Characterization of a unique technique for culturing primary adult human epithelial progenitor/"stem cells".
Characterization of a unique technique for culturing primary adult human epithelial progenitor/"stem cells".
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DOI：
10.1186/1471-5945-12-8
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发表时间：
2012-06-24
期刊：
影响因子：
--
通讯作者：
Feinberg SE
中科院分区：
文献类型：
--
作者：
Marcelo CL;Peramo A;Ambati A;Feinberg SE
来源于表皮、口腔粘膜和尿道的原代角质形成细胞用于构建基于细胞的伤口愈合装置和再生医学。这项研究提出了一种体外技术，该技术通过在每24小时更换一次的大量无血清、无必需脂肪酸、低钙培养基下生长单层来快速扩增培养中的角质形成细胞。通过胰蛋白酶消化组织，从表皮皮肤、口腔粘膜和输尿管产生原代细胞培养物。使用具有生长因子的Epilife培养基在高培养基体积下培养细胞。一旦密集汇合，角质形成细胞单层在覆盖培养基中产生悬浮细胞，其可以每24小时收获一次。在7 - 10天的时间内。将细胞悬液（约8 X 105个细胞/ml）倒入新烧瓶中，在2 - 4天内形成另一个融合单层。这种新的培养物又产生额外的细胞悬浮液，当连续传代时，在2 - 3个月内扩增细胞株，而不使用酶来分裂培养物。分析称为上皮弹出角质形成细胞（ePUK）的细胞悬浮液的培养扩增、细胞大小和葡萄糖利用、与载体珠的附着、微球体形成、角质形成细胞分化的诱导，并通过免疫组织化学表征。ePUK在培养物中大大扩增，附着于载体珠粒，不形成微球体，在24小时内使用约50%的培养基葡萄糖，含有更大部分的较小直径细胞（8 - 10微米），当返回到常规饲养时间表（48小时，和15ml/T-75烧瓶），并且可以通过加入1.2mM培养基钙或必需脂肪酸来分化。ePUK细胞被鉴定为循环（表达Ki67）基底细胞（表达p63、K14）。使用这种原代培养技术，可以产生大量的上皮细胞，而不使用胰蛋白酶来分裂培养物。这些细胞直径小，具有基底细胞祖细胞/"干"（P/SC）细胞特征，这些特征是通过每天喂食大于正常培养基体积诱导的。ePUK上皮细胞具有用于再生医学和上皮P/SC表型的基础研究的潜力。
Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that rapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty acid free, low calcium medium that is replaced every 24 hrs. Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of tissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely confluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested every 24 hrs. over a 7–10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask to form another confluent monolayer over 2–4 days. This new culture, in turn produced additional cell suspensions that when serially passed expand the cell strain over 2–3 months, without the use of enzymes to split the cultures. The cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size and glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte differentiation, and characterized by immunohistochemistry. The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used approximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells (8–10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and 15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The ePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing). Using this primary culture technique, large quantities of epithelial cells can be generated without the use of the enzyme trypsin to split the cultures. The cells are small in diameter and have basal cell progenitor/”stem” (P/SC) cell characteristics induced by daily feeding with larger than normal medium volumes. The ePUK epithelial cells have the potential to be used in regenerative medicine and for basic studies of epithelia P/SC phenotype.
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影响因子：
7.6
作者：
Izumi, K;Terashi, H;Feinberg, SE
通讯作者：
Feinberg, SE
DOI：
10.1073/pnas.82.16.5390
发表时间：
1985-01-01
影响因子：
11.1
作者：
BARRANDON, Y;GREEN, H
通讯作者：
GREEN, H
影响因子：
64.5
作者：
MEHREL, T;HOHL, D;ROOP, DR
通讯作者：
ROOP, DR
DOI：
10.1152/ajprenal.90733.2008
发表时间：
2009-09-01
影响因子：
4.2
作者：
Tseng-Rogenski, Stephanie;Liebert, Monica
通讯作者：
Liebert, Monica
DOI：
10.1073/pnas.1102454108
发表时间：
2011-05-10
影响因子：
11.1
作者：
Chaffer, Christine L.;Brueckmann, Ines;Weinberg, Robert A.
通讯作者：
Weinberg, Robert A.