KR20100079571A - Method of external cultivation for cow's egg cell by using cultivation composition derived from undifferentiated human stem cell - Google Patents
Method of external cultivation for cow's egg cell by using cultivation composition derived from undifferentiated human stem cell Download PDFInfo
- Publication number
- KR20100079571A KR20100079571A KR1020080138099A KR20080138099A KR20100079571A KR 20100079571 A KR20100079571 A KR 20100079571A KR 1020080138099 A KR1020080138099 A KR 1020080138099A KR 20080138099 A KR20080138099 A KR 20080138099A KR 20100079571 A KR20100079571 A KR 20100079571A
- Authority
- KR
- South Korea
- Prior art keywords
- culture
- stem cell
- embryonic stem
- vitro
- human embryonic
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
본 발명은 소 난자의 체외 배양 방법에 관한 것으로서, 더욱 상세하게는 소 난자의 체외 배양 환경을 개선하고 배양 효율을 증가시킬 수 있는 소 난자의 체외 배양 방법에 관한 것이다. The present invention relates to a method for in vitro culture of bovine eggs, and more particularly, to a method for in vitro culture of bovine eggs capable of improving the in vitro culture environment of bovine eggs and increasing the culture efficiency.
배아줄기세포는 남성의 생식세포인 정자와 여성의 생식세포인 난자가 결합하여 생성된 수정란에서 유래한다. 그리고 배아줄기세포는 착상 전 배반포기배의 내부 세포괴로부터 획득되며 모든 조직의 세포로 분화할 수 있는 능력을 지녔으나 배양조건만 주어진다면 분화되지 않은 상태로 무한대로 증식이 가능한 아직 분화되지 않은 세포를 말한다. 다시 말해서 특수 분화 배양 환경에서는 원하는 방향으로 세포 분화가 유도되어 신경, 당뇨, 혈액과 같은 질환의 세포 치료제로 이용될 수 있어서 한 개의 배아줄기세포주만으로도 수많은 환자의 치료에 이용될 수 있다고 기대되는 만능세포이다.Embryonic stem cells are derived from fertilized eggs produced by combining sperm, a male germ cell, and an egg, a female germ cell. Embryonic stem cells are obtained from internal lumps of blastocysts before implantation and have the ability to differentiate into cells of all tissues, but given the culture conditions, they have yet to differentiate into undifferentiated cells that can proliferate indefinitely. Say. In other words, pluripotent cells that are expected to be used for the treatment of numerous patients with only one embryonic stem cell line can be used as a cell therapeutic agent for diseases such as nerves, diabetes, and blood in a specific differentiation culture environment. to be.
수정란의 체외 배양은 체내 배양에 비해서 배양효율이 떨어진다. 체외 배양 환 경을 개선 시키기 위해서 배양용 배지의 조성과 알부민 또는 우태아혈청 같은 단백질 첨가제 그리고 항산화제와 성장인자 등과 같은 다양한 물질에 대한 연구가 이루어지고 있다. 그러한 예로써, 일반적으로 배양환경에서 생성되는 활성산소종 (reactive oxygen species; ROS) 감소에 베타 메르캅토에탄올과 비타민 E (Kitagawa 등 2004), 알파토코페롤과 아스코르비산 (Hossein 등 2007), 플라보노이드 (Ohshima H 등 1998; Williams RJ 등 2004) 같은 항산화제 첨가가 유효하게 작용하여 체외 수정란 발달효율이 향상된다고 보고된 바 있다. 우태아 혈청 및 화학적으로 정제된 합성 혈청 대사물과 상피세포 성장인자를 같이 넣은 배양액을 이용하여 소 난자의 배 발달율 증가와 성장관련 유전자 증가 등 긍정적인 영향을 얻었다고 보고한 바 있다. 또한 소 복제수정란을 체외배양 할 때 배양액에 생쥐의 상피세포 성장 인자 (EGF; epidermal growth factor)와 생쥐의 과립세포-대립세포 집락자극인자 (GM-CSF: granulocyte macrophage-colony stimulating factor) 를 첨가 하였을 경우 세포 수 증가 등 발달에 있어서 긍정적인 영향을 주었다고 보고된 바 있다. 이러한 체외 배양 환경 개선에 대한 최종 연구 목적은 최대한 체내 환경과 유사한 환경을 제공함으로써 건강한 배아 즉 정상적인 생명체 발생이 용이한 배양 환경을 만들고자 함에 있다. In vitro culture of fertilized eggs is less effective than culture in vitro. In order to improve the in vitro culture environment, various materials such as the composition of the culture medium, protein additives such as albumin or fetal bovine serum, antioxidants and growth factors have been studied. For example, beta mercaptoethanol and vitamin E (Kitagawa et al. 2004), alphatocopherol and ascorbic acid (Hossein et al. 2007), flavonoids (DL) in reducing the reactive oxygen species (ROS) generally produced in the culture environment. It has been reported that the addition of antioxidants such as Ohshima H et al. 1998; Williams RJ et al. It was reported that fetal calf serum and chemically purified synthetic serum metabolites and epidermal growth factor were used to obtain positive effects such as increased embryonic development and growth-related genes. In addition, epidermal growth factor (EGF) and granulocyte macrophage-colony stimulating factor (GM-CSF) of mice were added to the culture medium when bovine cloned embryos were in vitro cultured. In this case, it has been reported to have a positive effect on the development such as an increase in the number of cells. The purpose of the final study on the improvement of the in vitro culture environment is to create a healthy embryo, that is, a culture environment in which normal life is easily generated by providing an environment similar to the in vivo environment.
