 ⁶ cells per 1 ml of composition 1 to prepare a cell suspension. After transferring the prepared suspension to one vial, it was stored in a freezing container. At this time, the freezing container was used containing isopropanol, and the vial was stored in the freezing container for one day while lowering the temperature to -80 ° C at a rate of about -1 ° C / min. Thereafter, the freezing vessel was transferred to a liquid nitrogen environment at about -196° C. and cryopreserved.

After about 16 weeks of cryopreservation, frozen vials were thawed in a 37°C environment. The cells preserved in the thawed vial were added in MEM alpha medium containing 10% Fetal Bovine Serum (FBS) to create a cell suspension. After the resulting suspension was centrifuged at 1200 rpm for 4 minutes, the supernatant was removed, and the cells were suspended in a new medium, which was dispensed into a cell culture plate, and cultured at 37°C and 5% CO ₂ conditions.

At this time, the control group includes a commercial composition for cryopreservation containing DMSO (Cryostor CS10/Cat.No.210102/biolifesolutions) (Control 1) and a commercial composition for cryopreservation containing no DMSO (Stemcellbanker Cat.No. 11890 amsbio) (control 2) was used.

(2) cell viability and cell viability after cell freezing/thawing proliferation rate OK 1

As described above in (1), after culturing cryopreserved cells in the composition of 1, control 1, and control 2, respectively, images of stem cells were obtained.

1 is an image showing the shape, viability and proliferation rate of stem cells after freezing/thawing them using the cell cryopreservation composition of the present invention. As shown in FIG. 1, it can be seen that the cell shape, viability, and proliferation degree are similar to those of a commercially available cryopreservation composition when using the cell cryopreservation composition of the present invention.

(3) After cell freezing/thawing, cell viability, cell proliferation rate OK 2

Cryopreservation was performed as described above in (1), and after about 1 week, about 4 weeks, and about 16 weeks of cryopreservation (respectively), the frozen vials were thawed in a 37°C environment. The cells preserved in the thawed vial were added in MEM alpha medium containing 10% Fetal Bovine Serum (FBS) to create a cell suspension. After the resulting suspension was centrifuged at 1200 rpm for 4 minutes, the supernatant was removed, the cells were suspended in a new medium, and this was dispensed into a cell culture plate, at 37°C and 5% CO ₂ for about 24 hours. cultured. After culturing for 24 hours, cell viability was measured to confirm cell recovery.

Figure 2 is a graph showing the survival rate and proliferation rate of stem cells after freezing/thawing them using the cell cryopreservation composition of the present invention. As shown in Figure 2, in the case of short-term and / or long-term (1 week, 4 weeks and 16 weeks) cryopreservation, when using the cell cryopreservation composition of the present invention, when using a commercially available cryopreservation composition, viability and It can be seen that the degree of proliferation is similar.

(4) Cell size analysis after cell freezing/thawing

In (3), the size of the cell was obtained as an image, and the size was measured. 3 is a graph showing the size of stem cells after freezing/thawing them using the cell cryopreservation composition of the present invention. As shown in FIG. 3, it can be seen that the cell size is similar to that of a commercially available cryopreservation composition when using the cell cryopreservation composition of the present invention.

Conventionally, for the storage of stem cells, the same and similar preservation methods as those of conventional cells have been used. However, since cells have different physiological characteristics depending on their origin and harvested tissue, when cryopreserving each cell, a cryopreservation method suitable for the target cell must be applied. In particular, in the case of stem cells, since they maintain their inherent regenerative and differentiated abilities, optimized cryopreservation conditions are required, and when using the composition, a high survival rate can be obtained upon thawing.

Claims (10)

A cell cryopreservation composition comprising glycols having 1 to 6 carbon atoms, glucose, ascorbic acid, glutamine, and tetramethylchromancarboxylic acid or tocopherol,
The composition does not contain dimethyl sulfoxide (DMSO) and animal-derived serum,
The composition is a cell cryopreservation composition that is added to 0.5 x 10 ⁴ cells to 1x10 ⁸ cells per 1ml. The method according to claim 1, wherein the glycols having 1 to 6 carbon atoms are ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, hexylene glycol or a combination thereof. delete delete The method according to claim 1, wherein the composition of the cell is a stem cell. The composition according to claim 5, wherein the stem cells are embryonic stem cells, mesenchymal stem cells, or dedifferentiated stem cells. Cell cryopreservation method comprising the following steps:
Adding cells to a preservative solution containing the composition of any one of claims 1, 2, 5 and 6; and
A step of cryopreserving the preservation solution to which the cells were added at -50 ° C to -300 ° C. The method according to claim 7, wherein the cells are stem cells. The method according to claim 8, wherein the stem cells are embryonic stem cells, mesenchymal stem cells, or dedifferentiated stem cells.

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