Isolation and Handling of Mouse Embryonic Fibroblasts

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.