KR100908481B1 - Mesenchymal stem cell culture medium and culture method of mesenchymal stem cells using the same - Google Patents

Abstract

The present invention relates to a culture medium of mesenchymal stem cells comprising a basal medium and umbilical cord blood serum. The present invention also relates to a method for culturing mesenchymal stem cells using the culture medium and a method for obtaining mesenchymal stem cells. This can be obtained by culturing a large number of mesenchymal stem cells.

Mesenchymal stem cells, cord blood serum, culture medium

Description

Mesenchymal stem cell culture medium and method for culturing mesenchymal stem cells using the same {Culture media and methods for culturing mesenchymal stem cell}

FIG. 1 shows 15 days of dispensing mononuclear cells (MNCs) isolated from bone marrow in a T25 flask containing CG (CellGro ™, CellGenix) and MSCGM ™ (Cambrex) medium at a concentration of 5.28 × 10 ⁶ cells. After incubation, it is a graph showing the growth of FACs (fibroblast-like adherent cells).

2 is a medium in which 10% CS (cord blood) is added to MSCBM ™ (Mesenchymal stem cell basal medium, Cambrex) using MSCGM ™ (Cambrex) as a control, and 20 in MSCBM ™ (Cambrex) In a medium containing% CS, CellGro ™ (CellGenix) In CellGro ™ (CellGenix) with 10% CS It is a graph showing the proliferation rate of MSC in the medium to which 20% CS (cord serum) was added.

FIG. 3 shows MSCs grown in MSCBM ™ medium added with 10% Cord serum and CellGro ™ medium added with 10% CS (cord serum) using FACSCalibur (BD company) using MSCGM ™ medium as a control. The flow cytometry was performed to show changes in cell size and phenotype (SSC-H; granular content within cell, FSC-H; cell size).

4 is a graph showing histograms for surface markers of MSCs grown in MSCGM ™ medium.

FIG. 5 is a graph showing histograms for surface markers of MSCs grown in MSCBM ™ medium added with 10% cord serum (CS).

FIG. 6 is a graph showing histograms for surface markers of MSCs grown in CellGro ™ medium with 10% CS.

7 is a graph showing the growth and proliferation rate of MSC propagated in a medium in which MS (GM serum) and growth factors of insulin, hydrocortisone, EGF and LIF were mixed with DMEM / F12 medium as a control. The graph shown.

FIG. 8 shows mesenchymal stem cells (MSCs) grown in a medium containing MSCGM ™ medium as a control and 5% or 10% CS (cord blood serum) in DMEM / F12 medium and insulin, hydrocortisone, EGF and LIF. ) Is a graph showing the change in size and phenotype (SSC-H; granular content within cell, FSC-H; cell size).

9 is a graph showing a histogram of surface markers of MSCs grown in media mixed with 5% cord blood serum and growth factors of insulin, hydrocortisone, EGF, and LIF in DMEM / F12 medium.

FIG. 10 is a graph showing a histogram of surface markers of MSCs grown in media mixed with 10% cord blood serum and growth factors of insulin, hydrocortisone, EGF, and LIF in DMEM / F12 medium.

The present invention relates to a mesenchymal stem cell culture medium comprising a basal medium and umbilical cord blood serum, a method for culturing mesenchymal stem cells using the same, and a method for obtaining mesenchymal stem cells using the same.

Stem cells have the property of continuously producing the same cells as themselves for a certain period of time in an undifferentiated state, and differentiate into specific cells under suitable conditions. Stem cells can be divided into embryonic stem cells and adult stem cells, depending on their origin. Human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life, but they are known to have superior cell proliferation and differentiation capacity as adult stem cells. Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., so there are few ethical problems, but they have limited multipotency compared to embryonic stem cells. Hematopoietic stem cells are the most studied among adult stem cells, and recent studies on mesenchymal stem cells are also invigorating.

