KR20160050412A - Composition of Media for Culturing Stem Cell - Google Patents

Abstract

The present invention relates to a composition of a culture medium for stem cells, and more specifically, to a composition of a culture medium for mesenchymal stem cells, wherein the composition comprises a basal medium in which DMEM and defined keratinocyte-SFM are mixed, L-ascorbic acid 2-phosphate, fetal bovine serum, basic fibroblast growth factors (b-FGF), insulin, N-acetyl-L-cysteine, calcium chloride, and hydrocortisone. The present invention can improve the differentiation and proliferation capacities of mesenchymal stem cells, and culture the mesenchymal stem cells at a lower cost than conventional methods, thereby making it possible to economically produce cell therapeutics using the mesenchymal stem cells.

Description

[0001] The present invention relates to a culture medium for culturing stem cells,

The present invention relates to a culture medium for stem cell culture, and more particularly, to a culture medium for culture of stem cells, which comprises a basic medium in which DMEM and Defined Keratinocyte-SFM are mixed, L-ascorbic acid 2-phosphate, fetal bovine serum, ), Insulin, N-acetyl-L-cysteine, calcium chloride and hydrocortisone.

Cells that have the ability to differentiate into two or more cells while maintaining self-replicating ability are called stem cells. Stem cells can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells, which can be differentiated into specific tissues and organs. As stem cells, adult stem cells are collectively referred to as adult stem cells, which are involved in the development of tissues and organs of the fetal period, neonatal period and adult period, maintenance of adult tissue homeostasis and regeneration of tissue damage.

In recent years, adult stem cells have been attracting attention in the development of new regenerative medical treatment techniques such as regeneration and function restoration of living tissues and organs that are dysfunctional or incompatible with various diseases or accidents. In addition, the development of stem cell-based therapies will lead to greater use of stem cells as the development of alternative methods of cell and tissue replacement is expected beyond conventional classical drug therapy or surgical treatment.

Therefore, various functions of stem cells have been studied at present. Among them, cell therapy techniques using mesenchymal stem cells have begun to receive the spotlight, and mesenchymal stem cells isolated from human body are suitable for treatment (WO 2006/019357, Korean Patent No. 0795708, Korean Patent No. 0818214).

     Currently, the research and development process related to cell therapy using adult stem cells involves collecting stem cells, blood-derived mononuclear cells or bone marrow-derived mononuclear cells from a patient, inducing cell proliferation and / or differentiation through in vitro culture, Progenitor cells) and / or inducing the differentiation of the differentiated cells into the patient's own body. However, the presently used mesenchymal stem cell culture medium, which is most optimized for mesenchymal stem cell stem cell survival, high fragmentation ability, and compact morphology maintenance, This is a financial burden (Korean Patent No. 0795708).

The present inventors have made intensive efforts to develop a culture medium for stem cell culture that can reduce the production cost of mesenchymal stem cells and enhance the culture efficiency. As a result, the present inventors have found that high-cost, high-efficiency KSFM- The medium was mixed with a relatively low-cost DMEM (Dulbecco Modified Eagle Medium), which is commonly used for culturing, at a constant ratio. When the mesenchymal stem cells were cultured using the mixed medium, the culture efficiency of the stem cells was increased And the present invention was completed.

It is an object of the present invention to provide a culture medium composition for culturing stem cells which is capable of maintaining the pluripotency while having a high proliferation rate.

It is another object of the present invention to provide a method for culturing mesenchymal stem cells using the medium composition.

In order to achieve the above object, the present invention provides a culture medium comprising a basic medium in which DMEM and Defined Keratinocyte-SFM are mixed, L-ascorbic acid 2-phosphate, fetal bovine serum, basic fibroblast growth factor (b-FGF) The present invention provides a medium composition for mesenchymal stem cell culture containing N-acetyl-L-cysteine, calcium chloride and hydrocortisone.

The present invention also provides a method for culturing mesenchymal stem cells, wherein mesenchymal stem cells are cultured in the medium composition.

According to the present invention, it is possible to improve the pluripotency and proliferative capacity of mesenchymal stem cells, and to cultivate mesenchymal stem cells at a lower cost than conventional culturing methods, so that a cell therapeutic agent using mesenchymal stem cells can be produced more economically have.

Fig. 1 shows morphological changes of adipose-derived mesenchymal stem cells in a mixed medium.
Fig. 2 shows the yields after culturing of adipose-derived mesenchymal stem cells in a mixed medium.
FIG. 3 shows changes in cell size after culturing of adipose derived mesenchymal stem cells in a mixed medium.

