혈청이 마우스 근육세포 증식에 미치는 효과
Effect of Serum Concentration on Growth of Mouse Skeletal Muscle Cells In Vitro
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이 연구의 목적은 근육 세포의 증식 배양을 위해 필요한 핵심 인자인 혈청이 혈청 대체물의 첨가에 의해 대체될 수 있는지를 분자 생물학적측면에서 검증하는 것이다. 혈청 대체물이 첨가된 low serum (5% FBS) 기반의 promo cell 배지는 성장 배양액으로 사용되었고, 마우스 하지 골격근의근육세포는 pre-plating (pp) 방법에 의해 분리되었다. Pre-plating 4의 세포는 작고 둥근 형태의 굴절성을 가진 Myoblast/satellite like cells의 형태가관찰되었으며, 근육 줄기세포 전사인자들(Pax3/7, Myf5, Myod1)과 골격근 발달 전사인자(Myog)의 발현량이 섬유아세포와 비교하여 높게 나타났다. 따라서 그들을 Myoblast derived cells로 명명하고, 기본 세포주로 사용하였다. 분리된 MDCs는 5%, 10% 혹은 20% 혈청이 첨가된 배양액에서2주간 배양되었다. 배양 6일째부터 대조군(5% 혈청)과 비교하여 20% 혈청은 세포 수가 증가하였으며, 양적 혈청 농도 의존성이 확인되었다. 증식및 세포사멸 관련 유전자들은 대조군과 비교하였다. 배양 1주 차에 20% FBS군은 세포증식 촉진 유전자인 Myc 발현이 증가한 반면 pro-apoptosis 유전자인 Bax의 발현량이 감소했고, 2주 차에는 세포주기정지 인자인Cdkn1a의 감소와 Myc의 지속적인 증가와 Bax/Bcl의 감소가 나타났다. 각기다른 FBS 처리농도에서 배양된 Myoblast derived cells을 동일한 Myotube유도 배양액에서 2주간 분화하였다. 대조군과 비교하여 20% FBS군은Myod1, Myog, Myf6, Myh1의 유의적 증가가 1주부터 확인되었다. 결론적으로 혈청 대체 물질은 근육세포 배양액에서 혈청의 효과를 완벽히모사할 수 없음이 증명되었고, 따라서 체외에서 상업적 목적의 근육 세포 대량 증식 및 분화를 위해서는 적절한 혈청 대체물 개발이 선행돼야 할것으로 사료된다.
Abstract
This study was performed to examine whether serum used for growth of skeletal muscle cells in vitro can be replaced by serum substitutes or not. Briefly, It was used commercial Promo cell growth medium with serum replacements (5% FBS) as a basal growth medium (GM). Mouse skeletal muscle cells were isolated from hind limb by Pre-plating (pp) technology. The isolated cells at pp 4 stage showed small and round morphology with refractive looks like myoblast/satellite like cells, and showed increase of Pax3/7, Myf5, Myod1 and Myog expression compared to fibroblasts. The cells were then named as myoblast derived cells (MDCs), and were used for the further experiments. MDCs were cultured into GM supplemented with 5, 10 or 20% FBS for 2 weeks. The total cells number were calculated after every 2 days using a hemocytometer. The number of cells increased significantly in 20% FBS cultured cells as compared to control (≥6 days) and was positively correlated with FBS concentration. Proliferation and apoptosis related genes were analyzed by real time PCR. It was shown the upregulation of Myc expression and downregulation of Bax by 20% FBS treatment at 1 week. Furthermore, there was reduction of Cdkn1a/ Bax/Bcl2 and increase of Myc expression at 2 weeks in 20% FBS cultured cells compared to control. The MDCs were differentiated with Promocell differentiation media (serum free) for 2 weeks, where 20% FBS compared to control showed significantly increased in expression of Myod1/Myog/Myf6/ Myh1 from 1 to 2 weeks. In conclusion, serum substitutes could not fully mimic the properties of the serum in the muscle cell culture. Therefore, for mass production of muscle cells for commercial purposes, it is necessary to develop the suitable serum to overcome cost and the ethical implications of extracting FBS.
