Nair Rekha, Ngangan Alyssa V, McDevitt Todd C
The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
J Biomater Sci Polym Ed. 2008;19(6):801-19. doi: 10.1163/156856208784522056.
The ability of embryonic stem cells (ESCs) to differentiate into all somatic cell types makes them an attractive cell source for regenerative medicine and tissue-engineering applications. In addition to their potential to restore cellularity of injured or diseased tissues, molecular factors produced by stem cells may also directly influence tissue morphogenesis, thereby providing therapeutic benefit independent of stem cell differentiation. In order to examine this hypothesis, it is necessary to separate the cells from the molecular factors they are capable of producing. One potential method of separation is to acellularize clusters of differentiating ESCs, referred to as embryoid bodies (EBs), from the extracellular matrix they synthesize. Thus, the objective of this study was to examine the effectiveness of different reagents, including peracetic acid, sodium dodecyl sulfate, Triton X-100 and DNase, to acellularize EBs. The efficiency of acellularization was assessed based on cell viability and retention of overall mass, DNA and protein, as well as histological examination of the resulting acellular matrix. Initial studies suggested that sequential treatments of Triton X-100 and DNase successfully yielded a cohesive acellular product that retained protein content and significantly reduced levels of DNA. Additional optimization studies were performed with combinations of Triton X-100 and DNase to assess the specific effects of reagent concentration, treatment duration and solvent volume/EB ratios. These results establish methods to effectively obtain novel acellular matrices from differentiating ESCs that may contain morphogenic cues and have potential applications in regenerative medicine.
胚胎干细胞（ESC）分化为所有体细胞类型的能力，使其成为再生医学和组织工程应用中极具吸引力的细胞来源。除了具有恢复受损或患病组织细胞数量的潜力外，干细胞产生的分子因子也可能直接影响组织形态发生，从而提供独立于干细胞分化的治疗益处。为了验证这一假设，有必要将细胞与其能够产生的分子因子分离。一种潜在的分离方法是从分化的胚胎干细胞簇（称为胚状体，EB）合成的细胞外基质中去除细胞。因此，本研究的目的是检验包括过氧乙酸、十二烷基硫酸钠、Triton X-100和脱氧核糖核酸酶在内的不同试剂使EB脱细胞的有效性。基于细胞活力、总体质量、DNA和蛋白质的保留情况以及对所得脱细胞基质的组织学检查来评估脱细胞效率。初步研究表明，依次用Triton X-100和脱氧核糖核酸酶处理成功产生了一种粘性脱细胞产物，该产物保留了蛋白质含量并显著降低了DNA水平。使用Triton X-100和脱氧核糖核酸酶的组合进行了额外的优化研究，以评估试剂浓度、处理持续时间和溶剂体积/EB比例的具体影响。这些结果建立了从分化的胚胎干细胞中有效获得新型脱细胞基质的方法，这些基质可能含有形态发生线索，并在再生医学中具有潜在应用。
