Isolation and culture of mouse newborn skin fibroblast cells in a simple and inexpensive method

Authors
Alireza Kheirollah
Mostafa Chashmpoosh
Asma Mohammadi
Neda Abdovis
Hamide Ghasempoor
10.29252/jmj.14.3.35
Abstract
Background  and  objective

Cell culture is one of the main techniques used in scientific researches that done in three ways: Primary culture, secondary culture or cell passage and purchase Cell Lines of Cell banks. Fibroblast cell is an important component of connective tissue that is found almost everywhere in the body. Fibroblast cells based on their location have functional and morphology roles. The aim of this project was simplification through innovation and localization, and to obtain mouse newborn skin fibroblast cells in order to use these cells for further study.

Methods

For this project were used from mouse neonatal that is one to two days. Initially all devices, including surgical materials, culture dishes, buffer and etc were sterilized by autoclaving at 121 ° C and intense pressure. As well as workplace were sterilized by 70% alcohol and UV. Initially mouse neonatal under the biology hood and in 70% alcohol were killed and were sterilized with sterile instruments and then skin fibroblast cells were isolated from these mice. In addition skin fibroblast cells for further growth were transferred of the primary environment to a fresh and new medium and first passages was performed on these cells.

Results

Fibroblast cells in different days were evaluated in terms of growth and morphology by fluorescent microscope (Olympus) with a magnification of 10, that this cells in terms of morphology were appropriate.

Conclusion

According to the results of this study, the simplicity of this method, no need to high costs and sterile conditions without the use of antibiotics and anti-fungal, makes reasonable use of this method for isolation and cell culture in different studies.



Keywords: Cell culture, Fibroblast cells, Mouse newborn
Keywords
Cell culture
Fibroblast Cells
Mouse newborn
1. Chang HY, Chi J-T, Dudoit S, Bondre C, van de Rijn M, Botstein D, et al. Diversity, topographic differentiation, and positional memory in human fibroblasts. Proceedings of the National Academy of Sciences. 2002;99(20):12877-82. 2. Borna company. Layers of skin[Internet]. [available date25/1/2016];[one page]. Available from: http://www.borna-ray.com/JA933101.html. 3. Rittié L, Fisher GJ. Isolation and culture of skin fibroblasts.Methods in molecular medicine. 2005;117:83. 4. Xi X, McMillan DH, Lehmann GM, Sime PJ, Libby RT, Huxlin KR, et al. Ocular fibroblast diversity: implications for inflammation and ocular wound healing. Investigative ophthalmology & visual science. 2011;52(7):4859-65. 5. Kuro-o M. Klotho in health and disease. Current opinion in nephrology and hypertension. 2012;21(4):362-8. 6. Stenmark KR, Frid MG, Yeager M, Li M, Riddle S, McKinsey T, et al. Targeting the adventitial microenvironment in pulmonary hypertension: A potential approach to therapy that considers epigenetic change. Pulmonary circulation. 2012;2(1):3. 7. Tacke F, Weiskirchen R. Update on hepatic stellate cells: pathogenic role in liver fibrosis and novel isolation techniques. 2012. 8. Zeisberg EM, Kalluri R. Origins of cardiac fibroblasts. Circulation research. 2010;107(11):1304-12. 9. Golpour M, Niaki HA, Khorasani HR, Hajian A, Mehrasa R, Mostafazadeh A. Human Fibroblast Switches to Anaerobic Metabolic Pathway in Response to Serum Starvation: A Mimic of Warburg Effect. International journal of molecular and cellular medicine. 2014;3(2):74. 10. Tripathi M, Billet S, Bhowmick NA. Understanding the role of stromal fibroblasts in cancer progression. Cell adhesion & migration. 2012;6(3):231-5. 11. Philp A, Macdonald AL, Watt PW. Lactate–a signal coordinating cell and systemic function. Journal of Experimental Biology. 2005;208(24):4561-75. 12. Wesselschmidt, R. L. Mouse Embryonic Feeder Cell Protocol: Mitotic Inactivation of MEFs by Mitomycin C.2009. Available at: http://www.thermo.com/ 13. Pekkanen-Mattila M, Ojala M, Kerkelä E, Rajala K, Skottman H, Aalto-Setälä K. The Effect of Human and Mouse Fibroblast Feeder Cells on Cardiac Differentiation of Human Pluripotent Stem Cells.Stem Cells International. 2012;2012. 14. Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hamp A, Dvorak P. Comparative study of mouse and human feeder cells for human embryonic stem cells. International Journal of Developmental Biology. 2008;52(4):353. 15. Ryan JA. Introduction to Animal Cell Culture. Corning Incorporated, Life Sciences, Chelmsford St. 2008. 16. Seluanov A, Vaidya A, Gorbunova V. Establishing primary adult fibroblast cultures from rodents. Journal of Visualized Experiments.2010(44). 17. Jozefczuk J, Drews K, Adjaye J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. Journal of visualized experiments: JoVE. 2012(64). 18. Xu J. Preparation, culture, and immortalization of mouse embryonic fibroblasts. Current protocols in molecular biology. 2005 May:28-1. 19. Los M, Mozoluk M, Ferrari D, Stepczynska A, Stroh C, Renz A, et al. Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling. Molecular biology of the cell. 2002;13(3):978-88.