In the present invention, DMEM medium, which has been conventionally used for culture of mesenchymal stem cells, and DMEM medium, which is relatively cheaply available in the form of a semi-finished product, are mixed with KSFM-P medium (Korean Patent No. 0679642) When adipose-derived mesenchymal stem cells were cultured, there was almost no change in the characteristics of the stem cells, and it was confirmed that the size of the cells could be maintained constantly while effectively multiplying the cells.

Therefore, the present invention relates to a pharmaceutical composition comprising a basic medium in which DMEM and Defined Keratinocyte-SFM are mixed, L-ascorbic acid 2-phosphate, fetal bovine serum, basic fibroblast growth factor (b-FGF), insulin, N- L-cysteine, calcium chloride, and hydrocortisone. The present invention also relates to a culture medium for culturing mesenchymal stem cells containing N-acetyl-L-cysteine, calcium chloride and hydrocortisone.

As used herein, the term "stem cell" refers to a cell having the ability to self-replicate and to differentiate into two or more cells, and "adult stem cells" Or stem cells appearing in the adult stage.

As used herein, the term "mesenchymal stem cells" is undifferentiated stem cells isolated from human or mammalian tissues and may be derived from various tissues. In particular, mesenchymal stem cells derived from umbilical cord, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose derived mesenchymal stem cells, muscle derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, , Amniotic membrane-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells, and techniques for separating stem cells from each tissue are well known in the art.

The term " fat-derived stem cell "used in the present invention refers to an undifferentiated stem cell isolated from adipose tissue, and the separation method thereof may be, for example, as follows. That is, after culturing a fat-containing suspension suspended in physiological saline obtained from liposuction, the stem cell layer adhered to the culture container such as a flask is treated with trypsin and then scraped off or scraped off to be suspended in a small amount of physiological saline And recovering the mesenchymal stem cells from the adipose-derived mesenchymal stem cells.

In the present invention, the medium may be selected from the group consisting of 0.05 to 1 mM of ascorbic acid 2-phosphate, 2 to 20% fetal bovine serum, 10 to 1 ng / ml of basic fibroblast growth factor (b-FGF) Insulin, 0.2 to 20 mM N-acetyl-L-cysteine, 0.01 to 1 mM calcium chloride and 5 ng / ml to 1 μg / ml of hydrocortisone.

In the present invention, the mixing ratio of DMEM to Defined Keratinocyte-SFM medium is preferably 1: 0.1-10, more preferably 1: 0.25-3.

In one embodiment of the present invention, DMEM and KSFM-P medium are mixed at a ratio of 1: 0.5; 1: 1; 1: 2; 1: 3 and 1: 4 were used, similar cell morphology was obtained as compared with the case of using KSFM-P alone medium and it was remarkably superior to that of KSFM-P alone medium Cell yield.

In another aspect, the present invention relates to a method for culturing mesenchymal stem cells, which comprises culturing mesenchymal stem cells in the medium composition.

In the present invention, the mesenchymal stem cells may be characterized in that they are derived from human fat.

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 examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Isolation of human adipose tissue-derived mesenchymal stem cells

Human adipose tissue obtained from abdominal fat was separated by liposuction and washed with PBS. The tissue was minced and digested with DMEM medium supplemented with collagenase type 1 (1 mg / ml) for 2 hours at 37 ° C. Washed with PBS and centrifuged at 1000 rpm for 5 minutes. The supernatant was suctioned and the pellet remaining on the bottom was washed with PBS and centrifuged at 1000 rpm for 5 minutes. The cells were washed with PBS and then cultured in DMEM (10% FBS, 2 mM NAC, 0.2 mM ascorbic acid).

After one night, unattached cells were washed with PBS and incubated with Keratinocyte-SFM containing 5% FBS, 2mM NAC, 0.2mM ascorbic acid, 0.09mM calcium, 5ng / ml rEGF, 5g / ml insulin and 74ng / ml Hydrocortisone media were replaced every 2 days and subcultured to prepare four differentiated multipotent mesenchymal stem cells derived from adipose tissue.

Example 2: Culture of human adipose tissue-derived mesenchymal stem cells in a mixed medium

The human adipose-derived mesenchymal stem cells (P4) obtained in Example 1 were cultured in a medium containing KSFM-P (Table 1) and DMEM (Gibco, BRL) alone or mixed at the following ratio, Density, morphology, and yield.

