KR100662706B1 - Culture method for human embryonic stem cells by using culture medium collected after amniotic fluid cells culture - Google Patents

Abstract

The present invention relates to a method for culturing human embryonic stem cells using amniotic cells.

The cell culture method of the present invention includes a method of using amniotic cells as a feeder cell layer for culturing human embryonic stem cells, and a method of using the culture medium collected after culturing amniotic cells as a culture medium of human embryonic stem cells.

Human embryonic stem cells cultured by the cell culture method of the present invention retain the cytological, immunological and genetic characteristics of undifferentiated cells. In addition, human embryonic stem cells cultured by the cell culture method of the present invention have the ability to form well embryos under differentiation conditions and differentiate into various tissues.

Therefore, the cell culture method of the present invention can be usefully used in the field of treatment and research using human embryonic stem cells.

Description

Culture method for human embryonic stem cells by using culture medium collected after amniotic fluid cells culture}

1 is a diagram showing the morphological characteristics of human embryonic stem cells cultured using amniotic cells as feeder cell layers.

Figure 2 is a diagram showing the morphological characteristics of human embryonic stem cells cultured using culture medium collected after the amniotic cell culture.

3 is a karyotyping diagram of human embryonic stem cells cultured using amniotic cells as feeder cell layers.

Figure 4 is a diagram showing the expression of embryonic-specific cell membrane antigen in human embryonic stem cells cultured using amniotic cells as feeder cell layer.

Figure 5 shows the expression of Oct-4 mRNA in human embryonic stem cells cultured using amniotic cells as feeder cell layers.

Figure 6 shows the telomerase activity of human embryonic stem cells cultured using amniotic cells as feeder cell layers.

7 is a diagram showing the result of differentiating human embryonic stem cells cultured using amniotic fluid cells as feeder cell layers.

The present invention relates to a method for culturing human embryonic stem cells using amniotic cells.

Human embryonic stem cells are cells derived from the inner cell mass of the human blastocyst. These cells have the ability to differentiate into the trioderm cells that make up the human body. After Thomson et al. Succeeded in culturing human embryonic stem cells in 1998, the field of cell therapy using stem cells Be in the spotlight.

Human embryonic stem cells, unlike mouse embryonic stem cells, require a feeder layer to maintain and propagate undifferentiated state. In the culture of human embryonic stem cells, mouse embryonic fibroblasts (MEF) have been mainly used as feeder cell layers.

However, when the human embryonic stem cells are cultured together with cells of other species, there is a high possibility of being infected with a virus, and there is a problem that later acts as an obstacle for cell therapy.

Xu et al. (2001) succeeded in culturing human embryonic stem cells without the feeder cell layer by using the culture medium of mouse embryonic fibroblasts as a culture medium of human embryonic stem cells. However, this method also used a culture solution collected after the culture of mouse embryonic fibroblasts, which are cells of a different species than humans, and thus cannot be considered to exclude the problem of exposure to heterologous cells.

Therefore, in recent years, studies have been made on a culture method using cells derived from humans without using cells of other species as feeder cell layers.

Amit et al. (2003) disclose a method for culturing and maintaining human embryonic stem cells in undifferentiated state using foreskin cells of newborns.

Cheng et al. (2003) disclose a method of culturing human embryonic stem cells using human adult marrow cells.

Richarde et al. (2002, 2003) also reported that the culture of human embryonic stem cells using various cells was able to maintain undifferentiated cells derived from muscle and skin of aborted fetuses.

On the other hand, it has been reported to express Oct-4, a marker of undifferentiated cells, in amniotic fluid cells, which are cells that are eliminated from the fetus during pregnancy (Prusa et al., 2003).

Therefore, the present inventors completed the present invention by confirming that the amniotic cells may be used as a feeder cell layer, or the culture medium may be used as a culture medium for human embryonic stem cells to stably culture the stem cells while maintaining their undifferentiated characteristics.

In the present invention, by using amniotic cells, to provide a cell culture method that can stably culture human embryonic stem cells while maintaining undifferentiated properties.

The present invention provides a method for culturing human embryonic stem cells using amniotic cells.

The cell culture method of the present invention includes a method of using amniotic cells as feeder layer for culturing human embryonic stem cells.

The method comprises the steps of 1) culturing amniotic cells, 2) coculture of amniotic cells and human embryonic stem cells.

Amniotic cells are fetal cells that are released into amniotic fluid when the fetus is growing in the mother's stomach. Amniotic cells have a very low content ratio in amniotic fluid, but since they are vigorous in dividing capacity, a large amount of cells can be obtained by initial culture under appropriate conditions.

In step 1), medium is added to the cell precipitate obtained by centrifugation of amniotic fluid, and an appropriate amount is planted in a culture dish.

Usable media in this step include Chang media, AmnioMax, and the like.

Subculture is performed while confirming the density of the cell population, and the cells are sufficiently grown, and the cells are divided and frozen and stored in liquid nitrogen until used for the next step.

When frozen amniotic fluid is used as a feeder cell layer for culturing human embryonic stem cells, it is required to maintain the undifferentiated state, so it is treated with mitomycin C, a drug that inhibits cell division.

In step 2), the amniotic fluid cells and the embryonic stem cell SNUhES cell line (obstetrics and gynecology, Seoul National University College of Medicine) prepared above are co-cultured.

As the medium that can be used in the step, KO-DMEM medium containing 10% fetal bovine serum (FBS), penicillin (Penicillin), streptomycin (Streptomycin), etc. may be used.

When embryonic stem cell colonies are formed, the cells are fractionated to have 100-200 stem cells per clump, and the cells are passaged at 7-day intervals to observe the state of the cells.

In addition, the cell culture method of the present invention includes a method of using the culture medium (conditioned media) collected after culturing the amniotic cells as a culture medium of human embryonic stem cells.

The method comprises the steps of 1) culturing the amniotic cells and collecting the culture solution, and 2) culturing human embryonic stem cells with the culture solution.

In step 1), the method of culturing the amniotic cells is the same as step 1) of the method using the amniotic cells as the feeder cell layer.

After treating the myocytes with mitomycin C, and cultured in a flask for cell culture using DMEM medium containing FBS. When the cell density is 70-80% or more, the medium is replaced with fresh medium and the medium is removed after 24 hours every day for one week. The collected medium is filtered using a sterile filter and then frozen and stored until use in the next step.

In the second step, human embryonic stem cells are cultured using the conditioned media collected above.

When the embryonic stem cell group is formed, the cells are fractionated to have 100-200 stem cells per cell mass and passaged at 7-day intervals to observe the state of the cells.

Human embryonic stem cells cultured by the cell culture method of the present invention have a normal karyotype and exhibit the morphological characteristics of undifferentiated cells.

Human embryonic stem cells cultured by the cell culture method of the present invention are alkaline phosphatase, stage specific embryo antigen (SSEA-3), SSEA-4, Tra-1-60, which are cell membrane markers of undifferentiated cells. , Expressing Tra-1-81.

Human embryonic stem cells cultured by the cell culture method of the present invention is characterized in embryonic stem cells (ESC), embryonic germ cells (EGC) and embryonic carcinoma cells (ECC). To express Oct-4 (octamer-binding transcription factor-4) mRNA is known as an essential element to establish a multipotential stem cell.

Human embryonic stem cells cultured by the cell culture method of the present invention exhibits telomerase activity, an enzyme showing high activity in cells that are not aging and continue to proliferate. Telomerase is an enzyme that maintains the length of telomeres at the end of chromosomes. It is involved in the infinite proliferation and survival of cells and is known as a marker of embryonic tissue.

In the cell culture method of the present invention, even when culturing human embryonic stem cells without the feeder cell layer using culture medium collected after the amniotic cell culture, human embryonic stem cells are maintained while being undifferentiated until the initial 4-5 passages. can do.

Therefore, the cell culture method of the present invention enables to stably culture human embryonic stem cells while maintaining undifferentiated properties.

Human embryonic stem cells cultured by the cell culture method of the present invention form an embryoid body well under differentiation conditions. Embryos are basically forms that should be formed in differentiation experiments using human embryonic stem cells.

Therefore, human embryonic stem cells cultured by the cell culture method of the present invention are considered to have no problem in the future for differentiation into various tissues.

In addition, human embryonic stem cells cultured by the cell culture method of the present invention showed the same results consistently regardless of whether or not mitomycin C was treated to amniotic fluid. That is, the cell culture method of the present invention can stably culture human embryonic stem cells in an undifferentiated state regardless of propagation of vegetative cells, thereby reducing the time and cost required for the culturing of stem cells.

Since the cell culture method of the present invention cultures human embryonic stem cells using human-derived cells, it can be usefully used for cell transplantation for cell therapy without fear of infection due to heterologous feeder cell layers.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Culture of Human Embryonic Stem Cells by the Method of the Present Invention

Human embryonic stem cells were cultured as follows by the method of using amniotic cells as a feeder cell layer in the cell culture method of the present invention.

1) Isolation and Culture of Amniotic Cells

Amniotic cells were cultured by the method of the present invention.

10 ml of amniotic fluid collected for chromosome examination for prenatal genetic diagnosis was centrifuged at 1000 rpm for 8 minutes.

1 ml of Chang medium (Irvine scientific) was added to the cell precipitate obtained by centrifugation, and the suspension was planted in 0.5 ml portions on a cover glass slide placed in an in situ culture dish.

After culturing for 2 days in a cell incubator maintained at 37 ° C. and 5% CO ₂ condition, 2 ml of nutrient medium was added the next day, and the medium was exchanged by checking the density of the attached cell group through cell observation under a phase contrast microscope for about a week. gave. After confirming that 4-5 cell groups were attached to the slides, subcultures were performed on the cover slides of two culture grafts treated with 0.25% trypsin-EDTA.

After that, the density of the cell population attached to the cover slide and the density of the cells in the cell division were checked and harvested for harvesting and karyotyping. After completion of cell harvest and karyotyping, the remaining passaged amniotic cells were checked for cell status, treated with 0.5 ml of 0.25% trypsin-EDTA, and passaged to one 25 cm ² T-flask per 35 mm dish.

Two days later, the medium was added to the 25T-flask, and on the third day, one 25T flask was subdivided into two 75 cm ² T-flasks. These two 75 cm ² T-flasks were subdivided into five 75 T-flasks each. The cultured cells were again treated with 0.25% trypsin-EDTA, and the cells were aliquoted to 2 X 10 ⁶ cells in a medium containing 5% DMSO and stored in a liquid nitrogen container.

The amniotic fluid cells stored as described above are used as a feeder cell layer for organ culture in vitro in an undifferentiated state of human embryonic stem cells, and the concentration of mitomycin C (mitomycin C), which inhibits cell division, is 0.01 mg / ml. Treated for 150 minutes. Amniotic cells not treated with mitomycin C were also used as feeder cell layers.

2) Human Embryonic Stem Cell Culture Using Amniotic Cell Nutrient Layer

Human embryonic stem cells were cultured using the amniotic fluid cells obtained in 1) as nutrient layers.

Prepare a culture dish pretreated with matrigel diluted to 20: 1, and use the amniotic cells treated with mitomycin C to inhibit division and the amniotic cells not treated with mitomycin C as embryonic stem layers. Cell SNUhES cell line (Gynecology, Seoul National University) was cultured.

Cultures consisted of 20% serum replacement (SR, Gibco), 0.4ng / ml basic fibroblast growth factor (bFGF), 1% non-essential amnio acid, 0.1 mM beta-mercaptoethanol (MEM-F12 containing β-mercaptoethanol), 0.5% penicillin / streptomycin was used.