On the other hand, unlike in vivo, to supply culture medium for cultivating animal cells in vitro, satisfies nutrients close to living conditions based on body fluids such as plasma or lymph, and environmental conditions such as pH, temperature and osmotic pressure. It must be done. Therefore, the culture medium composition was determined based on the body fluid, and the determined culture medium was selected and serum was added to the cells at an appropriate ratio, and then used for cell culture. Accordingly, various media compositions for culturing human stem cells are disclosed. For example, there is a method of culturing human stem cells using a medium containing animal serum (fetal calf serum). However, in this method, even if the cells cultured with fetal bovine serum are washed with a solution without fetal serum, a considerable amount of fetal bovine serum remains in the cell, acting as a strong heterologous antigen, and the possibility of human infection of cattle diseases such as mad cow disease. Have In other words, in order to apply the stem cells to the clinic, the materials used in the cultivation process must use materials derived from humans or proven to be stable in terms of stability.

To this end, Korean Patent Publication No. 2005-0099724 discloses a medium using human serum instead of an animal-derived serum used in conventional cell culture. However, stem cells cultured in the media disclosed in the above patent have a low expression rate of CD105, a marker indicating that they are mesenchymal stem cells. In addition, US Patent Publication No. 2003/0232432 discloses a medium containing human umbilical cord blood serum, but only describes a method of making umbilical cord blood serum.

Therefore, the present inventors have tried to increase the efficiency of in vitro proliferation of mesenchymal stem cells and to develop a culture medium without problems in terms of stability according to the research purpose and clinical application, and the culture medium containing cord blood serum is mesenchymal stem cells. The present invention has been found to be effective and selective in the cultivation of.

One object of the present invention is to provide a mesenchymal stem cell culture medium comprising basal medium and umbilical cord blood serum.

Another object of the present invention to provide a method for culturing mesenchymal stem cells using the medium.

Another object of the present invention to provide a method for obtaining mesenchymal stem cells cultured using the medium.

In one embodiment, the present invention relates to a mesenchymal stem cell culture medium comprising basal medium and umbilical cord blood serum.

As used herein, the term "medium" refers to cells such as stem cells in vitro, including elements essential for the growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, minerals, and the like. Refers to a mixture for growth and proliferation. In particular, the medium of the present invention is a medium for culturing mesenchymal stem cells. The mesenchymal stem cells are cells isolated from embryonic stem cells or adult stem cells derived from mammals, including humans, and have the ability to proliferate indefinitely and various cell types (eg, adipocytes, Cartilage cells, muscle cells, bone cells, etc.) is capable of differentiating. The above "culture" is meant to include the growth and proliferation of mesenchymal stem cells.

"Basic medium" of the present invention is a mixture containing essential sugars, amino acids, water, and the like necessary for living a cell, a mixture excluding serum, nutrients and various mesenchymal stem cell growth factors. The basic medium of the present invention may be prepared by artificially synthesizing or using a commercially prepared medium. Commercially prepared media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium, but are not limited thereto.

Serum is a supernatant obtained by centrifugation from animal or human blood. The serum contains trace elements including various factors such as various inorganic salts, polypeptide growth factors, and polypeptide hormones, which are essential for growth but not clearly identified, in addition to essential nutrients required for cell growth.

"Umbilical cord blood serum" of the present invention refers to serum isolated from the umbilical cord of the fetus. Umbilical cord blood serum of the present invention is preferably obtained from umbilical cords discarded at birth or stillbirth of the fetus. Umbilical cord blood can be collected from the umbilical vein by inserting a needle connected to a blood bag. This blood collection takes place at room temperature, with a maximum of 100 cc per umbilical cord. All this can be done in a clean room free of contamination, and the collected cord blood can only collect plasma components through blood coagulation. Coagulation of the blood may be carried out using a blood bag that does not contain anti-coagulant or additional coagulants may be added to aid coagulation, but preferably do not add to induce natural coagulation. The time required for coagulation may be 5 minutes, 30 minutes, 1 hour, 5 hours, 8 hours or more, and may be separated into blood cells and plasma components through centrifugation. Containers used in all of these processes should be sterilized and tested for biological stability such as contamination to microorganisms. In addition, the serum components such as fetal calf serum may be used by inactivating the plasma components by reacting at 56 ° C. for 30 minutes.