본 발명은 전술한 바와 같이, 소 난자의 체외 배양 기술을 개선하고자 이루어진 것으로서, 소 난자의 체외 배양 환경을 개선하여 체외 배양 효율을 증가시킬 수 있는 소 난자의 체외 배양 방법을 제공함을 목적으로 한다. As described above, an object of the present invention is to improve in vitro culture technology of bovine eggs, and an object of the present invention is to provide an in vitro culture method of bovine eggs which can increase the culture efficiency of bovine eggs in vitro.
또한, 본 발명은 소 난자의 체외 배양 시 체외 배양 환경을 개선하고 배양 효율을 증가시킬 수 있는 첨가제를 제공함을 목적으로 한다. In addition, an object of the present invention is to provide an additive that can improve the in vitro culture environment and increase the culture efficiency during in vitro culture of bovine eggs.
본 발명의 일 측면에 따른 소 난자의 체외 배양 방법은 증식 단계의 인간 배아줄기세포 콜로니를 제1 배양액으로 배양하는 줄기세포 배양 단계, 상기 줄기세포 배양 단계로부터 생성된 용액 상의 미분화 인간 배아줄기세포 유래 배양 조성물을 회수하는 단계, 상기 회수된 배양 조성물을 소 난자의 체외 배양 시 체외 배양액으로 사용되는 제2 배양액에 첨가하여 상기 소 난자를 체외 배양하는 단계를 포함한다. In vitro culture method of bovine egg according to an aspect of the present invention is a stem cell culture step of culturing the human embryonic stem cell colony of the proliferation step with a first culture medium, derived from undifferentiated human embryonic stem cells on the solution generated from the stem cell culture step Recovering the culture composition, and adding the recovered culture composition to a second culture medium used as an in vitro culture solution for in vitro culture of bovine eggs, thereby culturing the bovine eggs in vitro.
상기 줄기세포 배양 단계에서는 적어도 90% 이상 증식된 인간 배아 줄기세포를 1 내지 3일 배양할 수 있다. 또한, 상기 미분화 인간 배아줄기세포 유래 배양 조성물은, 제1 배양액 및 상기 줄기세포 배양 단계에서 발생한 상기 인간 배아줄기세포의 대사 산물을 포함한다. 한편, 상기 제1 배양액은 불-필수 아미노산을 포함할 수 있다.In the stem cell culture step, human embryonic stem cells grown at least 90% or more may be cultured for 1 to 3 days. In addition, the undifferentiated human embryonic stem cell-derived culture composition comprises a first culture medium and a metabolite of the human embryonic stem cells generated in the stem cell culture step. On the other hand, the first culture may include non-essential amino acids.
본 발명의 다른 측면에 따른 소 난자 체외 배양용 첨가제는 증식 단계의 인 간 배아줄기세포 콜로니를 제1 배양액으로 배양하는 줄기세포 배양 단계 및 상기 줄기세포 배양 단계로부터 생성된 용액 상의 물질을 회수하는 단계로부터 수득 되고, 제1 배양액 및 상기 줄기세포 배양 단계에서 발생한 상기 인간 배아줄기세포의 대사 산물을 포함한다. The bovine egg in vitro culture additive according to another aspect of the present invention is a stem cell culture step of culturing a human embryonic stem cell colony of the proliferation step with a first culture solution and recovering the material on the solution generated from the stem cell culture step Obtained from the first culture medium and the metabolite of the human embryonic stem cells generated in the stem cell culture step.
