背景:用于人间充质干细胞/基质细胞(hMSC)的传统大规模培养系统使用固体微载体作为附着基质。尽管由于高的表面与体积比,使用这种基底是有利的,从相同的底物中收获细胞是一个挑战,因为它需要酶处理,经常与激动相结合。这里,我们研究了用于扩增和非酶回收hMSCs的两相系统。将全氟化碳液滴分散在富含蛋白质的生长培养基中,并用作hMSC培养的临时液体微载体。
结果:hMSCs成功附着在这些液体微载体上,与在固体上培养的形态相似。在第9天实现3.03±0.98(hMSC1)和3.81±0.29(hMSC2)的倍数增加。然而,在第4天记录最大扩张倍数(4.79±0.47(hMSC1)和4.856±0.7(hMSC2))。这种减少是由由于两相之间的界面收缩而在达到汇合时的细胞聚集引起的。细胞质量,通过差异化评估,细胞表面标记表达和克隆能力,在液体微载体上膨胀后保留。细胞收获通过两个步骤非酶促实现:首先通过诱导液滴聚结,然后抽吸界面。即使在诱导液滴聚结后,hMSC的质量特征仍继续保留。
结论:一种临时微载体的前景是非常令人兴奋的,该载体可用于扩增细胞,然后在不使用蛋白水解酶的情况下\'消失\'以进行细胞释放。这里,我们已经证明,hMSCs可以附着并增殖在这些全氟化碳液体微载体上,非常重要的是,保持其质量。
BACKGROUND: Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid microcarriers for hMSC culture.
RESULTS: hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence.
CONCLUSIONS: The prospect of a temporary microcarrier that can be used to expand cells and then \'disappear\' for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality.