WO2013008960A1 - Human amniotic fluid stem cell culture medium and method for culturing amniotic fluid stem cells using same - Google Patents

Abstract

The present invention relates to amniotic fluid stem cell (AFSC) culturing, and more particularly, to AFSC culturing characterized by separating amniotic fluid cells from amniotic fluid through centrifugal separation, and culturing the separated amniotic fluid cell precipitate for seven days in an AmnioMax culture, after which subculturing is performed from a first subculture in a serum-free medium, wherein the amniotic fluid stem cells in the serum-free culture medium have a higher growth rate when compared to a conventional subculturing in a serum-added culture medium, and exhibit positive results for SSEA-4 and HLA-ABC expression, stem cell marker and myocyte marker expression, etc.

Description

Human Amniotic Stem Cell Culture Medium and Amniotic Stem Cell Culture Method Using the Same

The present invention relates to a human amniotic stem cell culture medium and a stem cell culture method using the same.

When the fertilized egg is fertilized in the human uterus, it implants on the wall of the uterus and after several days the embryo is surrounded by amniotic sac filled with amniotic fluid. These amniotic fluid secretes and mixes various synthetic compounds synthesized from the body of the fetus. Therefore, a number of research reports on the culture of amniotic cells have been published since amniotic fluid obtained from pregnant women can produce various cells in the human body.

Amniotic fluid cells are fetal cells that are eliminated as amniotic fluid as the fetus grows in the womb of the mother. The contents of the amniotic fluid are very small, but because of their high dividing capacity, a large amount of cells can be obtained with good initial culture. Many methods and culture media of human embryonic stem cells using amniotic cells have been published. Medium is added to the cell precipitate obtained by centrifugation of amniotic fluid, and an appropriate amount is taken to culture the incubator. Examples of a medium commonly used in this step include Chang medium and AmnioMax.

In this step, the scientists carry out subculture while confirming the density of the amniotic cell population. In this way, the cells are cultured sufficiently and the cultured amniotic cells are frozen and stored in the liquid nitrogen tank until the cells are divided and used for the next step. In order to use frozen amniotic fluid as a feeder cell layer for human embryonic stem cells, it is necessary to maintain the undifferentiated state. Therefore, mitomycin C is used for this purpose.

In the step of culturing the embryonic stem cells and the embryonic stem cells in the cryopreservation, fetal bovine serum (FBS), penicillin (Penicillin), streptomycin (Steptomycin) is usually included to synthesize the embryonic stem cell group (colony) KO-DMEM medium and DMEM / F12 medium are commonly used.

Stem cells are cells that have the ability of self-proliferation to differentiate into various cells through suitable environment and stimulation. Embryonic stem cells (ES cells) isolated from early embryos, from embryonic primitive germ cells Three types of embryonic germ cells (EG cells) isolated and multipotent adut progenitor cells (MAPC cells) isolated from adult bone marrow are well known. In particular, adult stem cells were first isolated from bone marrow (Science 276, 71-74, 1997) and then from skeletal muscle (Proc. Natl Acad. Sci. USA 96, 1999), adipose tissue ( fat tissue) (Tissue Eng. 7, 2001), each of which was found to be capable of similar lineage differentiation. However, since the adult stem cells were difficult to isolate and culture, it is urgent to research and develop a replaceable stem cell source.

Mother's amniotic fluid has the advantage that it can be separated harmlessly to both mother and fetus. After fertilized egg implanted on the uterine wall, it is surrounded by amniotic sac after a few days. At this time, amniotic fluid is collected and amniotic fluid is analyzed. It can be easily cultured. By analyzing amniotic fluid, abnormalities in the chromosomes of the fetus can be confirmed, and pathogen infection can be confirmed. In addition, the amniotic fluid is collected before the birth of the fetus to check the health of the fetus. If the amniotic fluid is used for research purposes with the consent of the mother, there is an advantage in that a large amount of cells can be easily obtained in comparison with other adult stem cells. In addition, mesenchymal stem cells (MSCS) derived from amniotic fluid can be cultured for a certain period of time to extract the medium and put into fibroblasts to induce cell growth. Mesenchymal stem cells are differentiated from cartilage, bone, fat, myeloid epilepsy, muscle, and nerves. Primitive cells are maintained in the bone marrow in adults, but in fetuses and umbilical cord blood, peripheral blood is present in the amniotic fluid.

