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Growth of primary embryo cells in a microculture system
Max Villa1, Sara Pope, Joanne Conover
1Department of Mechanical Engineering, University of Connecticut, Storrs, CT 06269, USA.
Abstract:
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 microm/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.
Insights
Optimal microfluidic perfusion rates support mouse embryonic cell growth. A 10 nL/min flow rate enhances primary mouse embryonic fibroblast (mEF) proliferation, maintaining cell viability and morphology comparable to traditional methods.
Area of Science:
- Cell Biology
- Biotechnology
- Microfluidics
Background:
- Microfluidic systems offer controlled environments for cell culture.
- Optimizing perfusion is key to balancing nutrient supply and waste removal.
- Primary cells, like mouse embryonic fibroblasts (mEFs), have specific culture requirements.
Purpose of the Study:
- To determine optimal perfusion conditions for culturing primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) in a microfluidic system.
- To investigate the role of perfusion rate and seeding density on primary cell proliferation.
- To compare cell morphology and viability in microfluidic culture versus traditional petri dish culture.
Main Methods:
- Utilized a microfluidic perfusion culture system.
- Identified optimal perfusion rate (10 nL/min) and flow velocity (0.55 microm/s) for mEFs.
- Cultured mEFs and mESCs for one week.
- Assessed cell proliferation and protein expression using fluorescence immunoassays.
Main Results:
- Optimal perfusion rate for mEFs identified as 10 nL/min with 0.55 microm/s flow velocity.
- Primary mEFs showed higher dependence on secreted factors than 3T3 fibroblasts.
- Seeding density and perfusion rate critically impact primary cell proliferation.
- Microfluidic culture maintained mEF and mESC morphology and viability similar to petri dish cultures.
- Perfusion-based microculture supported the high proliferation of pluripotent mESCs.
Conclusions:
- Established optimal perfusion conditions for mEF and mESC microfluidic culture.
- Demonstrated the efficacy of microfluidic perfusion for maintaining cell viability and proliferation.
- Highlighted the potential of microfluidic systems for stem cell research and culture.
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