Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.
Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.
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DOI：
10.1016/j.actbio.2015.04.013
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发表时间：
2015-08
影响因子：
9.7
通讯作者：
Bryant SJ
中科院分区：
文献类型：
--
作者：
Amer LD;Holtzinger A;Keller G;Mahoney MJ;Bryant SJ
本研究旨在开发一种用于人胚胎干细胞（hESC）衍生的胰腺祖细胞聚集体的三维培养平台，该平台能够长期培养，保持聚集体的大小和形态，不会对分化产生不利影响，并提供聚集体回收的方法。开发了一个平台，其中含有I型胶原蛋白的聚（乙二醇）水凝胶用于细胞-基质相互作用，肽交联剂用于聚集体的容易回收。该平台首先用RIN-m5 F细胞进行了证明，显示了单细胞和聚集体的包封和随后的释放，而不会对活力产生不利影响。将有效直径为82（15）μm的hESC衍生的胰腺祖细胞的聚集体包封在水凝胶中或在悬浮液中培养28天。在第14天，聚集体活力在水凝胶中得以维持，但在悬浮培养物中显著降低（88%）。然而，到第28天，两种培养条件下的活力均降低。聚集体尺寸在水凝胶中得以维持，但在悬浮液中到第28天显著更高（约2倍）。释放聚集体，然后进行第二次酶处理以获得单细胞的能力使得能够通过流式细胞术进行评估。在包封之前，有39%的Pdx 1 +/Nkx6.1+细胞，这是β细胞成熟所需的关键内分泌标志物。双阳性细胞的分数在水凝胶中不受影响，但在悬浮培养28天时轻微且显著降低。总之，我们证明，MMP-敏感的PEG水凝胶含有胶原蛋白I型是一个有前途的平台，人胚胎干细胞衍生的胰腺祖细胞，保持可行的聚集体，聚集体的大小，和祖细胞的状态，并提供容易恢复的聚集体。
This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15) μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced in both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (~2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1+/Nkx6.1+ cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.