Purification of Mouse Embryonic Fibroblasts (MEFs)

Abstract

Mouse embryonic fibroblasts (MEFs) are primary fibroblasts purified from mouse embryos at a defined time post‐fertilization. MEFs have versatile applications, including use as feeder cell layers or sources of untransformed primary cells for a variety of biological assays. MEFs are most commonly isolated between embryonic day (E)12.5 and E13.5 but can be isolated from embryos as early as E8.5 and as late as E15.5. The individual embryos are harvested by carefully removing uterine tissue, yolk sac, and placenta. The embryos are euthanized, and non‐mesenchymal tissues, such as the fetal liver and heart, are removed before tissue homogenization. The remaining fetal tissue is homogenized by mechanical mincing using a sterile blade, followed by enzymatic digestion and resuspension. During tissue dissociation, the duration of trypsin‐EDTA/DNase digestion and enzyme concentration are critical parameters to produce high‐quality MEFs with the highest rates of cell viability and proliferation potential. MEFs can be cryopreserved at passage (P) 0 if >80% confluent, passaged for further expansion before freezing down, or directly utilized for downstream applications, i.e., preparation as feeder cell layers. Primary MEFs possess a limited proliferation capacity of ∼20 cell divisions, beyond which the percentage of senescent cells rapidly increases; thus, cultures should only be expanded/passaged to a maximum of P5. Critical for cell viability during cryopreservation and thawing of MEFs is the slow decrease in temperature when freezing, the rapid increase when thawing, the use of a cryoprotective agent, and an optimal cell density. While it is critical to generate high‐quality MEFs to standardize and optimize preparation procedures and utilize fresh reagents, some variability in proliferation capacity and cell viability between MEF preparations remains. Thus, MEF preparation, culture, and cryopreservation procedures are continuously being optimized. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Purification, passaging, and expansion of MEFsSupporting Protocol: Cryopreservation and thawing of MEFs

Culture and expansion of murine proximal airway basal stem cells

“…However, the mouse ABSCs could not expand in this culture system, which suggested that the CR culture method might not be directly applied to the culture of mouse ABSCs, and it needs to be further improved and optimized. NIH/3T3, MEFs, and RbEFs are representative feeder cells [ 17 , 27 – 29 ]. Therefore, 3T3-CM, MEF-CM, and RbEF-CM were subsequently used for further exploration.…”

“…NIH/3T3 mouse embryonic fibroblasts were purchased from the Guangzhou Jennio Biotech Co., Ltd. and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 9% newborn calf serum (NBCS; Gibco). Primary mouse embryonic fibroblasts (MEFs) were isolated from CF-1 mice at 12.5 days of gestation, and primary rabbit embryonic fibroblasts (RbEFs) were isolated from 20 to 22 days pregnant New Zealand rabbits according to the previously described method [ 17 ]. MEFs and RbEFs were cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin.…”