Biological properties of C57BL/6 mouse embryonic fibroblasts and preparation of feeder layers

doi:10.3969/j.issn.2095-4344.2014.11.016

Abstract

Abstract:

BACKGROUND: Kunming mouse embryonic fibroblasts are the most common feeder layers at present, and there are rare reports addressing C57BL/6 mouse embryonic fibroblasts as feeder layers.

OBJECTIVE: To separate and culture C57BL/6 mouse embryonic fibroblasts in vitro, and produce feeder layers to enlarge the resources of mouse embryonic fibroblasts.

METHODS: C57BL/6 mouse embryonic fibroblasts were isolated and cultured by trypsin digestion method in vitro. The biological characteristics and growth rule of the fibroblasts were investigated, then the feeder layers for the cell culture were produced. The growth of cell colonies on the prepared feeder layer was tested.

RESULTS AND CONCLUSION: C57BL/6 mouse embryonic fibroblasts grew well with a large amount, by trypsin digestion method at different concentrations. There was no significance in the survival rate after cryopreservation for 1 week, 2 weeks, 1 month, 3 months and 6 months. The cells were proliferative from the second to fifth passage and declined sharply after the sixth passage. The planted mouse embryonic fibroblasts feeder layers had a high activity within 3 days, but got a sharp decline after 4 days. So it is best to use C57BL/6 mouse embryonic fibroblast feeder layers within 3 days after they’re inactivated. C57BL/6 mouse embryonic fibroblast feeder layer can support embryonic stem cells and induce pluripotent stem cells to grow as Kunming mouse embryonic fibroblasts.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: mice, embryonic development, fibroblasts, cell proliferation, trophoblasts, stem cells

CLC Number:

R318

Li Ying, Gong Ya-fei, Chen Xin-jie. Biological properties of C57BL/6 mouse embryonic fibroblasts and preparation of feeder layers[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(11): 1737-.

Figures/Tables 5

References

[1] Biswas A,Hutchins R.Embryonic stem cells.Stem Cells Dev.2007; 16(2):213-222.

[2] Takahashi K,Tanabe K, Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by definedfactors. Cell.2007;131(5):861-872.

[3] Maherali N, Hochedlinger K.et al.Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells.Cell Stem Cell.2008;3(6):595-605.

[4] Tavernier G, Mlody B, Demeester J,et al. Current methods for inducing pluripotency in somatic cells. Adv Mater.2013;25(20): 2765-2771.

[5] Balasubramanian S,Jasty S,Sitalakshmi G,et al.Influence of feeder layer on the expression of stem cell markers incultured limbal corneal epithelial cells.Indian J MedRes.2008;128(5): 616-622.

[6] Page RL, Ambady S, Holmes WF,et al.Induction of stem cell gene expression in adult human fibroblasts withouttransgenes. Cloning Stem Cells.2009;11(3):417-426.

[7] Camarasa M,Brison D,Kimber SJ,et al.Naturally immortalised mouse embryonic fibroblast lines support human embryonic stem cell growth. Cloning Stem Cells. 2009;11(3):453-462.

[8] Xue F,Ma Y,Chen YE,et a1.Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells. Cell Reprogram.2012;14(4):364-376．

[9] Gepstein L.Derivation and potential applications of human embryonic stem cells.Circulation Res.2002;91 (10): 866-876.

[10] Thomson JA, Itskovitz-Eldor J, Shapiro SS,et al.Embryonic stem cell lines derived from human blastocysts.Science. 1998;282(5391):1145-1147.

[11] Reubinoff BE,Pera MF,Fong CY,et al. Embryonic stem cell lines from human blastocysts:somatic differentiation invitro. Nat Biotechnol.2000;18(4):399-404.

[12] Cowan CA, Klimanskaya I, McMahon J, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med.2004;350(13):353-356.

[13] Chen H,Qian K,Hu J,et al.The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores. Hum Reprod.2005;20(8)2201-2206.

[14] Lin S,Talbot P.Methods for culturing mouse and human embryonic stem cells. Methods Mol Biol.2011;690:31-56.

[15] 黄治军,吕中华,郑鹏,等.昆明小白鼠胚胎干细胞分离培养的影响因素[J].黑龙江畜牧兽医.2010;52(1):16-18.

[16] 林真,沈晓丽,王孟丽,等.昆明小鼠胚胎成纤维细胞培养条件的研究[J].临床和实验医学杂志,2010,9(3):161-163.

[17] Lim J W, Bodnar A.Proteome 1analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics. 2002;2(9):1187-203.

[18] Li Z,Leung M,Hopper R,et al.Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds. Biomaterials.2010;31(3):404-412.

[19] Awan A,Oliveri RS, Jensen PL,et al.Immunoflourescence and mRNA analysis of human embryonic stem cells(hESCs) grown under feeder-free conditions.Methods Mol Biol.2010; 584(4): 195-210.

[20] Braam SR,Denning C,Mummery CL．Genetic manipulation of human embryonic stem cells in serum and feeder-free media．Methods Mol Biol.2010;584(42):413-423．

[21] Hernandez D,Ruban L,Mason C.Feeder-free culture of human embryonic stem cells for scalable expansion in a reproducible manner.Stem Cells Dev.2011;20(6):1089-1098.

[22] 方驰华,胡海北,郝建志,等.ICR小鼠胚胎成纤维细胞的分离培养及饲养层制备[J].中国组织工程研究与临床康复,2010,14 (23): 4299-4302.

[23] 胡嘉波,麻全慧,胡三强,等.人胚胎干细胞饲养层的制备及生物学活性[J].中国组织工程研究与临床康复, 2011,15(23): 4233-4236.

[24] Zeng C, Pan F, Jones LA, et al. Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system. Brain Res. 2010;1319:21-32.

[25] 韩建群,修瑞娟.两种细胞增殖分析方法的比较性研究[J].山东医药,2012,52(12):70-72.

[26] [26]Buck SB,Bradford J,Gee KR,et al. Detection of S-phase cell cycle progression using 5-ethynyl-2′-deoxyuridine incorporation with click chemistry,an alternative to using 5-bromo-2′-deoxyuridine antibodies. Biotechniques.2008; 44(7):927-929.

[27] ]Warren M,Puskarczyk K,Chapman SC. Chick embryo proliferation studies using EdU labeling. Dev Dyn. 2009;238 (4):944-949.

[28] 刘雷,扬立业.EdU在检测细胞增殖中的应用[J].医学综述, 2010,16(19):2901-2904.

[29] Khademhosseini A, Ferreira L, Blumling J 3rd, et al.Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates. Biomaterials.2006;27(36):5968-5977.

[30] 腊晓琳,田海清,蔡霞.高效制备昆明鼠胚胎成纤维细胞饲养层[J].中国组织工程研究与临床康复,2011,15(27):5002-5006.

[31] 王志强,梁锐,陈明清,等.胰蛋白酶消化法和组织块法原代培养包皮成纤维细胞的比较[J].生命科学研究,2012,16(3):242-247.

[32] 张万里,李伟,林学科,等.制作胚胎干细胞饲养层细胞条件的优化[J].中国组织工程研究与临床康复,2010,14(36):6768-6771．