Chen Chen,Liu Xin,Liu Ge,et al.Comparison of ceiling culture versus insert culture for dedifferentiated fat cells in rats[J].Journal of Clinical Pediatric Surgery,,22():72-78.[doi:10.3760/cma.j.cn101785-202001032-014]
Comparison of ceiling culture versus insert culture for dedifferentiated fat cells in rats
- Abstract:
- ObjectiveTo explore the differences between ceiling culture and insert culture for dedifferentiated fat (DFAT) cells in rats to provide research rationales for primary culture of DFAT cells.MethodsMature fat cells were isolated from rat inguinal adipose pad of eight 4-6-week-old Sprague-Dawley (SD) rats and DFAT cells harvested in vitro by dedifferentiation culture using ceiling and insert cultures.Cell morphology,time and amount of complete culture medium required from isolation to first passage were compared.Purity and function of differentiation into smooth muscle cells of DFAT cells were compared by flow cytometry analysis and cell immunofluorescence.ResultsDFAT cells obtained by two methods both were fibroblast-like,spindle-shaped and peak-valley-like.The time required for DFAT cells cultured by ceiling culture and insert culture to grow to the first passage were (16.00±1.41) and (16.25±0.96) days respectively and the difference was not significant (P>0.05).The surface antigen phenotypes of DFAT cells cultured by two methods were positive for CD29,CD90 and negative for CD31,CD45;flow cytometric results of DFAT cells cultured by ceiling culture showed CD29 was expressed in (97.63±3.15)% cells,CD90(98.63±0.83)%,CD31(0.11±0.12)%,CD45(0.18±0.11)%;the results of DFAT cells cultured by insert culture showed CD29 was expressed in (96.93±3.61)% cells,CD90 (98.65±0.84)%,CD31 (0.16±0.13)%,CD45 (0.16±0.10)%.And the differences were not significant (P>0.05).DFAT cells cultured by ceiling and insert cultures could differentiate into smooth muscle cells and express αSMA and SMMHC after induction in vitro.The rates of αSMA positive cells were (73.00±7.88)% and (73.56±8.44)% respectively and the difference was not significant (P>0.05).Ceiling culture for DFAT cell growth to the first passage required more complete medium than insert culture[93.75(90.00,93.75) vs 48.50(48.50,48.50) ml]and the difference was significant (P<0.05).ConclusionNo significant differences exist in time expense,cell morphology,purity or function of induction differentiation into smooth muscle cells in vitro of DFAT cells cultured by ceiling and insert culture.However,from the perspective of economic expense,insert culture offers some advantage.
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Memo
收稿日期:2020-01-14。
基金项目:国家自然科学基金(81701531)
通讯作者:杨屹,Email:yangy2@sj-hospital.com