본 발명에 따르면, 소 난자의 체외 배양 시 배양 환경을 개선함으로써 배양 효율을 극대화할 수 있다. 나아가 소뿐만 아니라 다른 동물들에도 유사하게 적용이 가능할 것으로 기대하고 있다. According to the present invention, it is possible to maximize the culture efficiency by improving the culture environment during in vitro culture of bovine eggs. Furthermore, it is expected that similar applications can be made not only for cattle but also for other animals.
이하 본 발명을 상세하게 설명하도록 한다. Hereinafter, the present invention will be described in detail.
본 발명에 따르면, 소 난자의 체외 배양 단계에서 소 난자의 체외 배양액에 추가로 미분화 인간 배아줄기세포 유래 배양 조성물을 첨가함으로써 소 난자의 체외 배양 효율을 증가시킬 수 있다. According to the present invention, in vitro culture efficiency of bovine egg may be increased by adding an undifferentiated human embryonic stem cell-derived culture composition to bovine egg in vitro culture.
상기 미분화 인간 배아줄기세포 유래 배양 조성물은 증식 중인 인간 배아줄기세포 콜로니를 줄기세포 배양액에 바람직하게는 1 내지 3일 정도 배양함으로써 수득될 수 있다. 상기 인간 배아줄기세포 유래 배양 조성물은 상기 줄기세포 배양액뿐만 아니라 상기 인간 배아줄기세포의 배양 과정에서 생성되는 대사 산물을 포함한다. 상기 인간 배아줄기세포 콜로니는 증식 중인 인간 배아줄기세포로서, 적어도 90% 이상 증식이 완료된 인간 배아줄기세포 콜로니를 사용하는 것이 바람직하다. 전술한 바와 같이 상기 인간 배아줄기세포 콜로니를 1 내지 3일 배양한 후, 인 간 배아줄기세포 콜로니를 제외한 용액 상의 물질을 회수함으로써 상기 인간 배아줄기세포 유래 배양 조성물을 수득할 수 있다. The undifferentiated human embryonic stem cell-derived culture composition may be obtained by culturing human embryonic stem cell colonies in proliferation in a stem cell culture medium, preferably about 1 to 3 days. The human embryonic stem cell-derived culture composition includes not only the stem cell culture medium but also metabolites generated during the culture of the human embryonic stem cells. The human embryonic stem cell colony is a human embryonic stem cell in proliferation, and it is preferable to use human embryonic stem cell colonies in which at least 90% or more proliferation is completed. As described above, after culturing the human embryonic stem cell colony for 1 to 3 days, the human embryonic stem cell-derived culture composition may be obtained by recovering the material in solution except for the human embryonic stem cell colony.
상기 인간 배아줄기세포 유래 배양 조성물은 소 난자의 체외 배양시 일종의 첨가제로서 기본 배양액에 혼합되어 사용된다. The human embryonic stem cell-derived culture composition is used as a kind of additive in in vitro culture of bovine eggs and mixed with the basic culture solution.
이하, 구체적인 실시예들을 들어 본 발명을 더욱 상세하게 설명하도록 한다. Hereinafter, the present invention will be described in more detail with reference to specific embodiments.
[실시예]EXAMPLE
[실시예 1]Example 1
1. 미분화 인간배아줄기세포배양 유래 조성물의 준비1. Preparation of undifferentiated human embryonic stem cell culture-derived composition
미분화 인간배아줄기세포 유래 배양 조성물은 5% 마트리젤이 코팅된 배양 용기 내에 90% 이상 증식된 인간 배아줄기세포 콜로니를 줄기세포 배양액으로 2일 동안 배양하여 획득하였다. 줄기세포 배양액은 DMEM/F12 (Gibco)에 1% 불-필수 아미노산(non-essential amino acid), 0.1 mM의 β-머캅소에탄올(β-mercaptoethanol), 1%의 페니실린/스트렙토마이신(Pennicilin/streptomycin), 20%의 혈청대체물질(ㄴserum replacement 와 8ng/ml의 파이브로블라스트 성장 인자 II(fibroblast growth factor II) 가 첨가된 것을 사용하였다. Undifferentiated human embryonic stem cell-derived culture composition was obtained by culturing human embryonic stem cell colonies grown more than 90% in 5% Matrigel-coated culture vessel with stem cell culture for 2 days. Stem cell cultures contained 1% non-essential amino acid, 0.1 mM β-mercaptoethanol, and 1% penicillin / streptomycin in DMEM / F12 (Gibco). 20% serum replacement (b Serum replacement and 8ng / ml fibroblast growth factor II) was used.
2. 소 난자의 채취 및 성숙배양2. Collection and maturation of bovine eggs
소 난소의 표면에 육안으로 보이는 3-6 mm크기 다수의 난포로부터 난구세포-난자 복합체 (cumulus-oocyte complexes) 를 18 게이지(gauge) 주사기를 이용하여 채취하였다. 회수된 난구세포-난자 복합체는 1mg/ml 소혈청 알부민이 들어있는 TL- HEPES 버퍼로 3회이상 충분히 세척하고 우태아혈청 10%와 0.2mM 피루브산 나트륨, 1㎍/ml 난포자극 호르몬, 1㎍/ml 에스트라디올, 25㎍/ml 젠타마이신이 들어간 TCM-199 체외성숙용 배양액에 넣어 38.8℃ 온도와 5% 이산화탄소 조건의 배양기내에서 24시간 동안 성숙 배양하였다. Cumulus-oocyte complexes were collected from 3-6 mm sized follicles visible to the surface of bovine ovary using an 18 gauge syringe. The recovered oocyte-oval complexes were washed three or more times with TL-HEPES buffer containing 1 mg / ml bovine serum albumin, and 10% fetal calf serum and 0.2 mM sodium pyruvate, 1 μg / ml follicle stimulating hormone, 1 μg / ml In estradiol, 25μg / ml gentamycin containing TCM-199 in vitro culture medium was matured for 24 hours in an incubator at 38.8 ℃ temperature and 5% carbon dioxide conditions.
3. 소 난자의 단위발생 및 체외 배양3. Unit Generation and In Vitro Culture of Bovine Oocytes
소 난자의 단위발생을 위해 체외 성숙 배양된 난구세포-난자 복합체는 1 mg/ml 의 히알루로니다제를 이용해서 먼저 난구세포를 제거하고 TL-HEPES버퍼로 충분히 세척한 다음 10 μM 칼슘아이오마이신이 들어있는 CR1aa 배양액에서 5분 동안 활성화를 유도하고 2 μM 6-디메틸아미노퓨린(6-dimethylaminopurine)이 들어있는 CR1 aa 배양액에서 3시간 동안 배양하였다. 이때에 사용된 CR1aa 배양액 내에는 3mg/ml 지방산이 제거된 (fatty acid free) 소혈청 알부민이 함유되어 있고 본 실험의 기본 배양액으로 이용되었다. 활성화시킨 난자는 CR1aa 기본 배양액에 넣어 2일간 배양시키고 4-8세포로 분열된 난자를 다시 CR1aa 기본 배양액 (대조군)과 10% 미분화 배아줄기세포 유래 조성물이 들어있는 배양액 (실험군) 으로 나누어 38.8℃ 온도와 5% 이산화탄소 조건의 배양기내에서 6일간 추가 배양하여 체외 배양 8일째 배반포기배까지의 배 발달상태를 비교 조사하였다. In vitro maturation of the oocyte-bearing oocyte complex for the oocyte development of bovine oocytes, the oocytes were first removed using a 1 mg / ml hyaluronidase, washed thoroughly with TL-HEPES buffer, and then 10 μM calcium-iomycin Activation was induced for 5 minutes in the containing CR1aa culture and incubated for 3 hours in CR1 aa culture containing 2 μM 6-dimethylaminopurine. The CR1aa culture used at this time contained 3 mg / ml fatty acid free bovine serum albumin and was used as the basic culture solution of this experiment. Activated eggs were incubated in CR1aa basal culture for 2 days, and the eggs divided into 4-8 cells were divided into CR1aa basal culture (control) and culture solution containing 10% undifferentiated embryonic stem cell-derived composition (experimental group). The embryos were further cultured for 6 days in an incubator at 5% CO2 and examined for the developmental status of blastocysts up to 8 days of in vitro culture.
