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Updated: Apr 26, 2026
Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Published on: June 21, 2012
Isolation and handling of mouse embryonic fibroblasts
Kanika Jain1, Paul J Verma, Jun Liu
1Stem Cells and Reprogramming Group, Biological Engineering Laboratories, Faculty of Engineering, Monash University, Clayton, VIC, 3800, Australia.
Abstract:
Primary mouse embryonic fibroblasts (MEFs) are the most commonly used feeder layers that help to support growth and maintain pluripotency of embryonic stem cells (ESC) in long-term culture. Feeders provide substrates/nutrients that are essential to maintain pluripotency and prevent spontaneous differentiation of ESC. Since embryonic fibroblasts stop dividing after a few passages, care must be taken to isolate them freshly. Here, we provide a protocol to derive MEFs and describe the method to inactivate the cells using mitomycin C treatment. The protocol also describes freezing, thawing, and passaging of MEFs. This basic protocol works well in our laboratory. However, it can be modified and adapted according to any user's particular requirement.
Insights
This study details a protocol for deriving and preparing mouse embryonic fibroblasts (MEFs) for use as feeder layers. These inactivated MEFs support embryonic stem cell (ESC) growth and pluripotency in culture.
Area of Science:
- Cell Biology
- Developmental Biology
- Stem Cell Research
Background:
- Primary mouse embryonic fibroblasts (MEFs) are crucial feeder layers for maintaining embryonic stem cell (ESC) pluripotency and growth in vitro.
- MEFs require careful handling due to limited proliferative capacity, necessitating fresh isolation or proper inactivation and storage.
- Existing methods for MEF preparation can be resource-intensive or require specific expertise.
Purpose of the Study:
- To provide a standardized and reproducible protocol for deriving and inactivating primary mouse embryonic fibroblasts (MEFs).
- To outline methods for the cryopreservation, thawing, and passaging of MEFs for long-term use.
- To ensure a reliable supply of feeder cells essential for consistent embryonic stem cell culture.
Main Methods:
- Isolation of primary mouse embryonic fibroblasts (MEFs) from mouse embryos.
- Inactivation of MEFs using mitomycin C treatment to prevent proliferation.
- Detailed procedures for freezing, thawing, and passaging MEFs for subsequent use as feeder layers.
Main Results:
- Successful derivation and inactivation of MEFs using the described protocol.
- Demonstrated viability and effectiveness of cryopreserved MEFs in supporting ESC culture.
- The protocol is adaptable for specific laboratory requirements and modifications.
Conclusions:
- The presented protocol offers a reliable method for preparing mouse embryonic fibroblasts (MEFs) for embryonic stem cell (ESC) culture.
- Properly inactivated and stored MEFs are essential for maintaining ESC pluripotency and preventing differentiation.
- This protocol serves as a valuable resource for researchers working with ESCs and feeder layer technology.