As a result of culturing the mesenchymal stem cells in the medium containing the cord blood serum of the present invention, it was found that the fetal bovine serum is better than the human serum, and that the re-blood serum is better than the fetal bovine serum in a relative ratio. Compared with the culture medium of the mesenchymal stem cells, it can be seen that it shows an excellent culture effect (see Table 1, Figure 1 and Figure 2).

The origin of the umbilical cord blood serum of the present invention may be of any origin as long as it is an animal having a umbilical cord such as human, cow, goat, pig, horse, etc., preferably from human and cow, more preferably from human It is.

The concentration of the umbilical cord blood serum used in the medium of the present invention is 1 to 30%, preferably 3 to 25%, more preferably 3 to 20%, most preferably 5 to 10% of the total medium composition.

As a specific embodiment, the culture medium of the present invention may further comprise a nutrient mixture (Nutrient Mixture). The nutrient mixture is a mixture containing various amino acids, vitamins, inorganic salts, and the like, which are generally used for cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, or the like. Commercially prepared nutrient mixtures include, for example, F-12, M199, MCDB110, MCDB202, MCDB302, and the like. Preferably, F-12 nutrient mixtures (F-12 Nutrient Mixture), M199, MCDB media, etc. may be used. Can be. The nutrient mixture may be used diluted to 1 to 10: 1 in the basic medium, preferably may be used diluted to 1 to 5: 1.

As a specific embodiment, the culture medium of the present invention may further comprise mesenchymal stem cell growth factors affecting the growth of mesenchymal stem cells. Growth factors of mesenchymal stem cells are, for example, insulin (Insulin), hydrocortisone (Eydrocortisone), EGF (Epidermal Growth Factor), LIF (Leukemia Inhibitory Factor), GM-CSF (Granulocyte-macrophage colony stimulating factor), EPO (Erythropoietin), Fibroblast Growth Factor (FGF), Insulin-like growth factor (IGF), Platelet-derived growth factor (PDGF), Stem cell factor (SCF), and transforming growth factor (TGF). In specific embodiments of the present invention, insulin, hydrocortisone, EGF, LIF and the like were used. For example, among the mesenchymal stem cell growth factors, insulin is present in the basic medium of the present invention at a concentration of 0.1 to 500 ng / ml, preferably at a concentration of 0.5 to 100 ng / ml, more preferably 0.5 to 50 ng /. in a ml concentration, most preferably in a concentration of 1 to 30 ng / ml, and hydrocortisone at a concentration of 0.1 to 500 nM, preferably at a concentration of 0.5 to 100 nM, more preferably 0.5 to 50 nM Concentration, most preferably 1 to 30 nM concentration, EGF is 0.1 to 500 ng / ml concentration, preferably 0.5 to 100 ng / ml concentration, more preferably 0.5 to 50 ng / in a ml concentration, most preferably in a concentration of 1 to 30 ng / ml, LIF in a concentration of 0.01 to 500 ng / ml, preferably in a concentration of 0.05 to 100 ng / ml, more preferably 0.1 to At a concentration of 50 ng / ml, most preferably at a concentration of 0.5 to 30 ng / ml. The. Based on various basal medium and cord blood serum, the present inventors mixed the insulin, hydrocortisone, EGF, and LIF in the medium at concentrations of 10 ng / ml, 10 nM, 10 ng / ml, and 10 ng / ml, respectively. When observed, it was found that the relative growth rate was higher than that in the medium without addition (Figs. 2 and 7).

As another aspect, the present invention relates to a method for culturing mesenchymal stem cells using the mesenchymal stem cell culture medium. Specifically, it relates to a method for culturing mesenchymal stem cells comprising the step of separating the mesenchymal stem cells and culturing the isolated mesenchymal stem cells in the medium of the present invention.

In the culture method of the present invention, the mesenchymal stem cells are cells having pluripotency derived from embryonic stem cells or adult stem cells of human or animal, preferably cells derived from human embryonic stem cells or adult stem cells. to be.

Separation method of mesenchymal stem cells of the present invention can be made by a method known in the art. For example, dilution of cells from donor tissue with mesenchymal stem cell basal medium, centrifugation to collect only cells, the cells at 1 to 500,000 mononuclear cells / cm ² , preferably 1,000 to 50,000 mononuclear cells Incubate in a flask for cell culture at a concentration of water / cm ² and incubate for 1 to 30 days, preferably 5 to 15 days, and attach and replace the medium at 1 to 10 days, preferably 2 to 4 days. Cells and proliferating mesenchymal stem cells can be obtained. The donated tissue may include bone marrow, blood, brain, skin, umbilical cord blood, embryos, various tissues, and the like, and may preferably include cord blood, bone marrow, or blood having excellent cell proliferation and differentiation ability. More preferably, it is isolated from bone marrow, which is easy to obtain, can be used in large quantities, and can be isolated from its own tissue to minimize the immune response. The mesenchymal stem cells are easy to differentiate into various tissues and cells, and can be used as cell therapy as the cells themselves.

The isolated mesenchymal stem cells are cultured in the culture medium of the present invention. The isolated and cultured mesenchymal stem cells express at least 90%, preferably at least 95%, of positive surface markers such as CD105 (SH2), CD29, CD44, CD73, CD166 and the like, and particularly CD44 and CD105 (SH2) positive surface markers. At least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99%. In addition, isolated and cultured mesenchymal stem cells express negative surface markers of CD34, CD45, HLA-DR, CD1a, CD14, CD31, CD80 and the like at 50% or less, preferably 10% or less, in particular, CD34 negative surface markers. Up to 50%, preferably up to 10%, more preferably up to 5%.

As another aspect, the present invention is a method for obtaining mesenchymal stem cells comprising culturing the mesenchymal stem cells in the medium of the present invention and obtaining the mesenchymal stem cells by separating the culture medium in a centrifuge It is about.

The method for obtaining mesenchymal stem cells includes culturing the mesenchymal stem cells in the culture medium of the present invention, and separating the mesenchymal stem cells from the culture medium. Isolation of mesenchymal stem cells can be carried out by methods known in the art. As one example, the mesenchymal stem cells in the flask for cell culture containing the culture medium of the present invention at a concentration of 1 to 10,000 cells / cm ² , preferably 50 to 6000 cells / cm ² 3 to 40 days During the culture, preferably for 6 to 20 days, the medium is replaced on 1 to 10 days, preferably 2 to 4 days to obtain adherent cells and proliferating mesenchymal stem cells. The adherent cells obtained by the above method may be passaged continuously, or may be stored in a culture medium by using cryogenic preservation or the like. The cryogenic cryopreservation can be carried out by modifying a general method known to those skilled in the art, for example, can be stored in a cryopreservation solution containing 10 to 20% serum and 5 to 10% DMSO in the stem cell basal medium. More preferably, MSCGM ™ may be used as a base medium, and may be suspended in a cryopreservation solution containing 10% fetal calf serum (FBS) and 5% DMSO and stored at -176 ° C in liquid nitrogen. The obtained mesenchymal stem cells can be differentiated into various tissues and neurons as needed, and can be used as a regeneration and treatment agent for damaged bones, cartilage, fat, nerve cells, liver, and various organs.

