Embryoid Body-Explant Outgrowth Cultivation from Induced Pluripotent Stem Cells in an Automated Closed Platform

Abstract

Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system "P 4C S" for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 10(7). There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells.

Figure 1

Histology of teratoma generated by Edom-iPS#S23 cells. (a) Teratoma (low-power view). (b) Epidermis. (c) Immature neuroepithelium. (d) Intestinal epithelium. (e) Intestinal epithelium with goblet and Paneth cells (high-power view of panel (d)). (f) Smooth muscle.

Figure 2

Time-course images of teratoma-derived cells in automated culture. (a) Scheme for automated culture of teratoma-derived cells. ((b), (c), (d)) Phase-contrast photomicrography of day 1 (b), day 4 (c), and day 7 (d) after the start of the automated culture. The experiments were repeated four independent times. The scale bars indicate 500 μm.

Figure 3

Time-course images of keratinocytes in automated culture. (a) Scheme for automated culture of keratinocytes. ((b), (c), (d)) Phase-contrast photomicrography of day 1 (b), day 4 (c), and day 6 (d) after the start of the automated culture. The experiments were repeated three independent times. The scale bars indicate 500 μm.

Figure 4

Time-course images of iPSC-derived EB-explant outgrowth cells in automated culture. (a) Scheme for automated culture of EB-explant outgrowth cells. ((b), (c), (d), (e)) Phase-contrast photomicrography of day 1 (b), day 4 (c), day 8 (d), and day 23 (e) after the start of the automated culture. The EBs adhered to the culture surface (on day 1) and the cell outgrowth was clearly confirmed surrounding the EBs (on day 4), after which we performed the subculture manipulation (on day 7). The uniform cell distribution after passage could be confirmed by an image on day 8. The cells were cultured until reaching subconfluent state (on day 23). The experiments were repeated three independent times. The scale bars indicate 500 μm.