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Updated: Jan 20, 2026
Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts
Published on: March 22, 2017
A comparison of strategies for immortalizing mouse embryonic fibroblasts
Melissa M St Amand1,2, John A Hanover2, Joseph Shiloach1
1Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 14-A Room 176, Bethesda, MD 20892, USA.
Abstract:
The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.
Insights
Immortalizing mouse embryonic fibroblasts (MEFs) streamlines research by extending their lifespan and enabling consistent protein lysate generation. Several methods were evaluated, with SV40 T-antigen infection proving most reliable for creating stable, rapidly dividing MEF cell lines.
Area of Science:
- Cell Biology
- Developmental Biology
- Genetics
Background:
- Mouse embryonic fibroblasts (MEFs) are crucial research tools derived from genetically defined mouse models.
- Primary MEFs have limited lifespans and are time-consuming to generate, hindering extensive experimentation.
- Immortalization of MEFs offers a solution to overcome these limitations, facilitating larger-scale studies.
Purpose of the Study:
- To evaluate and compare different strategies for immortalizing mouse embryonic fibroblasts (MEFs).
- To identify optimal methods for generating stable, long-lived MEF cell lines for research applications.
- To provide recommendations for researchers seeking to immortalize MEFs.
Main Methods:
- Cultivation of primary MEFs under reduced oxygen (3% O2) to assess spontaneous immortalization.
- Supplementation of culture media with ROCK inhibitor Y-27632 to enhance proliferation and survival.
- Immortalization of MEFs using Simian virus 40 Large T-antigen (SV40 T) infection.
- Characterization of immortalized MEF lines, including proliferation rates and viability.
Main Results:
- Prolonged culturing at 3% O2 allowed some MEF lines to reach over 40 passages, but spontaneous immortalization success varied.
- Supplementing with 5 µM ROCK inhibitor Y-27632 aided proliferation and spontaneous immortalization in MEFs nearing their Hayflick limit.
- SV40 T-antigen infection reliably generated immortalized MEF lines with a median doubling time of 25 ± 9 hours and >90% viability.
- Different immortalization strategies yielded MEF lines with distinct characteristics, pros, and cons.
Conclusions:
- MEF immortalization significantly reduces culture maintenance and increases experimental throughput.
- Both reduced oxygen culturing and ROCK inhibitor supplementation can promote MEF immortalization, though with variable success.
- SV40 T-antigen infection provides a robust and efficient method for generating high-quality immortalized MEF cell lines.
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