The method for culturing human embryonic stem cells using human adult bone marrow cells is described by Cheng et al. (2003). The method for culturing and maintaining human embryonic stem cells in undifferentiated state using fore skin of newborns is Amit. (2003), a report on the expression of Oct-4, a marker of undifferentiated cells, by culturing amniotic cells derived from pregnant fetuses has been reported by Prusa et al. (2003).

In general, DMEM medium containing Fetal bovine serum (FBS) (Giboco BRL Co., Ltd.) has been used for the tissue cell culture (Korean Patent Publication No. 2001-56100), and Umbilical Cord Matrix (Umbilical Cord Matrix) Dulbecco's Modified Eagle's Medium (DMEM) has generally been used to isolate and culture UCM cells and to differentiate Umbilical Cord blood (UCB) cells.

Proliferation of human embryonic stem cells using human bone marrow stromal cells is disclosed in Korean Patent Publication No. 2007-79587, which isolates mononuclear cells from any one of human peripheral blood, bone marrow and umbilical cord blood. Method of coculture with monolayer and monocultured mononuclear cell in the presence of adult stem cells (coculture), in particular, the method of culturing and propagating hematopoietic stem cells derived from umbilical cord blood is disclosed in Korean Patent Registration No. 10-773253 have. In addition, Korean Patent Registration No. 10-1037002 is disclosed as a method of culturing and proliferating chondrocytes for transplantation.

However, DMEM medium or ABM medium containing fetal bovine serum (FBS) has been consistently used in the culture proliferation of hematopoietic stem cells or chondrocytes.

In Korean Patent Registration No. 10-908481, CNT- containing mesenchymal stem cells containing umbilical cord blood serum and No. 10-1026070 was added 10% fetal bovine serum (FBS) of epidermal sstem cells. Cell culture and proliferation in 57 medium and Fetal Bovine Serum free medium are disclosed, respectively.

In this way, fetal bovine serum (FBS) or human serum (HS) has been commonly added to the culture medium for various stem cells or mesenchymal stem cells, but in order to develop stem cells into pharmaceutical compositions or cosmetic compositions, they are contaminated. There is a need for stable culture growth with minimal contamination.

The object of the present invention is to conceive in view of the above points, the culture propagation of amniotic cells isolated from amniotic fluid and coculture with human embryonic stem cells using amniotic cells as feeder cell layer (coculture step in the subculture) The present invention provides a medium and culture method for culturing without adding fetal bovine serum (FBS) or human serum (HS).

The object of the present invention is to separate the amniotic fluid stem cell (AFSC); Subcultured the isolated amniotic stem cells in a serum free medium; Measure and compare the growth rate of amniotic stem cells cultured in a conventional serum supplement medium (Chang's medium), and react the amniotic stem cells with a PE-conjugated single antibody to perform a Cytometric analysis, the stem of the amniotic stem cells This was accomplished by confirming cell marker expression and confirming the differentiation capacity into muscle cells.

According to the present invention, since the amniotic stem cells not only effectively proliferate in a serum-free medium MesemCult-XF Medium, but also express all the characteristics of the amniotic fluid-derived stem cells as compared to the conventional serum supplement medium, the pharmaceutical composition. Or there is an excellent effect to minimize the possibility of contamination for the development of the cosmetic composition.

1 is a photomicrograph showing the amniotic stem cells cultured in a conventional serum addition medium and serum-free medium of the present invention, respectively.

2 is a graph comparing the growth curve of amniotic stem cells in the serum-free medium of the present invention with that in the conventional serum-added medium.

Figure 3 is a diagram showing the flow cytometry pattern of amniotic stem cells cultured in the serum-free medium and the conventional serum addition medium of the present invention, respectively.

Figure 4 is a photograph showing the results of RT-PCR showing the expression pattern of stem cell markers in the amniotic fluid cells cultured in serum-free medium and conventional serum-added medium, respectively.

