全神经球贴壁培养法体外长期扩增视网膜前体细胞

Long-term Expansion of Retinal Progenitor Cells in Vitro by Culturing Intact Neurospheres in Monolayer

目的:探寻一种既高效又能连续体外扩增视网膜前体细胞(RetinalProgenitorCell,RPC)的培养方法。方法:小鼠胚胎视网膜细胞采取神经球贴壁培养或直接贴壁培养法。神经球贴壁培养时,先经悬浮培养生成富含前体细胞的神经球,然后将低速离心分离出的神经球接种到Poly-D-Lysine包被的培养瓶,在前体细胞培养液中(含FGF-2、EGF和LIF)贴壁培养,头24h在培养液中添加10%的胎牛血清。直接贴壁培养法是将细胞悬液直接接种到预先包被Poly-D-Lysine的培养瓶。免疫组化和FACS分析神经球贴壁培养细胞的干细胞特性和分化能力,并与直接贴壁培养法得到的细胞进行比较。结果:神经球接种到预先包被Poly-D-Lysine的培养瓶后贴附到培养瓶底,逐渐形成贴壁生长的单层细胞。该法能够在体外长期扩增视网膜前体细胞,3个月内细胞能够传代10次以上。在RA诱导分化时,能够生成成熟视网膜细胞,表达视网膜细胞特异性标记。FACS分析和免疫组化染色显示神经球贴壁培养所得细胞在未分化时Nestin阳性率(92%)细胞率以及分化为成熟视网膜细胞的比率(53.7%),均高于直接贴壁培养法获得的细胞。结论:神经球贴壁培养法能够在体外长期大量扩增视网膜前体细胞,能较好地保持干细胞特性,并容易分化为成熟的视网膜细胞,较直接贴壁培养具有较大的优越性。

Purpose： To develop an efficient culture system for expansion of retinal progenitor cells for long-term in vitro. Methods： Retinal cells from embryonic mice were cultured as neurospheres-adhesive or as simple monolayer culture. For neurospheres-adhesive culture, spheres collected by low centrifugation from floating cultures were re-seeded onto Poly-D-Lysine coated flasks containing retinal progenitor cells（RPC） medium plus 10% FBS for the first 24 hours, then cultured in RPC medium （including FGF、EGF and LIF）. For simple monolayer culture, on the other hand, cell suspension were directly plated on culture flasks pre-coated with Poly-D-Lysine. Proliferation and differentiation capability of the RPC from these two culture systems were analyzed by immunocytochemistry and FACS analysis. Results： Intact neurospheres-adhesive culture system was successful in expansion of RPC which maintain in uncommitted state. Immunochemistry staining and FACS analyzing demonstrated that the rate of nestin positive cells in uncommitted state （92%） and mature retinal neuron upon differential（53.7% ） were higher than their counterparts in simple-adhesive cultures. Conclusion ： In this study we acquired a stable expansion of RPC which preferentially differentiate into mature retinal neurons.