참고문헌 (25)
- Bentzinger CF, "Building muscle : Molecular regulation of myogenesis", Cold Spring Harb. Perspect. Biol, 4, pp.a008342~ (2012)
- Bjare U, "Serum-free cell culture", Pharmacol. Ther, 53, pp.355~374 (1992)
- Cosgrove BD, "A home away from home : challenges and opportunities in engineering in vitro muscle satellite cell niches", Differentiation, 78, pp.185~194 (2009)
- Ding S, "Maintaining bovine satellite cells stemness through p38 pathway", Sci. Rep, 8, pp.10808~ (2018)
- Fang CY, "Long-term growth comparison studies of FBS and FBS alternatives in six head and neck cell lines", PLoS ONE, 12, pp.e0178960~ (2017)
- Gstraunthaler G, "A plea to reduce or replace fetal bovine serum in cell culture media", Cytotechnology, 65, pp.791~793 (2013)
- Hildyard JC, "Identification and validation of quantitative PCR reference genes suitable for normalizing expression in normal and dystrophic cell culture models of myogenesis", PLoS Curr., 6 (2014)[DOI]
- Hildyard JCW, "Determination of qPCR Reference Genes Suitable for Normalizing Gene Expression in a Canine Model of Duchenne Muscular Dystrophy", J. Neuromuscul. Dis, 5, pp.177~191 (2018)
- Kolkmann AM, "Serum-free media for the growth of primary bovine myoblasts", Cytotechnology, 72, pp.111~120 (2020)
- Laitinen A, "A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells", Cytotechnology, 68, pp.891~906 (2016)
- Lavasani M, "Isolation of muscle-derived stem", progenitor cells based on adhesion characteristics to collagen-coated surfaces, 2013, pp.976, 53~65
- Lawson MA, "Differentiation of myoblasts in serum-free media : Effects of modified media are cell line-specific", Cells Tissues Organs, 167, pp.130~137 (2000)
- Lindroos B, "Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro", Cytotherapy, 11, pp.958~972 (2009)
- Oster SK, "The myc oncogene : MarvelouslY Complex", Adv. Cancer Res, 84, pp.81~154 (2002)
- Pagani FD, "Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans : Histological analysis of cell survival and differentiation", J. Am. Coll. Cardiol, 41, pp.879~888 (2003)
- Pavyde E, "Skeletal muscle-derived stem", progenitor cells : A potential strategy for the treatment of acute kidney injury, 2016, pp.2016, 9618480~
- Saini A, "Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification", PLoS ONE, 13, pp.e0192384~ (2018)
- Schlosser I, "Dissection of transcriptional programmes in response to serum and c-Myc in a human B-cell line", Oncogene, 24, pp.520~524 (2005)
- Smaldone MC, "Muscle derived stem cell therapy for stress urinary incontinence", World J. Urol, 26, pp.327~332 (2008)
- Smith CK, "Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells", J. Cell Physiol, 159, pp.379~385 (1994)
- Stern-Straeter J, "Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis", J. Tissue Eng. Regen. Med, 5, pp.e197~206 (2011)
- Takahashi K, "Induction of pluripotent stem cells from adult human fibroblasts by defined factors", Cell, 131, pp.861~872 (2007)
- Tedesco FS, "Repairing skeletal muscle : Regenerative potential of skeletal muscle stem cells", J. Clin. Investig, 120, pp.11~19 (2010)
- Varlakhanova NV, "Myc maintains embryonic stem cell pluripotency and self-renewal", Differentiation, 80, pp.9~19 (2010)
- Wu X, "Muscle-derived stem cells : Isolation, characterization, differentiation, and application in cell and gene therapy", Cell Tissue Res, 340, pp.549~567 (2010)