5 is an Immunofluorescence staining result of observing the differentiation pattern of muscle cells in the amniotic fluid stem cells cultured in the serum-free medium and the conventional serum addition medium of the present invention, respectively.

Figure 6 shows the Western blotting results of observing myocyte differentiation patterns of amniotic stem cells of the present invention.

In the present invention, the term "stem cells" includes all stem cells derived from humans. Stem cells derived from humans have the property of continuously producing the same cells as themselves in an undifferentiated state for a certain period of time and differentiating specific cells under appropriate conditions. Stem cells are classified into embryonic stem cells and adult stem cells, depending on their origin. Since human embryonic stem cells are made from embryos that can occur as human life, ethical issues arise, and cell proliferation and differentiation ability are superior to adult stem cells. On the other hand, adult stem cells can be obtained from bone marrow, umbilical cord blood, skin, amniotic fluid, and the like, but have fewer ethical problems, but have limited multipotnecy than embryonic stem cells. Hematopoietic stem cells are the most common research on adult stem cells, and recent studies on mesenchymal stem cells have been conducted.

However, in order to artificially culture stem cells in vitro unlike in vivo, it is necessary to satisfy nutrients close to body fluids such as plasma and lymph, and bioenvironmental conditions such as pH, temperature, and osmotic pressure. Therefore, a culture medium should be prepared based on body fluids, and serum (serum) should be added. To date, various culture medium compositions have been disclosed for the purpose of culturing human embryonic stem cells. Among them, medium containing fetal bovine serum has been used as animal serum. However, even if cells cultured in a medium containing fetal bovine serum are washed with a solution without fetal serum, a significant amount of serum remains in the cells, acting as a strong heterologous antigen, and possibly causing bovine disease. do. Therefore, in order to apply human stem cells in vivo, the culture medium component in terms of safety, and for the medium using human serum instead of bovine serum in the stem cell culture medium, Korean Patent Publication No. 2005-99724 The stem cells cultured in the human serum medium have a disadvantage of low expression rate of CD105, which is a mesenchymal stem cell marker, and US Patent Publication No. 2003/232432 discloses a medium containing human umbilical cord blood serum.

In the present invention, the "culture medium" of stem cells refers to a composition containing essential ingredients necessary for the growth and proliferation of cells in vitro. The culture medium of stem cells includes various growth factors necessary for the culture of serum and mesenchymal stem cells in the basic medium. The basal medium may be used artificially synthesized or 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-Mineral Essential Medium). Gibco, Invitrogen, Newyork, G-MEM, Glasgow's Mineral Essential Medium, Iscove's Modified Dulbecco's Medium Vancouver, Canada).

Meanwhile, serum for culture of stem cells is a supernatant centrifuged from blood of an animal or human. In addition to being essential nutrients for stem cell growth, serum contains various inorganic salts, trace elements, polypeptide growth factors and hormones. For example, the origin of the serum for stem cell culture medium may be umbilical cords of humans, cattle, goats, pigs and horses. Human blood serum is an example of umbilical cord blood serum, which is collected by inserting a syringe connected to a blood bag from a vein of an umbilical cord that is discarded during childbirth or stillbirth of the fetus. Umbilical cord blood collection is performed at room temperature and up to 100 cc per umbilical cord is collected. The collection process is carried out in the Crean room and the cord blood is coagulated naturally to collect only plasma components, which are centrifuged to separate blood cells and serum. It has been reported that the fetal bovine serum than the human serum and the cord blood serum than the fetal bovine serum have superior cell growth and proliferation effects as mesenchymal stem cell culture medium additive components. There are many reports of improved stem cell growth rate in stem cell culture medium containing fetal bovine serum and cord blood serum, growth factors and hormones in various basic media. For example, dilution with the mesenchymal stem cell primary medium to which the concentration of cord blood serum 5-10% was added to the primary medium and centrifuged to collect only cells, and the cells were collected in a cell culture flask at a concentration of 1,000 to 50,000 mononuclear cells / cm 2. Incubate for 15 days, but the culture medium is replaced in 2-4 days to obtain adherent cells and proliferated mesenchymal stem cells. The donated tissues include bone marrow, blood, brain, umbilical cord blood, amniotic fluid, skin, embryos, and the like. The mesenchymal stem cells obtained above are easily differentiated into various tissues or organs and can be used as cell therapy as a pharmaceutical composition or a cosmetic composition as the cell itself.

