An Improved Method for Highly Efficient Isolation of Primary Mouse Embryonic Fibroblasts

Mouse embryonic fibroblasts (MEFs) play a vital role in stem cell research where they function as feeder cells to maintain mouse and human embryonic stem cells (ESC) or pluripotent stem cells (iPSC) in an undifferentiated state [1,2]. Successful culturing of either stem cell type depends on the quality of the feeder cells, since they secrete the extracellular matrix that directly affects the growth of ESCs and iPSCs. In addition, MEFs can be converted to a pluripotent state or directly transdifferentiated into a variety of more complex mature cell types such as functional neurons and cardiomyocytes which, in turn, are used in studies of development, neurological and cardiac disease modeling, drug discovery, and regenerative medicine [3,4,5,6]. MEFs isolated from different genetically altered mouse models can be used to study growth control and the DNA damage response [7,8,9].

In this article, we utilize an alternative procedure for gently dissociating fibroblasts from embryonic mouse tissue. The simple and reliable procedure significantly improves the yield and overall cell health and is applicable to a variety of mouse strains, including pathogen-free mice and genetically modified disease mice. Isolated MEF cells were monitored through four population doublings, and displayed morphologic and growth properties equivalent to fresh cells.

Result and discussion

We evaluated a panel of tissue-digestion enzymes and protocols to determine the enzyme or enzyme combination that yielded the highest number of viable cells. We then compared our optimal formulation and method, which we have commercialized as the Pierce Mouse Embryonic Fibroblast Isolation Kit (Part No. 88279) to a conventional do-it-yourself (DIY) trypsin method from the literature [3,10]. MEFs were isolated from mouse embryos of a pathogen-free CD1 mouse (Day 11 to 13) using the Pierce Kit (Figure 1) or the trypsin method. Cell yield and cell viability were determined from cell suspensions prepared from one embryo in a total volume of 1.5mL. We consistently obtained a 2-fold higher cell yield with the Pierce Kit compared to the trypsin protocol, and the average cell viability was greater than 90% (Figure 2).

Figure 1. Schematic diagram of the primary mouse embryonic fibroblast isolation procedure.

Figure 2. MEF cell yield and viability immediately after isolation. MEF cells were isolated from Mouse embryonic tissue at E11-13 using Pierce Mouse Embryonic Fibroblast Isolation Kit (Part No. 88279), and a do-it-yourself (DIY) trypsin method. Cell yield and cell viability were determined from cell suspensions isolated from duplicate samples of a single mouse embryo in a total volume of 1.5mL. Cell viability was determined by trypan blue exclusion assay and total cell yield was determined using an Invitrogen™ Countess™ Automated Cell Counter. n=5. Data represent the mean ± SD.

Isolated MEFs display their typical spread morphology by Day 3 in culture (Figure 3). By Day 7, MEFs looked elongated and their pseudopodia grew across each other to form a confluent monolayer culture. Over the culture period of one week, MEF cells were maintained in an optimal culture condition provided by specific media DMEM for Primary Cell Isolation. The high quality of cells was confirmed by consistently high levels of cell viability and purity of MEFs prepared by both our method and the DIY trypsin method (Figure 4).

Figure 3. Examples of MEFs in culture following isolation using the Pierce Mouse Embryonic Fibroblast Isolation Kit. Phase-contrast and fluorescent images of MEFs after 3 and 7 days in culture. Cultures were plated at a density of 2 x 10⁵ cells per well in a 12-well plate. For the fluorescent image (right panel), MEFs were stained with an antibody specific to β-actin. Images were taken at 20X and 60X magnification on a Carl Zeiss™ microscope (AxioVision™ Rel. 4.7).

Figure 4.MEF viability and purity over time in culture. Left. Cell viability at Day 1 and Day 7 was calculated as the ratio of total propidium iodide (PI) negative cells to total cells indicated by Hoechst nuclear staining. Right. Cell purity at Day 1 and Day 7 was calculated as the ratio of vimentin-stained cells to total cells indicated by Hoechst nuclear staining. A total of 200 cells were analyzed from two independent experiments. Data represent the mean ± SD.

To assess the morphology of MEFs over serial passages, MEF cells were isolated, plated at equal numbers (medium density) into six-well plates in triplicate, expanded every 5 days for a total of four passages. Then the morphology of each passage was observed by phase-contrast microscopy. The identity of the MEF cells was verified by immunocytochemical staining of the MEF marker protein vimentin (Figure 5A and B). The majority of cells from each culture passage display typical MEF morphology and are vimentin-positive cells.

A. Phase-contrast Images

B. Stained Images

Figure 5. Morphology of isolated and cultured MEFs over serial passages. MEFs were plated at a density of 2 x 10⁵ cells per well in a 12-well plate for each doubling. A. Phase-contrast images of MEFs from various passage numbers in culture. The typical morphology of MEFs from passage 0 (P0) to P4 were seen. B. Vimentin (red) expression of MEFs in P0 to P4 cultures. Green is beta-actin staining to indicate all cell populations in the cultures. Images were taken at 20X magnification on a Carl Zeiss microscope (AxioVision Rel. 4.7).

To assess the recover viability after the cryopreservation, MEF cells were isolated from Day 11 mouse embryos, expanded at Day 7, and then cryopreserved as frozen primary cell stocks of approximately 2 million cells each. The average cell viability of cells recovered from cryopreservation was 97%.

Conclusions

We have developed a reliable and convenient method for the isolation and culture of MEFs. The cell yield and viability obtained from our method are significantly better than that of obtained from the conventional trypsin-based method. The validated reagents and optimized procedure enable the establishment of a standardized MEF culture expressing fibroblast-specific protein markers. The isolated fibroblasts can be passaged at least four times without any observable changes in morphology and properties, and the fibroblasts can be cryopreserved for future use with very minimal loss of viability. MEFs isolated and cultured by this method can be used as a cell culture model for a diverse range of studies such as gene regulation and stem cell research.

Methods

MEF isolation:

Freshly dissected mouse embryos of pathogen-free CD1 mouse (Day 11-13) were minced and then incubated with Pierce MEF Isolation Enzyme (with papain)(Part No. 88290) for 25 to 30 minutes and washed twice with Hanks Balanced Salt Solution (HBSS). For do-it-yourself trypsin-based isolation (DIY Trypsin), mouse embryonic tissue was incubated with 0.25% trypsin for 25 to 30 minutes and washed twice HBSS. Remaining steps of the both procedures were the same. The tissue was disrupted in Complete DMEM for Primary Cell Isolation (Part No. 88287) by pipetting up and down 15 to 20 times with a pipette fitted with a 1000µL tip to generate a single cell suspension. Total cell yield was determined using an Invitrogen™ Countess™ Automated Cell Counter and cell viability was determined by trypan blue exclusion assay.

Immunostaining of cultured MEFs:

Cultured MEFs at the indicated days in culture were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton™ X-100 in HBSS for 10 minutes at room temperature and blocked with 3% BSA in HBSS for 30 minutes at room temperature. Cells were then labeled with primary antibodies overnight at 4°C and subsequently with the corresponding secondary antibodies at room temperature for 1 hour, then washed twice with HBSS.

Animal care:

Time-pregnant CD-1 mice were obtained from Charles River Laboratories and housed in the University of Illinois College of Medicine at Rockford animal facility. Experiments were performed exactly as approved by the Animal Care and Use Committee at the University of Illinois College Of Medicine in Rockford, IL, and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

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