The undifferentiated embryonic stem cell population (colony) is fractionated with a fine glass knife to have 100-200 stem cells per clump, and then passaged at 7-day intervals, and the morphology of the cells is observed. The results are shown in FIG. 1 (2 days (a), 7 days (b) when the myocytes were treated with mitomycin C and 2 days (c), 7 days when the mitomycin C was not treated with the amniotic cells) d)).

As shown in Figure 1, the human embryonic stem cells cultured according to the culture method of the present invention has a small round shape, a number of phosphorus is observed, the ratio of the nucleus is significantly higher than the intracellular cytoplasm typical stem cells Cell group morphology is shown.

This morphology is consistent with human embryonic stem cells cultured using mouse embryonic fibrous cells as feeder cell layers (Amit et al., 2003).

In particular, human embryonic stem cells cultured by the method of the present invention showed the same result regardless of whether or not mitomycin C was treated with amniotic fluid.

Therefore, it can be seen that the amniotic fluid cells used as the feeder cell layer in the method of the present invention can culture human embryonic stem cells in an undifferentiated state at a level similar to that of the previously used feeder cells.

Example 2 Culture of Human Embryonic Stem Cells by the Method of the Present Invention

Human embryonic stem cells were cultured as follows by using a amniotic fluid culture medium as a culture medium for human embryonic stem cells in the cell culture method of the present invention.

1) Culture of Amniotic Cells and Culture Media Collection

Isolation and culture of amniotic cells were performed by the same method as in Example 1).

After treating mitomycin C to the amniotic fluid prepared as above, it was incubated in a 25 cm ² T flask at a density of 2 X 10 ⁶ / ml. While culturing with DMEM medium containing FBS, the cells were exchanged with fresh medium when the cell density was 70-80% or more. After removing the medium for 24 hours every day for one week, the culture medium (conditioned media) for culturing human embryonic stem cells was prepared by filtration with a sterile filter.

2) Culture of Human Embryonic Stem Cells Using Amniotic Cell Culture

A culture dish pretreated with 0.1% gelatin was prepared and embryonic stem cell SNUhES cell line (Gynecology, Seoul National University College of Medicine) was cultured using the culture solution prepared in 1) above.

The undifferentiated embryonic stem cell group was fractionated to have 100-200 stem cells per cell mass with a fine glass knife, and then subcultured at 7 day intervals, and the results were shown in FIG. 2.

As shown in Figure 2, human embryonic stem cells cultured according to the culture method of the present invention showed the morphological characteristics of undifferentiated cells similar to the cells cultured in Example 1.

This tendency lasted up to the initial 4-5 passages, allowing undifferentiated cells with active cell division.

Therefore, it can be seen that the amniotic fluid cells used as the feeder cell layer in the method of the present invention can culture human embryonic stem cells in an undifferentiated state at a level similar to that of the previously used feeder cells.

Experimental Example Confirmation of Undifferentiated Characteristics and Differentiation Ability of Human Embryonic Stem Cells Cultured by the Method of the Present Invention

(1) karyotyping

In order to analyze the undifferentiated characteristics of human embryonic stem cells cultured by the method of the present invention, karyotyping was performed on the human embryonic stem cells cultured in Example 1 as follows.

The 200 cell groups were physically separated from the amniotic feeder cell layer and collected in a 15 ml conical tube to a total volume of 1 ml using Chang medium, and the colcemid (Colcemid, 10 µg / ml) was 21G. 15 drops were collected with a needle and incubated in a cell incubator for 4 hours.

After a period of time, centrifuged at 1000 rpm for 8 minutes, the precipitate was washed with 1 ml dPBS, centrifuged again, and 200 µl of 0.25% trypsin-EDTA was added to the precipitate, mixed, and left for 3 minutes. 1 ml of medium was added and centrifuged, followed by bathing in 0.075 M KCl stock for 20 minutes.

Centrifugation was performed by adding 0.5 ml of Canoy fixing reagent (Methanol: Acetic acid = 3: 1), followed by primary fixation and secondary fixation. After making the sample slides, the slides were observed by performing Kimsa banding by trypsin and Giemsa (GTG). The chromosomal aberration notation followed the naming convention of the International System for Human Cytogenetic Nomenclature (ISCN), established in 1995, and the results are shown in FIG. 3 (when mitomycin C was treated on the amniotic cells: a, mito on the amniotic cells). If not treated with mycin C: b).

As shown in FIG. 3, human embryonic stem cells cultured by the method of the present invention show normal karyotype regardless of whether or not mitomycin C is treated in the amniotic fluid, having 44 autosomal and XX sex chromosomes. I could confirm it.

(2) immunohistochemical staining

Membrane proteins known as cell surface markers of undifferentiated embryonic stem cells with respect to human embryonic stem cells cultured in Example 1 to analyze the undifferentiated characteristics of human embryonic stem cells cultured by the method of the present invention Activity measurement and immunostaining were performed for the target.

The marker proteins used in this example were alkaline phosphatase, stage specific embryonic antigens SSEA-1, SSEA-3, SSEA-4, and Tra-1-60, Tra-1-81.

The determination of alkaline phosphatase was performed using the alkaline phosphatase detection kit (Sigma, USA) as follows.

After removing the existing culture solution was washed twice by adding PBS (phosphate buffered saline, pH 7.4). Citrate-acetone-formaldehyde solution (citrate-acetone-formaldehyde solution) was treated for 1 minute and fixed, and then washed twice with PBS for 2 minutes. Naphthol AS-BI alkaline solution (Naphthol AS-BI alkaline solution) was treated for 15 minutes. After 15 minutes, washed 3 times with PBS for 2 minutes to observe the color development. All the above process was carried out in a dark room at room temperature.

In addition, immunohistochemical staining was performed on stage specific embryonic antigens SSEA-1, SSEA-3, SSEA-4, and Tra-1-60 and Tra-1-81 as follows.

First, the culture medium was removed, and then fixed by 30% by adding 4% paraformaldehyde to ES cells. After fixation, 3% H ₂ O ₂ was treated for 10 minutes and washed three times with PBS for 5 minutes. Normal goat serum was treated for 1 hour for inhibition of nonspecific reaction.

After 1 hour, the primary antibody for each marker factor was treated for 1 hour and then washed three times with PBS for 3 minutes. The primary antibody against SSEA-1 was MC-480, the primary antibody against SSEA-3 was MC 631, and the primary antibody against SSEA-4 was MC-813-70 antibody. Diluted to a concentration of 100 was used.

The secondary antibody was then treated for 45 minutes. As a secondary antibody, mouse antibody (Mouse Ig) of Vectastaine ABC kit was used.

After the secondary antibody treatment was washed three times with PBS for 3 minutes, ABC (avidin biotin peroxidase complex, Vestor) solution was treated for 30 minutes. After washing three times with PBS again three times, the color reaction was performed for 20 minutes by treatment with the peroxidase substrate kit DAB (Vestor). After the reaction, washing with PBS three times for 3 minutes to observe the presence of color. All of the above was done in a dark room at room temperature.

As described above, the expression of each marker factor was confirmed and the results are shown in FIG. 4 (alkaline phosphatase activity (a), Tra-1-60 (b), when mitomycin C was treated to amniotic fluid) Alkaline phosphatase activity (g) without treatment with mitomycin C in Tra-1-81 (c), SSEA-1 (d), SSEA-3 (e), SSEA-4 (f) and amniotic fluid , Tra-1-60 (h), Tra-1-81 (i), SSEA-1 (j), SSEA-3 (k), SSEA-4 (l))

As shown in FIG. 4, human embryonic stem cells cultured by the method of the present invention were shown to express alkaline phosphatase on the cell surface, regardless of whether or not mitomycin C was treated with amniotic fluid.

