Growth of primary embryo cells in a microculture system
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We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 μm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.
KeywordsEmbryonic stem cells Embryonic fibroblasts Perfusion microculture system
This work was supported by the State of Connecticut under the Connecticut Stem Cell Research Initiative (Grant 06SCA05). M. Villa and S. Pope thank the UConn-Wesleyan Stem Cell Core for embryonic stem cell culture training.
- M.F. Brown, T.P. Gratton, J.A. Stuart, Metabolic rate does not scale with body mass in cultured mammalian cells. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R2115–R2121 (2007)Google Scholar
- C.P. Ng, M.A. Swartz, Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model. Am. J. Physiol. Heart Circ. Physiol. 284(5), 1171–1777 (2003)Google Scholar
- R.L. Panton, Incompressible flow (Wiley, New York, 1996)Google Scholar
- K. Schuster-Gossler et al., Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. Biotechniques. 31(5), 1022–1026 (2001)Google Scholar
- Y. Takagi et al., Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model. J. Clin. Invest. 115(1), 102–109 (2005)Google Scholar