4. 이중염색4. Double Dyeing
대조군과 실험군의 체외 배양 8일째 발달된 배반포기배의 질적인 차이를 조Qualitative differences between blastocysts developed on day 8 of in vitro culture of control and experimental groups
사하기 위해 영양배엽세포와 내부 세포괴 수를 동시에 검토할 수 있는 이중 염색을 실시하였다. 2중염색은 2단계로 실시되었다. 각 군의 배반포기배는 1% 트리톤-X(triton X-100)와 100㎍/ml 프로피디움 요오드화물이 포함된 TL-HEPES 용액에서 30초간 처리하고 (1단계) 25㎍/ml 비스벤자마이드 (Hoechst 33258)가 포함된 TL-HEPES 용액에 넣어 4℃에서 다음날까지 반응시켰다 (2단계). 염색된 배반포기배는 글리세롤로 세척한 다음 슬라이드 그라스에 올려 놓고 커버슬립으로 덮어 형광 현미경의 blue 필터 (excitation field-350 nm, emission field-461 nm) 하에서 관찰하여 세포 수를 세었다.For this purpose, double staining was performed to examine the number of trophoderm cells and internal cell mass at the same time. Double staining was performed in two stages. Blastocysts of each group were treated for 30 seconds in a TL-HEPES solution containing 1% triton-X and 100 μg / ml propidium iodide (step 1) and 25 μg / ml bisbenzamide (Hoechst 33258) was added to the TL-HEPES solution containing the reaction at 4 ℃ until the next day (step 2). Stained blastocysts were washed with glycerol, placed on slide glass, covered with coverslip, and observed under a blue filter (excitation field-350 nm, emission field-461 nm) under a fluorescence microscope to count the number of cells.
5. 실험 결과5. Experimental Results
미분화 줄기세포 배양 조성물이 소 Undifferentiated stem cell culture composition 단위발생란의Unit generation 발달에 미치는 영향 Development impact
*대조군; CR1aa +FAF-BSA 3mg/ml,Control; CR1aa + FAF-BSA 3 mg / ml,
실험군; CR1aa +FAF-BSA 3mg/ml + 미분화배아줄기세포 조성물 10% Experimental group; CR1aa + FAF-BSA 3mg / ml + 10% undifferentiated embryonic stem cell composition
**p<0.01** p <0.01
도 1은 미분화 배아줄기세포 유래 배양 조성물 첨가 유무에 따른 소 단위발생란의 발달과정을 보여주는 사진이다. 도 1에서, 대조군은 A, C, E 및 G, 실험군은 B, D, F 및 H이며 체외배양 2일 째 (A, B), 체외배양 4일 째 (C, D), 체외배양 6일 째 (E, F), 체외배양 8일 째 (G, H)를 각각 나타내고 있다.Figure 1 is a photograph showing the development of subunit embryos with or without the addition of undifferentiated embryonic stem cell-derived culture composition. In Figure 1, the control group A, C, E and G, the experimental group is B, D, F and H and in vitro culture 2 days (A, B), in vitro culture 4 days (C, D), in vitro culture 6 days The (E, F) and the 8 th day of in vitro culture (G, H) are shown, respectively.
상기 표 1 및 도 1을 참조하면, 체외에서 생산된 소 단위발생란의 발달에 미분화 배아줄기세포 조성물이 미치는 영향을 총 3회에 걸쳐 조사하였던 바 체외 배양 4일, 6일과 8일로 진전됨에 따라 대조군과 실험군 간에 배 발달율이 현저하게 차이가 있음을 알 수 있었다. 본 실험에 사용된 실험군 배양액은 CR1aa 배양액에 미분화배아줄기세포 유래 배양 조성물 10% 가 첨가된 것으로 배양 8일째 배반포기배형성율(도 1의 H 참조)은 대조군 7.3%에 비해 34.1% 로서 유의하게 높았다 (p<0.01).Referring to Table 1 and FIG. 1, the effects of the undifferentiated embryonic stem cell composition on the development of subunit embryos produced in vitro were examined three times. As a result, in vitro culture was progressed to 4 days, 6 days, and 8 days, the control group. The embryo development rate was significantly different between the and experimental groups. In the experimental group culture used in this experiment, 10% of undifferentiated embryonic stem cell-derived culture composition was added to the CR1aa culture, and the blastocyst stage formation rate (see H in FIG. 1) was significantly increased as 34.1% compared to the control group 7.3% at 8 days. High (p <0.01).
