Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner

Takamichi Miyazaki,Takehisa Isobe,Norio Nakatsuji,Hirofumi Suemori

doi:10.1038/srep41165

Abstract

We describe highly effective adhesion culture of human pluripotent stem cells (hPSCs) using laminin fragments without precoating. Culture substrates have been generally thought to exert a cell adhesion effect when they are precoated onto culture vessels. However, simple addition of laminin fragments to a cell suspension during passaging accelerated the adhesion of single dissociated hPSCs onto culture vessels that were not precoated with any culture substrate. Interestingly, similar to conventional precoating, the uncoated addition of laminin fragments supported robust adhesion of single hPSCs and maximum adhesion at a much lower concentration compared with precoating. Similar to precoating laminin fragments, hPSCs seeded with uncoated laminin fragments grew well without cell detachment and maintained pluripotency after continuous subculture. We tested other culture substrates, including full-length laminin and vitronectin, to support hPSC adhesion in the uncoated manner, but only laminin fragments had the potential for application in the uncoated manner. This cost-effective and time-efficient method may contribute to expansion of culture of hPSCs and accelerate the development of regenerative medicine using hPSCs.

Highlights

Their use is a time-consuming and laborious process, because preparation of a culture surface using these substrates requires precoating for several hours before seeding cells
Adhesion efficiency of human pluripotent stem cells (hPSCs) cultured in the uncoated manner
We demonstrated undifferentiated culture of hPSCs with iMatrix-511 in an uncoated manner that omits the precoating process of the culture substrate required in advance of passaging

Summary

Introduction

Their use is a time-consuming and laborious process, because preparation of a culture surface using these substrates requires precoating for several hours before seeding cells. We attempted to further improve the culture of hPSCs using the laminin fragment iMatrix-511, and found that addition of iMatrix-511 to the cell suspension was adequate during subculture, thereby omitting the procedure of precoating onto culture vessels. This finding was unexpected because hours of precoating culture substrates have been considered to be indispensable for adsorption onto culture vessels. By applying uncoated iMatrix-511, the expansion of hPSCs may be more efficient and less costly, time consuming, and labour intensive

Methods

Results

Conclusion