In addition, human embryonic stem cells cultured by the method of the present invention was positive for SSEA-4, Tra-1-60, Tra-1-81, SSEA-3 showed a partial positive response.

In contrast, no response to SSEA-1, a mouse embryonic stem cell-specific factor, was observed.

3) Oct-4 expression confirmed

In order to analyze the undifferentiated characteristics of human embryonic stem cells cultured by the method of the present invention, the expression of Oct-4 mRNA was observed for the human embryonic stem cells cultured in Example 1.

First, total RNA was isolated from human embryonic stem cells according to the method of Chomczynski and Sacchi (1987).

Pipette each tissue into a 1.5 ml tube, transfer 2-5 ml of RNA extraction buffer [5 M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0 per gram of sample]. ), 0.5% (w / v) sarkosyl, 2 mM EDTA, 5% (w / v)-mercaptoethanol] was shaken vigorously.

Add 0.5 ml 2M sodium acetate (pH 4.0), 5 ml phenol, 1 ml chloroform: isoamyl alcohol (v: v = 49: 1), shake vigorously for 10 seconds and mix thoroughly for 15 minutes at 4 ° C. It was left. The supernatant obtained by centrifugation at 10,000 × g, 4 ° C. was transferred to a new 50 ml tube and mixed by shaking gently up and down with the addition of 5 ml isopropanol.

After standing at 20 ° C. for at least 1 hour, the nucleic acid precipitate obtained by centrifugation at 10,000 × g, 4 ° C. and removing the supernatant was dissolved in 1.4 ml RNA extraction buffer. After adding 1.4 ml of isopropanol and leaving at least at -20 ° C for at least 1 hour, the nucleic acid precipitate obtained by centrifugation at 10,000 × g and 4 ° C was washed with 75% ethanol and dissolved in distilled water treated with DEPC (diethylpyrocabonate) RT. -PCR (Reverse Transcription-Polymerase Chain Reaction) experiment was used.

1 μg of total RNA isolated from ES cells was used as a template, and reverse transcription was performed at 42 ° C. for 60 minutes using a single strand cDNA synthesis kit (Roche).

After inactivating the reverse transcriptase at 99 ° C. for 5 minutes, cDNA was synthesized using 5 μl of the total reactant as a template in the reaction solution to which the following primers were added in the same tube. In this case, the primers used were β-actin primers (β-actin primer) in the forward direction, SEQ ID NO: 1 (5'-CGCACCACTGGCATTGTCAT-3 '), and reverse in SEQ ID NO: 2 (5'-TTCTCCTTGATGTCACGCAC-3'), Oct- In the case of 4 primers, the base sequence of SEQ ID NO: 3 (5'-GGCGTTCTCTTTGGAAAGGTGTTC-3 ') and the reverse direction of SEQ ID NO: 4 (5'-CTCGAACCACATCCTTCTCT-3') were used.

PCR reaction conditions were denatured at 94 ° C for 40 seconds, polymerized (annealing) at 61 ° C for 40 seconds, and PCR was performed to repeat the reaction for 35 times at 72 ° C for 40 seconds. The PCR product was electrophoresed on a 2% agarose gel to confirm the DNA bands, and the results are shown in FIG. 5 (when treated with mitomycin C in the amniotic cells, beta-actin (1) and Oct-4 (2). Beta-actin (3), Oct-4 (4), beta-actin (5), and amniotic fluid (6), if the myocytes were not treated with mitomycin C.

As shown in FIG. 5, it was confirmed that human embryonic stem cells cultured by the method of the present invention express Oct-4 mRNA, regardless of whether or not mitomycin C was treated with amniotic fluid.

4) Confirmation of telomerase activity

In order to analyze the undifferentiated characteristics of human embryonic stem cells cultured by the method of the present invention, telomerase activity was measured on the human embryonic stem cells cultured in Example 1.