도 2는 미분화 배아줄기세포 유래 배양 조성물 첨가 유무에 따른 소 단위발생란의 발달과정을 보여주는 이중 염색 사진이다. 도 2에서, 가)는 대조군 나)는 실험군이다. Figure 2 is a double staining picture showing the development of subunit embryos with or without the addition of undifferentiated embryonic stem cell-derived culture composition. 2, a) is a control group b) is an experimental group.
도 2를 참조하면, 대조군과 실험군의 체외배양 8일째 배반포기배의 질적인 차이를 조사하기 위해 이중염색으로 영양배엽세포수와 내부세포괴수를 조사하였던 바, 이 둘을 합한 총 세포수는 미분화배아줄기세포 유래 배양 조성물 10% 첨가된 실험군이 129.6±36.7로서 대조군 38.3±5.0 에 비해 3.3배 이상 유의하게 많았다 (p<0.01). 특히, 장차 태아로 발생될 내부세포괴 수의 경우 대조군 12.3±6.4 에 비해 44.1±5.7 로서(도 2의 B)) 미분화배아줄기세포 유래 배양 조성물 첨가 실험군에서 3.5배 이상 증가된 유의한 수치를 나타내었다 (p<0.01). 이러한 결과는 미분화배아줄기세포 유래 배양 조성물의 첨가가 영양배엽 세포와 내부세포괴 발생에 유의하게 작용하였음을 나타낸다.Referring to Figure 2, to investigate the qualitative difference between blastocyst blastocyst on day 8 in vitro culture of the control group and the experimental group was examined for the number of vegetative germ cells and internal cell mass by double staining, the total number of the combined cells is undifferentiated The experimental group to which 10% embryonic stem cell-derived culture composition was added was 129.6 ± 36.7, which was 3.3 times higher than the control group 38.3 ± 5.0 (p <0.01). In particular, the number of internal cell masses to be generated in the future fetuses was 44.1 ± 5.7 (Fig. 2B) compared to the control 12.3 ± 6.4 (Fig. 2B) and showed a significant increase of 3.5 times or more in the experimental group added to the undifferentiated embryonic stem cell-derived culture composition. (p <0.01). These results indicate that the addition of undifferentiated embryonic stem cell-derived culture composition acted significantly on the generation of trophoblast and internal cell mass.
이와 같은 결과를 종합해보면, 미분화 인간배아줄기세포 배양 유래 배양 조성물은 배반포기배 중 태아로 발생될 부위의 내부세포괴로부터 무한 증식된 배아줄기세포의 배양 산물로 태아발생에 유용한 성장인자, 싸이토카인, 아미노산 및 미지성분들이 다수 포함되어 있을 것으로 기대된다. 이러한 성분들이 체외에서 생산된 소 단위발생란의 배반포기형성율, 총세포수 및 내부세포괴의 세포수의 발달에 유의한 영향을 미친 것으로 사료된다. Taken together, the undifferentiated human embryonic stem cell culture-derived culture composition is a culture product of embryonic stem cells that have infinitely proliferated from internal cell masses of fetal blastocysts, which are useful for fetal development, cytokines, and amino acids. And many unknown components. These components were thought to have a significant effect on the development of blastocyst formation, total cell number and cell number of internal cell mass produced in vitro.
도 1은 미분화 배아줄기세포 유래 배양 조성물 첨가 유무에 따른 소 단위발생란의 발달과정을 보여주는 사진이다.Figure 1 is a photograph showing the development of subunit embryos with or without the addition of undifferentiated embryonic stem cell-derived culture composition.
