WO2013008960A1 - Milieu de culture de cellules souches de liquide amniotique humain et procédé de culture de ces cellules souches à l'aide de ce milieu - Google Patents

Milieu de culture de cellules souches de liquide amniotique humain et procédé de culture de ces cellules souches à l'aide de ce milieu Download PDF

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WO2013008960A1
WO2013008960A1 PCT/KR2011/005040 KR2011005040W WO2013008960A1 WO 2013008960 A1 WO2013008960 A1 WO 2013008960A1 KR 2011005040 W KR2011005040 W KR 2011005040W WO 2013008960 A1 WO2013008960 A1 WO 2013008960A1
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cells
stem cells
medium
serum
amniotic fluid
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PCT/KR2011/005040
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Korean (ko)
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손윤희
유지
서장수
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경북대학교병원
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    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0056—Xeno-free medium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly

Definitions

  • the present invention relates to a human amniotic stem cell culture medium and a stem cell culture method using the same.
  • 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.
  • the scientists carry out subculture while confirming the density of the amniotic cell population.
  • 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.
  • mitomycin C is used for this purpose.
  • fetal bovine serum FBS
  • penicillin Penicillin
  • streptomycin Y-DMEM
  • DMEM / F12 medium DMEM / F12 medium
  • Stem cells are cells that have the ability of self-proliferation to differentiate into various cells through suitable environment and stimulation.
  • Embryonic stem 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.
  • 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.
  • 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.
  • 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.
  • MSCS mesenchymal stem cells
  • 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).
  • 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.
  • FBS Fetal bovine serum
  • DMEM Umbilical Cord Matrix
  • UCM cells Umbilical Cord Matrix Dulbecco's Modified Eagle's Medium
  • 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.
  • Korean Patent Registration No. 10-1037002 is disclosed as a method of culturing and proliferating chondrocytes for transplantation.
  • DMEM medium or ABM medium containing fetal bovine serum (FBS) has been consistently used in the culture proliferation of hematopoietic stem cells or chondrocytes.
  • FBS fetal bovine serum
  • HS human serum
  • 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).
  • FBS fetal bovine serum
  • HS human serum
  • 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.
  • AFSC amniotic fluid stem cell
  • the amniotic stem cells 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • 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.
  • a culture medium should be prepared based on body fluids, and serum (serum) should be added.
  • serum serum
  • 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.
  • 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.
  • 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, ⁇ -MEM ( ⁇ -Mineral Essential Medium). Gibco, Invitrogen, Newyork, G-MEM, Glasgow's Mineral Essential Medium, Iscove's Modified Dulbecco's Medium Vancouver, Canada).
  • serum for culture of stem cells is a supernatant centrifuged from blood of an animal or human.
  • serum contains various inorganic salts, trace elements, polypeptide growth factors and hormones.
  • 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.
  • the mesenchymal stem cell primary medium 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.
  • 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.
  • 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 ⁇ -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.
  • 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 °C.
  • Example 2 Passage culture in serum free medium
  • Example 1 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).
  • control cells were cultured with AmnioMax II complete medium up to passage 2 and with ⁇ -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.
  • ⁇ -MEM medium Gibco, Invitrogen, newyork, USA
  • amniotic stem cells were subcultured in MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada), which is a serum free medium.
  • the cultured amniotic stem cells 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.
  • FACScan flow cytometry
  • CD44 hyaluronan receptor
  • CD73 marker of SH-3 and SH-4
  • CD90 marker of Thy-1
  • CD105 encodedoglin
  • SSEA SSEA in AFSC cells cultured in control and experimental medium -4 (stage-positive specific embryonic arod) showed an expression rate
  • CD45 marker of hematopoietic lineage
  • MHC major histocompatibility antigens
  • HLA-ABC major histocompatibility antigens
  • 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.
  • Example 2 of the present invention 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.
  • Matrigel BD Biosciences, Bedford, Mass., USA
  • differentiated medium 10% horse-serum (Gibco / BRL)
  • serum-free medium may be used in amniotic stem cell culture.
  • 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.
  • 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.
  • AFSC amniotic stem cells

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Abstract

La présente invention concerne la culture de cellules souches de liquide amniotique (AFSC) et, plus particulièrement, la culture d'AFSC caractérisée par la séparation des cellules de liquide amniotique du liquide amniotique par une séparation centrifuge, et par la culture du précipité des cellules de liquide amniotique séparées pendant sept jours dans une culture AmnioMax, une sous-culture étant ensuite effectuée à partir d'une première sous-culture dans un milieu exempt de sérum, les cellules souches de liquide amniotique dans le milieu de culture exempt de sérum ayant une vitesse de croissance supérieure par comparaison avec une sous-culture classique dans un milieu de culture additionné de sérum, et présentant des résultats positifs pour l'expression de SSEA-4 et HLA-ABC, l'expression d'un marqueur de cellules souches et d'un marqueur de myocytes, etc.
PCT/KR2011/005040 2011-07-08 2011-07-08 Milieu de culture de cellules souches de liquide amniotique humain et procédé de culture de ces cellules souches à l'aide de ce milieu WO2013008960A1 (fr)

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Cited By (2)

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CN111759864A (zh) * 2020-07-14 2020-10-13 中国人民解放军总医院 羊水干细胞在制备治疗狼疮肾炎的药物中的应用
US11077145B2 (en) 2015-04-03 2021-08-03 Stemmedicare Co., Ltd. Method for mass producing proteins in mesenchymal stem cells

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KR101975109B1 (ko) 2017-09-19 2019-05-03 경희대학교 산학협력단 스피룰리나를 포함하는 줄기세포 배양 및 분화용 배지 조성물
KR20230127938A (ko) * 2022-02-25 2023-09-01 주식회사 강스템바이오텍 무혈청배지를 이용한 줄기세포 배양 방법

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11077145B2 (en) 2015-04-03 2021-08-03 Stemmedicare Co., Ltd. Method for mass producing proteins in mesenchymal stem cells
CN111759864A (zh) * 2020-07-14 2020-10-13 中国人民解放军总医院 羊水干细胞在制备治疗狼疮肾炎的药物中的应用
CN111759864B (zh) * 2020-07-14 2022-09-13 中国人民解放军总医院 羊水干细胞在制备治疗狼疮肾炎的药物中的应用

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