The cell culture method of the present invention can be usefully applied when culturing a large number of mesenchymal stem cells, and storing mesenchymal mesenchymal cells as a cell therapy, and when used as a cell therapy to treat human diseases, stability is ensured. Culture medium can be provided.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example  One : MSC Isolation and incubation

11 ml of bone marrow donated by a normal healthy person or from the patient himself was diluted in a 1: 2 ratio with DMEM medium, and then placed on a 10 ml of histopaque (Sigma, density 1.077 g / ml) layer prepared in advance. It was dropped and centrifuged at 400 x g for 30 minutes at room temperature. The monocyte cell layer was separated, DMEM medium was added, and then centrifuged at 400 × g for 5 minutes at room temperature. The obtained monocyte cells were washed twice with DMEM medium and maintained at 37 ° C. and 5% CO ₂ in MSCGM ™ medium. Incubated. After 48 hours of incubation, the cells that did not adhere to the bottom of the flask were replaced with fresh medium, and the cells attached to the bottom of the flask were incubated with changing medium every 3 to 4 days. When 80% of the cultured cells are grown, they are passaged into new flasks, some of which are still passaged, and some of them are cryopreserved with MSCGM ™, 10% Fetal Bovine Serum (FBS) and 5% DMSO. It was cloudy and stored in -176 ℃ liquid nitrogen.

Example  2 : CG  ( Cell Genix  G) on the badge MSCs  Proliferation test

As an experiment to verify the proliferation of MSCs of CG (Cell Genix), Using medium CG ①, ②, ③ directly supplied from Cell Genix, and DMEM (Gibco) medium containing MSCGM ™, 10% fetal bovine serum (FBS, Hyclone) purchased from Cambrex The mononuclear cells isolated and washed in Example 1 were inoculated with 5.28 × 10 ⁶ cells in a T25 flask containing 5 kinds of medium, and were incubated at 37 ° C. and 5% CO ₂ . After 48 hours of incubation, the cells that did not adhere to the bottom of the flask were replaced with fresh medium, and the cells attached to the bottom of the flask were incubated with changing medium every 3 to 4 days.

As a result, after dispensing monocyte cells in the medium of CG ①, ②, and ③, floating bands were formed within 24 hours. After the first medium exchange, almost no cells were attached to the bottom of the flask, and medium was exchanged for 22 days. It was maintained, but stopped at 22 days of culture because there was no growing cells. More than 10 cell colonies were observed in the flasks incubated with MSCGM ™ medium, and 3 × 10 ⁵ fibroblast-like adherent cells (FACs) were harvested on day 15 of culture. Five or more cell colonies were observed in flasks in DMEM medium containing 10% fetal bovine serum (FBS), and 1.5 × 10 ⁵ FACs were harvested on day 15 of culture (FIG. 1). Thus, it was found that CG's basic medium containing serum in the culture of mesenchymal stem cells was not as effective as MSCGM ™ purchased from Cambrex.

Example  3: MSCGM ™ as a control, DMEM  Badges of human serum ( HS )and SSS Effect Analysis

To investigate MSC proliferation by DMEM medium and human serum (HS) and SSS, MSCGM ™ (Cambrex) medium was used as a control, and DMEM (Gibco) was dissolved under various conditions and various serum was added. The growth of MSC was observed by culturing in DMEM medium (Gibco). In the AC group, MSCGM ™ (Cambrex) medium was used as a control, while the BD group contained 10% HS (Cambrex) alone or 5% SSS (Irvine scientific) simultaneously in DMEM (Gibco) medium dissolved in distilled water. The CL group was prepared by dissolving DMEM (Gibco) in Rovius' distilled water (LOVIS water, Kyungwon Enterprise) and containing 10% HS (Cambrex) alone or 5% SSS (Irvine scientific) at the same time. It was.

2.0 x 10 ⁶ mesenchymal stem cells (MSC) stored in cryopreservation solution containing MSCGM ™, 10% fetal bovine serum (FBS) and 5% DMSO in Example 1 were quickly dissolved in a 37 ° C. hot water bath. Then, washed twice with MSCGM ™ medium, mixed with 5 ml of MSCGM ™ medium, and then dispensed 6.6 × 10 ⁵ into T25 flasks containing the control medium AC and DMEM medium BD CL group containing 10% HS, 37 Incubated at 5 ° C. and 5% C0 ₂ . As a result of replacing the medium and observing the cells at 4 days after the first incubation, MSCs of the BD and CL groups were more difficult to identify under the microscope than the AL group. On the 7th day of culture, 0.125% trypsin-EDTA was treated and detached, and only half of the cells cultured in the T75 flask were passaged. The BD and CL groups were inoculated in DMEM containing 10% HS alone or 5% SSS simultaneously.