도 2는 미분화 배아줄기세포 유래 배양 조성물 첨가 유무에 따른 소 단위발생란의 발달과정을 보여주는 이중 염색 사진이다.Figure 2 is a double staining picture showing the development of subunit embryos with or without the addition of undifferentiated embryonic stem cell-derived culture composition.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080138099A KR101010077B1 (en) | 2008-12-31 | 2008-12-31 | Method of external cultivation for cow's egg cell by using cultivation composition derived from undifferentiated human stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080138099A KR101010077B1 (en) | 2008-12-31 | 2008-12-31 | Method of external cultivation for cow's egg cell by using cultivation composition derived from undifferentiated human stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20100079571A true KR20100079571A (en) | 2010-07-08 |
| KR101010077B1 KR101010077B1 (en) | 2011-01-24 |
Family
ID=42640648
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020080138099A KR101010077B1 (en) | 2008-12-31 | 2008-12-31 | Method of external cultivation for cow's egg cell by using cultivation composition derived from undifferentiated human stem cell |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101010077B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020060052A1 (en) * | 2018-09-19 | 2020-03-26 | 메디칸 주식회사 | Self-culturing method and apparatus for autologous stem cell |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101446328B1 (en) * | 2013-11-27 | 2014-10-07 | 주식회사 미래셀바이오 | Composition for culture of embryo comprising mesenchymal stem cells medium and method for preparing the same composition |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100715188B1 (en) | 2005-12-08 | 2007-05-08 | 서울우유협동조합 | Medium composition for in vitro culture of bovine embryo and culture method using the same medium |
-
2008
- 2008-12-31 KR KR1020080138099A patent/KR101010077B1/en active IP Right Grant
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020060052A1 (en) * | 2018-09-19 | 2020-03-26 | 메디칸 주식회사 | Self-culturing method and apparatus for autologous stem cell |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101010077B1 (en) | 2011-01-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jin et al. | A novel two-step strategy for in vitro culture of early-stage ovarian follicles in the mouse | |
| EP1948786B1 (en) | Multipotent stem cells derived from human adipose tissue and cellular therapeutic agents comprising the same | |
| JP6918782B2 (en) | Culture medium | |
| JP7138359B2 (en) | In vitro maturation medium for immature oocytes and its application | |
| BRPI0706801A2 (en) | method of purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses | |
| KR20070104735A (en) | Culture media and methods for culturing mesenchymal stem cell | |
| CN104988110A (en) | Method for transforming umbilical cord mesenchymal stem cells into islet cells | |
| JP4202121B2 (en) | Mammalian gamete and embryo culture supplements and uses thereof | |
| US11840706B2 (en) | Composition and method for generating a desired cell type and/or tissue type from hair follicular stem cells | |
| KR20050084889A (en) | Composition for culturing multipotent stem cells and utilization of the same | |
| KR101010077B1 (en) | Method of external cultivation for cow's egg cell by using cultivation composition derived from undifferentiated human stem cell | |
| CN101225373A (en) | Bovine oocyte in vitro maturation serum-free medium | |
| CN111944745A (en) | Serum-free bovine oocyte in-vitro maturation culture solution and oocyte culture method | |
| CN104988111A (en) | Inducing liquid for converting UC-MSC into islet cells and application thereof | |
| CN106148269A (en) | A kind of mammalian cell culture and additive thereof | |
| KR102142400B1 (en) | Medium composition for culturing porcine pluripotent stem cell | |
| CN106754660B (en) | Method for in vitro differentiation of hair follicle stem cells into oocytes | |
| CN108823152A (en) | A kind of the In-vitro maturation liquid and in-vitro maturation culture method of oocyte of mouse | |
| JP5164439B2 (en) | Hair papilla cell culture method | |
| CN114410573A (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN113005076A (en) | Immature oocyte in-vitro culture solution and application thereof | |
| KR101590615B1 (en) | Medium composition for in vitro culture of porcine spematogonial stem cells and culture method using the same medium | |
| CN105039239A (en) | Cell transformation induction liquid and use thereof | |
| CN114369567B (en) | Method for constructing bovine expanded pluripotent embryonic stem cells and culture solution | |
| WO2012034352A1 (en) | Method for reverse differentiation of human somatic cells to produce autologous stem cells, kit and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| A302 | Request for accelerated examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20131224 Year of fee payment: 4 |
|
| FPAY | Annual fee payment |
Payment date: 20141224 Year of fee payment: 5 |
|
| FPAY | Annual fee payment |
Payment date: 20160114 Year of fee payment: 6 |
|
| FPAY | Annual fee payment |
Payment date: 20161227 Year of fee payment: 7 |
|
| FPAY | Annual fee payment |
Payment date: 20180115 Year of fee payment: 8 |
|
| FPAY | Annual fee payment |
Payment date: 20190115 Year of fee payment: 9 |
|
| FPAY | Annual fee payment |
Payment date: 20200114 Year of fee payment: 10 |