In the mesenchymal stem cell culture, mesenchymal stem cells are generally CD105, CD29, CD44, CD73, CD166. Equivalent positive surface markers are expressed.

The isolated mesenchymal stem cells express negative surface markers such as CD34, CD45, HLA-DR, CD29, CD14, CD31, and CD80. The mesenchymal stem cells isolated and cultured as described above are cryopreserved at cryogenic temperatures (-176 ° C. frozen nitrogen), and can be differentiated into various tissues and nerve cells, and thus, tissues such as damaged nerve cells, liver, bone, cartilage, fat, lung, Agents for organ regeneration and for the treatment of human diseases can be used.

Example 1 . Amniotic Stem Cell (AFSC) Isolation and Culture

Amniotic fluid stem cells were isolated through amniocentesis of a patient who visited the Department of Obstetrics and Gynecology, Kyungpook National University Hospital to pass the IRB. Amniotic stem cells were centrifuged at 1,200 rpm for 10 minutes in 10 mL of amniotic fluid donated from patients, and then washed with α-MEM Serum free medium. The washed monocytes were incubated for 7 days at 5% CO ₂ at 37 ° C. in AmnioMax II complete Medium (Gibco Newyork, USA). At this time, cells that did not adhere to the bottom of the flask were transferred to fresh medium and incubated separately for 7 days. The cells adhered to the bottom of the flask were incubated with a medium every 7 days. When 80% of the cultured cells were grown, the cells were passaged with a new flask, and some of the cultured cells were continuously passaged and some were suspended in cryopreservation solution. It was stored in a liquid nitrogen tank at 176 ℃.

Example 2 . Passage culture in serum free medium

Monocyte amniotic stem cells isolated and washed in Example 1 were subdivided into medium (control) and serum free medium (test) containing serum (Fetal Bovine Serum: FBS).

In this example, control cells were cultured with AmnioMax II complete medium up to passage 2 and with α-MEM medium (Gibco, Invitrogen, newyork, USA) based on Chang's medium in passage 3 for 2 to 3 days. Subcultured with fresh medium at intervals. On the other hand, in the experimental zone of the present invention, amniotic stem cells were subcultured in MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada), which is a serum free medium.

As a result, the results of examining the culture form of amniotic stem cells were grown while maintaining the morphology similar to fibroblast in both cells grown in both media as shown in FIG. It was confirmed that the cells grown in the xeno-free medium were somewhat rounded. As a result of analyzing the proliferative capacity of the stem cells, the growth rate was excellent and the growth rate was faster in the serum-free medium MesemCult-XF Medium than in Chang's medium (FIG. 2). These results confirm that both the safety and economics of the use of amniotic stem cells are advantageous in culturing in serum-free medium.

Experimental Example 1 Flow cytometric analysis

After the 4th and 6th passages in each culture medium according to Example 2, the cultured amniotic stem cells (AFSC) were reacted with PE-conjugated monoantibodies and analyzed by flow cytometry (FACScan, BD Biosciences, USA). . That is, the amniotic stem cells cultured in each culture medium were separated from the culture dish using 0.05% trypsin-EDTA, and centrifuged at 1,200 rpm for 5 minutes. This was washed with 1X PBS and 5 × 10 ⁵ cells were each well suspended in 100 mL of FACS buffer (2% BSA, PBS), followed by PE-conjugated monoclonal antibodies (control, CD44, CD73, CD45, CD90, CD105). , C-kit, SSEA4, HLA ABC, HLA DR) were added to each 10 mL and reacted at 4 ° C. for 30 minutes. After that, each expression was analyzed by flow cytometry.

As a result, CD44 (hyaluronan receptor), CD73 (marker of SH-3 and SH-4), CD90 (marker of Thy-1), CD105 (endoglin), SSEA in AFSC cells cultured in control and experimental medium -4 (stage-positive specific embryonic autigen) showed an expression rate, CD45 (marker of hematopoietic lineage) showed a negative response (Fig. 3).