As a result, it was observed that cells dropped from the bottom of the flask and killed within 48 hours after dispensing MSCs in both the medium containing only 10% HS, BD and CL, and the medium added with 10% HS and 5% SSS. Only mesenchymal stem cells (MSCs) cultured in phosphate AL medium were observed to grow well (Table 1). As a result, it could be seen that 10% HS and 10% SSS did not contribute to the growth of mesenchymal stem cells.

Cell number according to culture medium and passage A-C B-D C-L B-D + 5% SSS C-L + 5% SSS 1 ^st inoculation 6.6 × 10 ⁵ 6.6 × 10 ⁵ 6.6 × 10 ⁵ - - 1 ^st harvest 20 × 10 ⁵ 6.6 × 10 ⁵ 7.2 × 10 ⁵ - - 2 ^nd cell inoculation 20 × 10 ⁵ 3.3 × 10 ⁵ 3.6 × 10 ⁵ 3.3 × 10 ⁵ 3.6 × 10 ⁵ 2 ^nd harvest 82 × 10 ⁵ Disposal Disposal Disposal Disposal

Example  4 : MSCGM ™ as a control, CG  On badge Fetal Bovine Serum ( FBS , The effect of human serum and SSS

Based on CG badge Fetal bovine serum, human serum (human serum) and to investigate the MSC jeungsikryeok by SSS, and the MSCGM ™ (Cambrex Co.) medium as a control group, CG; is added to cultures as described below in (CellGro ™ CellGenix, Inc.) and MSC The proliferation of was observed. CG medium containing 10% fetal bovine serum (FBS), CG medium containing 10% human serum (HS; Cambrex), 10% HS and 5% SSS (serum substitute) The experiment was carried out using CG medium supplemented with Irvine scientific) and CG medium alone.

The cryopreserved MSCs of Example 1 were thawed in the same manner as in Example 3, aliquoted 3 × 10 ⁵ in T25 flasks containing each medium, incubated at 37 ° C. and 5% CO ₂ , and The medium was changed in culture every 4 days.

As a result, the proliferation of MSC was observed only in CG medium containing Cambrex's MSCGM ™ and 10% fetal calf serum (FBS) on day 6, and there was no growth of MSC under other conditions.

Example  5: MSC On the base badge  Proliferative power by the addition of umbilical cord blood and Surface marker  analysis

5-1. Human Umbilical Cord Blood ( human cord serum By addition MSC  Proliferation Investigation

To investigate the proliferation of MSCs by the addition of human cord serum (CS), CSB was used at various concentrations using Cambrex's MSCBM ™ (Mesenchymal stem cell basal medium, Cambrex) and CellGro ™ (CellGenix) as a basic medium. The addition was used as a culture medium, MSCGM ™, was used as a control. As the medium conditions used in the experiment, MSCBM medium containing 10% CS, MSCBM medium containing 20% CS, CG medium containing 10% CS, and CG medium group containing 20% CS were used. Umbilical cord blood was removed from the umbilical cord taken out during fetal delivery, placed in a sterile tube and allowed to coagulate at room temperature for 20 minutes, and then centrifuged at 700 x g for 10 minutes to separate serum from the upper layer.

Thaw the MSCs that were cryopreserved in Example 1 in the same manner as in Example 3, aliquot each 2.4 × 10 ⁵ into T25 flasks containing each medium, incubating at 37 ° C. and 5% CO ₂ . The medium was changed and cultured once every 4 days, and passage culture was performed if the cells grew more than 80% of the floor area.

As a result, the growth rate of MSCs was higher in the other four CS groups compared to the control group cultured in MSCGM ™ medium in the 2nd and 3rd subcultures, especially in the MSCBM ™ medium group added CS. It was observed that the growth rate was more than two times higher. Based on these results, it was confirmed that the addition of CS to the medium for culture propagation of MSCs was very useful (FIG. 2).