After the cells were collected in a 1.5 ml tube, 200 µl of the digestion buffer included in the Trapeze telomerase detection kit (Chemicon) was added. After allowing the buffer solution and the sample to mix well, it was left at 4 ℃ for 30 minutes. The sample was centrifuged at 12,000 x g and 4 ° C, and the obtained supernatant was transferred to a new tube.

The supernatant thus obtained was used for the telomerase reaction. 10 μl of the supernatant was denatured at 85 ° C. for 10 minutes to prepare an inactivated control.

Reaction conditions were carried out by reacting for 30 minutes at 30 ℃, denatured for 94 ℃, 30 seconds, and repeating the reaction to polymerize for 59 ℃, 30 seconds 33 times. PCR products were electrophoresed on 12.5% non-denatured polyacrylamide gels. After electrophoresis, a dye band (SYBR green I) was added to the polyacrylamide gel to detect protein bands, and the results are shown in FIG. 6 (when mitomycin C was treated to the amniotic cells, experimental group (1) and control group (2). ), If the amniotic cells were not treated with mitomycin C, experimental group (3), control group (3), amniotic cell (6), negative control group (7), positive control group (8).

As shown in Figure 6, human embryonic stem cells cultured by the method of the present invention was able to confirm high telomerase activity, regardless of whether or not the mitomycin C treated amniotic cells.

5) Confirmation of differentiation capacity

In order to confirm whether human embryonic stem cells cultured by the method of the present invention can be properly differentiated under differentiation conditions, differentiation was induced for human embryonic stem cells cultured in Example 1 above.

Human embryonic stem cells cultured in Example 1 were divided into about 500 embryonic stem cell masses and placed in an incubator for 2 hours in an in vitro baby culture dish. The cells were again taken out and transferred to a floating culture dish containing 4-5 ml of embryonic medium and cultured in a cell incubator.

Cultures used DMEM-F12 containing 20% serum replacement, 0.4 ng / ml bFGF, 1% non-essential amino acids, 0.1 mM beta-mercaptoethanol, 0.5% penicillin / streptomycin.

While culturing cells under differentiation-inducing conditions as described above, the formation of embryoid bodies was observed, and the results are shown in FIG. 7 (when mitomycin C was treated to amniotic cells (a), mitomycin C was treated to amniotic cells. (B)).

As shown in Figure 7, human embryonic stem cells cultured by the method of the present invention formed well embryos, regardless of whether or not the mitomycin C treated amniotic cells.

Therefore, it can be seen that human embryonic stem cells cultured by the method of the present invention can be differentiated into various tissues by appropriately adjusting the conditions.

6) Conclusion

In the above, it can be seen that the human embryonic stem cells cultured by the cell culture method of the present invention maintain the cytological, immunological and genetic characteristics of the undifferentiated cells both when inhibiting and not inhibiting the proliferation of amniotic cells. have.

Therefore, the cell culture method of the present invention can stably culture human embryonic stem cells in an undifferentiated state, without treating the drug that inhibits the proliferation of feeder cells.

Human embryonic stem cells cultured by the cell culture method of the present invention retain the cytological, immunological and genetic characteristics of undifferentiated cells.

Human embryonic stem cells cultured by the cell culture method of the present invention form an embryoid body under differentiation conditions, and have the ability to differentiate into various tissues.

In particular, the cell culture method of the present invention is capable of stably culturing human embryonic stem cells in an undifferentiated state regardless of propagation of vegetative cells, thereby reducing the time and cost required for the culturing of stem cells.

Therefore, the cell culture method of the present invention can be usefully used in the field of treatment and research using human embryonic stem cells.

Claims (2)

Culture method of human embryonic stem cells characterized in that the culture medium obtained after culturing the amniotic cells is used as a culture medium of human embryonic stem cells without feeder cells The method of culturing human embryonic stem cells according to claim 1, comprising the steps of: 1) harvesting the culture solution after culturing the amniotic cells, and 2) culturing human embryonic stem cells without the feeder cells with the collected culture medium.

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