Quantitative Efficiency Comparison of Mouse Embryonic Fibroblast Isolation Methods

سال انتشار: 1397
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 346

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شناسه ملی سند علمی:

NSCMRMED03_242

تاریخ نمایه سازی: 30 دی 1397

چکیده مقاله:

Background and Aim: Fibroblasts are spindle-shaped cells that derivedfrom mesenchymal stem cells. Because of their ability to producenutrients and growth factors such as LIF and b-FGF, these cells are used asa feeder layer for maintaining stem cell pluripotency and self-renewal. Inthis study, we compared the cell yield of two enzymatic and mechanicalmethods for mouse embryonic fibroblast isolation (MEF).Methods: All animal experiments of this study were approved by the ethicscommunity of the university. A pregnant BALB/c mouse was sacrificedand the embryos were isolated following uterine horn removing. Afterremoval of head and red organs, the embryos were divided into twogroups of equal numbers (n=5). The first embryo group was minced byrazor, incubated with trypsin 0.05% for 10 min and then centrifugedat 12 000 g for 5 minutes. Finally, the pellet was re-suspended in MEFmedium, and transferred to T25 culture flask. The second embryos groupwas minced and passed 10times through a syringe with needle 22G. Theobtained cells were washed by PBS, and then cultured as above. After24 hours, the cultured cells were trypsinized and counted using Neobarlam.Results: Viscous and clumpy materials were observed in the enzymaticmethod, but not in the mechanical method. In comparison to theenzymatic method, the mechanical method using needle 22G yieldeda significantly higher number of cells. The obtained cell number after24hours culture were 5×104 and 1.5×106 cells/flask for enzymatic andmechanical methods, respectively.Conclusion: The mechanical method provided higher numbers of cellsthan the enzymatic method. Probably, MEFs are trapped in the viscousmaterial of the mechanical method, due to nucleic acid-triggeredclotting, making it difficult to isolate these cells.

نویسندگان

Vahide Hosseini

Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Ashkan Kalantary-Charvadeh

Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Mahdi Totunchi

Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology a

Amir Mahdizadeh

Endocrine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran