MASS CULTIVATION OF HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS WITH MICROCARRIER
MASS CULTIVATION OF HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS WITH MICROCARRIER
复制标题
DOI：
10.1159/000310319
复制
发表时间：
1992-01-01
期刊：
影响因子：
2.6
通讯作者：
HONDA, Y
中科院分区：
文献类型：
--
作者：
KURIYAMA, S;NAKANO, T;HONDA, Y
微载体细胞培养允许大量培养锚定依赖细胞。本研究以Cytodex 3(R) （Pharmacia）为微载体，对人视网膜色素上皮细胞（RPE）进行了大规模培养。从流产胎儿中建立人RPE细胞，并在Dulbecco's modified Eagle's Medium （DMEM）中培养。第3 -5代传代后，将RPE细胞悬浮于50ml DMEM中，浓度为2 × 10(5)/ml，在旋转瓶中加入Cytodex 3，浓度为10mg /ml。在磁力搅拌器上以20-50 rpm（最终转速）保持培养物，加入DMEM至100 ml。每2天倒出50毫升DMEM并更换新鲜DMEM。1周后，细胞密度为10(6)/ml DMEM。相差显微镜显示微载体之间形成桥接，表明细胞粘附紧密。与传统的单层培养技术相比，微载体细胞培养具有多种优点，包括更大的细胞产量，使用更少的培养基和更少的污染风险，并且它还允许在不使用蛋白酶的情况下传代。使用这种培养系统，可以预期新的细胞培养物更广泛应用的可能性更大。
Microcarrier cell culture permits mass cultivation of anchorage-dependent cells. In this study, mass cultivation of human retinal pigment epithelial (RPE) cells was studied using Cytodex 3(R) (Pharmacia) as a microcarrier. Human RPE cells were established from aborted fetuses and cultured in Dulbecco's modified Eagle's Medium (DMEM). After the 3rd-5th passages, RPE cells were suspended in 50 ml of DMEM in a spinner flask at a density of 2 x 10(5)/ml, and Cytodex 3 was added to the spinner flask at a bead density of 10 mg/ml. Cultures were maintained at 20-50 rpm (final speed) on a magnetic stirrer, and DMEM was added up to 100 ml. Fifty milliliters of DMEM were decanted and replaced with fresh DMEM every 2 days. After 1 week, a cell density of 10(6)/ml DMEM was obtained. Phase contrast microscopy showed bridging formation between microcarriers, which suggests tight cell adhesion. Microcarrier cell culture has a variety of advantages which include greater cell production, use of less medium and less risk of contamination compared to the conventional monolayer culture technique, and it also allows passaging without using proteases. Using this culture system, greater possibilities for wider application of new cell cultures can be expected.