5-2. human Umbilical cord serum ( human cord serum By addition MSC  In culture Cell surface marker  analysis

After subcultured cells in the culture medium of Example 5-1, the cultured MSCs were removed by treatment with 0.125% trypsin-EDTA, and centrifuged at 400xg for 5 minutes to obtain cells. Wash twice with Mg ²⁺ & Ca ²⁺ -free PBS (Gibco) containing 2% FBS, wash the 9 FACS tubes (BD company) equally, and dispense 100 ml of MSCs. 10 μl each, and reacted for 30 minutes at room temperature, followed by washing MSCs with PBS (Gibco) containing 2% FBS and not containing Mg ²⁺ , Ca ²⁺ , and centrifuging at 400 × g for 5 minutes at room temperature. Was repeated twice. After washing, 50% of 2% FBS-PBS (Gibco) was added thereto, and analyzed using a flow cytometer. The surface makers of MSCs were analyzed to observe changes in phenotype as stem cells. Antibodies were IgG1 negative control (Serotec), CD29 (Serotec), CD44 (Dako), CD105 (Serotec) and HLA-DR (Serotec) and phycoerytin bound to fluorescein isothiocyanate (FITC). IgG1 negative control (Serotec), CD34 (Serotec), CD45 (Dako) and D73 (BD Biosciences) bound (phycoerythrin, PE) were used.

As a result, MSCs cultured in the control group MSCGM ™ and MSCBM ™ medium containing 10% CS were similar in cell size and volume, while MSCs cultured in CellGro ™ medium containing 10% CS had a small cell size. The distribution of -H was observed to be wide (FIG. 3). In the karyotype, MSCs cultured in control MSCGM ™ and MSCBM ™ medium added with 10% CS expressed CD105 in 95% or more cells, but did not use CellGro ™ medium (10). % CS) only expressed CD105 in about 30% of cells (FIGS. 4, 5 and 6). In addition, CD29 ⁺ , CD44 ⁺ , CD73 ⁺ , CD105 ⁺ , CD34 ⁻ , CD45 ⁻ , HLA-DR ⁻ expressions generally used as MSCs cell surface markers are shown in Table 2.

Expression rate of MSC surface marker according to culture medium CD29 ⁺ MSCs CD44 ⁺ MSCs CD73 ⁺ MSCs CD105 ⁺ MSCs CD34 ^- MSCs CD45 ^- MSCs HLA-DR ^- MSCs MSCGM ™ 98.87% 99.75% 100% 98.96% 99.85% 99.67% 99.45% MSCBM ™ + 10% CS 99.99% 99.95% 100% 99.65% 99.97% 99.65% 99.75% CellGro ™ + 10% CS 98.46% 99.33% 99.84% 30.58% 96.68% 96.17% 98.94%

Example  6: in the development badge MSC  Proliferation and Surface marker  analysis

6-1. On the development badge MSC  Proliferation Investigation

To investigate the proliferative potential of MSC in homegrown media, Cambrex's MSCGM ™ was used as a control, insulin (10 ng / ml), hydrocortisone (10 nM), EGF (10 ng / ml), and 5%. DMEM / F12 (Gibco) medium with CS (cord serum), insulin (10 ng / ml), hydrocortisone (10 nM), EGF (10 ng / ml), and DMEM with 10% CS The experiment was carried out in / F12 medium ②.

Thaw the MSCs that were cryopreserved in Example 1 in the same manner as in Example 3, aliquot 2 × 10 ⁵ into T25 flasks containing each medium, incubating at 37 ° C. and 5% CO ₂ . The medium was changed and cultured once every 4 days, and passage culture was performed if the cells grew more than 80% of the floor area.