1) KSFM-P (10% FBS)

② DMEM (10% FBS)

③ DMEM (10% FBS) + KSFM-P (10% FBS); Mixing ratio 1: 1

④ DMEM (10% FBS) + KSFM-P (10% FBS); Mixing ratio 1: 2

⑤ DMEM (10% FBS) + KSFM-P (10% FBS); Mixing ratio 1: 3

⑥ DMEM (10% FBS) + KSFM-P (10% FBS); Mixing ratio 1: 4

⑦ DMEM (10% FBS) + KSFM-P (10% FBS); Mixing ratio 2: 1

 Components of KSFM-P medium Constituent Availability density Defined Keratinocyte-SFM Invitrogen L-ascorbic acid 2-phosphate Sigma-aldrich 0.2 mM Insulin Millipore 5 [mu] g / ml N-acetyl-L-cysteine Sigma-aldrich 2 mM Calcium chloride Sigma-aldrich 0.09 mM Hydrocortisone Sigma-aldrich 74 ng / ml Fetal Bovine Serum Invitrogen 5%

1 × 10 ⁴ cells / well of adipose-derived mesenchymal stem cells of P4 were inoculated on a 6-well plate containing each medium for 5 days, and the morphology of the stem cells was measured on the 2 and 5 days of culture, respectively. Five days after the cultivation, the cultured stem cells were washed with DPBS, and then 200 μl of TriPLE solution was added thereto, and the cells were desorbed by a reaction at 37 ° C for 5 minutes. 800 μl of DMEM (10% FBS) was added to each well to neutralize the TriPLE solution, transferred to a sterile 1.5 ml EP tube, and centrifuged at 800 rpm for 3 minutes. After centrifugation, the supernatant was removed and the separated cells were resuspended by adding 500 ml of DPBS. After mixing 10 μl of suspended cells with DPBS and 10 μl of trypan blue, cell numbers, viability and cell size were measured.

(1) morphology of stem cells

Changes in morphology of the cells during culture in each medium were observed at days 2 and 5 of culture. As a result, as shown in Fig. 1, it was confirmed that the cells had an early adherence ability (two days of culture) superior to the KSFM-P or DMEM medium in the two culture medium mixed groups (groups 3 and 4).

The morphology of the cells was relatively large compared to the cells cultured in DMEM medium compared to the medium cultured in the other group of medium. On the fifth day of culture, the cell size was largest in DMEM medium In addition, the group was similarly identified. On the other hand, it was confirmed that the density of cells in the cultured medium was increased in the mixed medium compared with the culture in the KSFM-P or DMEM alone medium. Therefore, it was confirmed that the mixed medium containing KSFM-P and DMEM was more effective for cell size and growth.

(2) the number of stem cells (number)

The adipose derived mesenchymal stem cells were cultured for 5 days in each medium, and then the stem cells were collected using TriPLE solution and the number was measured by trypan blue technique.

As a result, as shown in FIG. 2 and Table 2, the cell yield in the KSFM-P medium, which is a medium of stem cell optimum, was increased about twice as much as that of the DMEM medium, indicating that KSFM- , Which is more suitable than DMEM medium.

In addition, when the stem cells were cultured in a culture medium in which KSFM-P and DMEM media were mixed at a certain ratio, it was confirmed that the cell yield was 2 to 3 times higher than the number of cells collected in the KSFM-P medium Respectively.

Therefore, it can be seen that by mixing KSFM-P medium and DMEM medium, the culture conditions required for cell growth are more effective than KSFM-P culture medium alone.

(3) Size of stem cells (size)

After obtaining the adipose-derived stem cells for 5 days in each culture medium, the average size of the cells was determined using a Luna ™ automated cell counter (Logos Biosystems, Inc. USA).

As a result, as shown in FIG. 3 and Table 3, the size of the stem cells cultured in each culture medium showed an average of about 15 to 17 μm with no significant difference. In other words, it was found that each culture medium had no significant effect on the cell size when it was obtained differently from the visual characteristics of the cells.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

(B-FGF), insulin, N-acetyl-L-cysteine (L-ascorbyl-L-cysteine) -cysteine, calcium chloride and hydrocortisone.
2. The method according to claim 1, wherein 0.05 to 1 mM of ascorbic acid 2-phosphate, 2 to 20% fetal bovine serum, 10 to 1 ng / ml of basic fibroblast growth factor (b-FGF), 0.1 to 100 ug / A culture medium for mesenchymal stem cell culture containing 0.2 to 20 mM of N-acetyl-L-cysteine, 0.01 to 1 mM of calcium chloride and 5 to 1 g / ml of hydrocortisone .
The culture medium according to claim 1, wherein the mixing ratio of DMEM to Defined Keratinocyte-SFM medium is 1: 0.1-10.
The culture medium according to claim 3, wherein the mixing ratio of DMEM to Defined Keratinocyte-SFM medium is 1: 0.25-3.
A method for culturing mesenchymal stem cells, wherein the mesenchymal stem cells are cultured in the medium composition according to any one of claims 1 to 4.
The method according to claim 5, wherein the mesenchymal stem cells are derived from human fat.

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