In addition, positive expression was observed in class I major histocompatibility (MHC) antigens (HLA-ABC), and the expression rate of MHC class II CHLA-DR was negative.

As a result of the experiment, it was confirmed that the expression in a similar pattern in the medium of the control and the experimental group.

Experimental Example 2 RNA Isolation and RT-PCR

According to Example 2, in order to confirm the expression of stem cell markers in the amniotic stem cells cultured in each culture medium, total RNA was extracted from the cells with Trizol reagent, and then cDNA was synthesized using a cDNA synthesis kit. Was performed. The control and experimental medium were used to confirm the expression of stem cell markers of similar levels in cultured amniotic stem cells, respectively (FIG. 4). Therefore, it has been verified that serum-free medium may be used in amniotic stem cell culture.

Experimental Example 3 Immunofluorescence Staining of Amniotic Stem Cells

In order to confirm the multipotency of the amniotic stem cells cultured according to the control and experimental groups to the muscle cells in Example 2 of the present invention, the expression patterns of the muscle cell markers MYOD and Desmin were performed. It was. Each amniotic stem cell was laid on a cell culture plate coated with Matrigel (BD Biosciences, Bedford, Mass., USA) and differentiated medium [10% horse-serum (Gibco / BRL) to induce differentiation into myocytes after 24 hours. , Carlsbad, CA, USA), DMEM (low-glucose formulation) with 0.5% chick embryo extract (Gibco / BRL, Carlsbad, CA, USA) and 1% penicillin / streptomycin (Hyclone, Logan, UT, USA)] And 5 mM 5-aza-2'deoxycytidine 3 (5-azaC; Sigma-Aldrich, St. Louis, Mo, USA). After incubation for 24 hours again, the medium was changed to differentiation medium, and medium was changed every 3 days to induce differentiation for 7 days. As a result, the expression of MyoD and Desmin was confirmed in both control and amniotic fluid cells cultured in the serum-free medium of the present invention (FIG. 5).

Therefore, it has been verified that serum-free medium may be used in amniotic stem cell culture.

Experimental Example 4 Western blotting of Amniotic Stem Cells

In order to confirm the differentiation capacity of amniotic stem cells into muscle cells of control and experimental groups according to Example 2 through Western blotting analysis, lysis buffer (150 mM NaCl, 20 mM TRIS, 1) 100 mL of% Tsiton X-100, 400U / mL RNase inhibitor, pH 8) was added to extract the protein and precipitated with methanol. 50 mg of protein was isolated from SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After attaching polyclonal anti-human MyoD (1: 500, Santa cruz Biotechnology, Santa Cruz, CA, USA) and desmin (1: 1000, Cell signaling, Danvers, MA, USA), secondary peroxidase-conjugated antibody (1: 1000, After reacting with Amersham Bioscience, Uppsala, Sweden, specific protein expression was confirmed using ECL fluorescence reagent (Amersham Bioscience, Uppsala, Sweden).

As a result, the expression of MyoD and Desmin in a similar manner in the amniotic stem cells cultured in each culture medium was confirmed (Fig. 6).

As can be seen from the above, the present invention has an excellent effect of subcultured amniotic stem cells (AFSC) using a serum-free medium is a very useful invention in the high-safety cosmetic industry and cell therapy medical industry.

Claims (7)

1. Human stem cell culture method characterized in that the passage in serum-free medium (Serum free medium).
2. The method of claim 1, wherein the passage is stem cell culture, characterized in that starting from passage 1.
3. The method of claim 1, wherein the serum-free medium is MesemCult-XF Medium.
4. The method of claim 1, wherein the human stem cells are stem cells, characterized in that the adult stem cells of any one of the amniotic stem cells, bone marrow cells, skin cells, umbilical cord blood cells.
5. Human stem cell culture subcultured by any one of claims 1 to 4.
6. Cosmetic composition, characterized in that containing the stem cells derived from the culture of claim 5 as an active ingredient.
7. Cell therapy composition with enhanced safety, characterized in that containing the stem cells derived from the culture of claim 5 as an active ingredient.

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