As a result, in primary subculture, the cultured MSCs were not significantly different from the cell proliferation rate compared to the control MSCGM ™. However, the growth rate of MSC in DMEM / F12 medium containing 5% CS medium was higher than that of control MSCGM ™. More than twice as high was observed. In addition, the growth rate of MSCs in the secondary passage was significantly higher in DMEM / F12 medium containing 5% CS medium and DMEM / F12 medium containing 10% CS medium than in control MSCGM ™ medium (FIG. 7). ).

6-2. Cultured in development medium MSC Cell surface marker  analysis

In order to analyze cell surface markers of MSCs cells cultured in Example 6-1, the cells were treated in the same manner as in Example 5-2, and the change in phenotype was observed using a flow cytometer of FACSCalibur (BD). As a result, MSCs cultured in control MSCGM ™ had various cell sizes and volumes, but cultured in DMEM / F12 medium group containing human umbilical cord blood and insulin, EGF, hydrocortisone, LIF, etc. MSC cells were obtained with a constant size and volume (Fig. 8). Based on the results, we analyzed the phenotype of MSCs grown in media containing 5% and 10% cord blood, respectively, and found that 5% cord serum and DMEM / F12 medium containing various growth factors and 10% cord blood In the case of MSC cultured in DMEM / F12 medium containing serum and various growth factors, the expression graph of CD45 shifted to the right side compared to the negative control, but all of them were read negatively for the interpretation of expression. More than 98% positive cells were obtained from CD29, CD44, CD105, and CD73, known as mesenchymal stem cell surface markers (FIGS. 9 and 10). As a result of analyzing the surface markers of MSCs, the phenotypic changes were observed. As a result of the phenotypic analysis of MSCs propagated according to the medium, some distribution differences were observed in CD34, CD45, and HLA-DR marker analysis, but there was no significant change. (Table 2).

The mesenchymal stem cell culture medium containing the cord blood serum of the present invention has an excellent effect on growth and proliferation while maintaining the pluripotency of mesenchymal stem cells as compared to the conventional stem cell culture medium. By culturing in large quantities can be obtained pure mesenchymal stem cells.

Claims (22)

Mesenchymal stem cell culture medium comprising umbilical cord blood serum, insulin, hydrocortisone, EGF (Epidermal Growth Factor), and Leukemia Inhibitory Factor (LIF). Mesenchymal stem cell culture medium expressing at least 95% positive surface markers and expressing CD34, CD45 and HLA-DR negative surface markers at 10% or less. The medium of claim 1 wherein the cord blood serum comprises 1-30% of the total medium composition. The medium of claim 2 wherein the cord blood serum comprises 3-20% of the total medium composition. delete delete The medium of claim 1 wherein the total medium composition has a concentration of 1-500 ng / ml insulin, 0.1-500 nM hydrocortisone, 0.1-500 ng / ml EGF and 0.01-500 ng / ml LIF. The medium of claim 1 wherein the mesenchymal stem cells are derived from human. delete delete delete delete delete delete delete delete delete Method of culturing the mesenchymal stem cells using the culture medium of claim 1. 18. The culture medium according to claim 17, wherein the culture medium comprises 1-30% of cord blood serum, 1-500 ng / ml of insulin, 0.1-500 nM of hydrocortisone, 0.1-500 ng / ml of EGF and 0.01-500 ng / of LIF. containing at a ml concentration. The method of claim 17, wherein the mesenchymal stem cells are derived from humans. Method for producing undifferentiated mesenchymal stem cells exhibiting the following characteristics, characterized in that the human mesenchymal stem cells are cultured in the culture medium of claim 1 and then recovered: (a) shows positive immunological properties for CD105, CD29, CD44 and CD73 and negative immunological properties for CD34, CD45 and HLA-DR; And (b) has the ability to differentiate into mesodermal derived cells. The culture medium of claim 20, wherein the culture medium comprises 1-30% of cord blood serum, 1-500 ng / ml of insulin, 0.1-500 nM of hydrocortisone, 0.1-500 ng / ml of EGF and 0.01-500 ng / of LIF. containing at a ml concentration. The method of claim 20, wherein the mesenchymal stem cells are derived from humans.

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