Article

Biomedical Microdevices

, Volume 12, Issue 6, pp 1097-1105

Enhanced effects of secreted soluble factor preserve better pluripotent state of embryonic stem cell culture in a membrane-based compartmentalized micro-bioreactor

  • Mohammad Mahfuz ChowdhuryAffiliated withInstitute of Industrial Science, The University of Tokyo Email author 
  • , Takeshi KatsudaAffiliated withInstitute of Industrial Science, The University of Tokyo
  • , Kevin MontagneAffiliated withInstitute of Industrial Science, The University of TokyoLIMMS/CNRS-IIS, The University of Tokyo
  • , Hiroshi KimuraAffiliated withInstitute of Industrial Science, The University of Tokyo
  • , Nobuhiko KojimaAffiliated withInstitute of Industrial Science, The University of Tokyo
  • , Hidenori AkutsuAffiliated withDepartment of Reproductive Biology, National Research Institute for Child Health and Development
  • , Takahiro OchiyaAffiliated withSection for Studies on Metastasis, National Cancer Center Research Institute
  • , Teruo FujiiAffiliated withInstitute of Industrial Science, The University of TokyoLIMMS/CNRS-IIS, The University of Tokyo
  • , Yasuyuki SakaiAffiliated withInstitute of Industrial Science, The University of TokyoLIMMS/CNRS-IIS, The University of Tokyo

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Abstract

Pluripotent stem cells are under the influence of soluble factors in a diffusion dominant in vivo microenvironment. In order to investigate the effects of secreted soluble factors on embryonic stem cell (ESC) behavior in a diffusion dominant microenvironment, we cultured mouse ESCs (mESCs) in a membrane-based two-chambered micro-bioreactor (MB). To avoid disturbing the cellular environment in the top chamber of the MB, only the culture medium of the bottom chamber was exchanged. Cell growth in the MB after 5 days of culture was similar to that in conventional 6-well plate (6-WP) and membrane-based Transwell insert (TW) cultures, indicating adequate nutrient supply in the MB. However, the cells retained higher expression of pluripotency markers (Oct4, Sox2 and Rex1) and secreted soluble factors (FGF4 and BMP4) in the MB. Inhibition of FGF4 activity in the MB and TW resulted in a similar cellular response. However, in contrast to the TW, inhibition of BMP4 activity revealed that autocrine action of the upregulated BMP4, which acted cooperatively with leukemia inhibitory factor (LIF), upregulated the pluripotency markers expression in the MB culture. We propose that BMP4 accumulated in the diffusion dominant microenvironment of the MB upregulated its own expression by a positive feedback mechanism—in contrast to the macro-scale culture systems—thereby enhancing the pluripotency of mESCs. The micro-scale culture platform developed in this study enables the investigation of the effects of soluble factors on ESCs in a diffusion dominant microenvironment, and is expected to be used to modulate the ESC fate choices.

Keywords

Embryonic stem cell Soluble factors Diffusion Microenvironment Micro-bioreactor