Purification of Mouse Embryonic Fibroblasts (MEFs)

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.


INTRODUCTION
Mouse embryonic fibroblasts (MEFs) are embryo-derived primary fibroblasts with limited proliferation potential (Todaro & Green, 1963).MEFs have versatile applications.Most commonly, MEFs are prepared as feeder cell layers to maintain embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in an undifferentiated state in prolonged culture (Evans & Kaufman, 1981;Jozefczuk et al., 2012).To be used as feeder cells, MEFs are expanded and mitotically inactivated by either chemical treatment with, i.e., Mitomycin C or γ-irradiation (Conner, 2001;Martin & Evans, 1975).Mitotic inactivated MEFs can then be seeded to form a confluent layer of cells (feeder layer) before seeding ESCs or iPSCs.The feeder layer provides a niche that inhibits cell differentiation.Inactivated MEFs remain metabolically active and support ESCs and iPSCs through the release of growth factors and extracellular matrix components, expression of adhesion molecules, and the support of cell-cell interaction (Llames et al., 2015;Saitoh et al., 2012).One critical MEF-secreted factor that maintains ESCs in their undifferentiated state is leukemia inhibitory factor (LIF) (Smith et al., 1988;Williams et al., 1988).Beyond feeder cells, MEFs derived from transgenic mice are used as in vitro cell systems to functionally test and analyze the impact of genetic alteration (Hannan et al., 2022).Primary non-transformed MEFs are a popular cell system to characterize "normal" cellular activities and signaling in response to various external stimuli or to investigate cellular processes such as mesenchymal differentiation in a non-cancerous background (Dastagir et al., 2014).Due to their limited expansion capacity, MEFs are best cryopreserved at a low passage number until required.The purification procedure and intrinsic batch-tobatch variation impact MEF quality.To minimize these effects and ensure high cell viability and expansion potential, procedures must be standardized, and only fresh reagents should be used.The following protocol describes the purification of MEFs at E13.5, passaging and expansion (Basic Protocol), cryopreservation and thawing (Supporting Protocol).
NOTE: Access to a laminar flow hood or equivalent, a tissue culture incubator, inverted microscope, bench-top centrifuge, −80°C freezer, and ultralow storage is essential for the following protocol.NOTE: All steps after uterine horn dissection and reagent preparation must be performed under aseptic conditions.

STRATEGIC PLANNING
Depending on the expected litter size and mating success, a sufficient number of female mice must be set up for timed mating.If pregnancy rates are low, other steps outlined in the Troubleshooting section can be introduced to increase mating success (Byers et al., 2012) (https:// www.jax.org/news-and-insights/ jax-blog/ 2014/ september/ six-steps-forsetting-up-timed-pregnant-mice).NOTE: All animal protocols must be reviewed and approved by the appropriate Animal Care and Use Committee and regulations followed for the care and use of laboratory animals.

PURIFICATION, PASSAGING, AND EXPANSION OF MEFS
This protocol describes the generation of MEFs from mouse embryos at E13.5.Mouse embryos are dissected from the pregnant female by removing the uterine horns under non-sterile conditions.The uterine horns are transferred to a laminar flow hood for further preparation under aseptic conditions.First, the uterine horns are rinsed twice in sterile DPBS-/-with antibiotic-antimycotic, followed by removing individual embryos from the

of 16
Current Protocols uterine tissue.The yolk sac and placenta are then removed, and the exposed embryos are rinsed in sterile DPBS-/-with antibiotic-antimycotic.Before tissue dissociation, the embryo is decapitated, and fetal organs are dissected.The embryonic tissue is finely minced in trypsin-EDTA with DNAse, resuspended multiple times, and digested for 15 min at 37°C.Digestion is stopped by adding MEF harvest medium.If large tissue aggregates remain, the tissue suspension can be passed through a cell strainer or directly transferred to a 15-ml Falcon tube, spun, and the supernatant aspirated.The tissue pellet is resuspended in MEF harvest medium and transferred to a T25 flask.The following day, the medium is refreshed to remove non-attached cells, tissue aggregates, and debris.The T25 flask should be between 70% to 90% confluent.

NOTE:
As mentioned above, all animal work must comply with national and institutional requirements for the use of animals in research.

NOTE:
Pregnant female mice are humanely euthanized by cervical dislocation.This procedure is performed according to approved standard operating procedures.The uterine horns are immediately placed on ice until the embryos have been decapitated according to the approved institutional protocols.

NOTE:
To minimize the risk of contamination, the female mouse is extensively sprayed with ethanol, and tissue harvest and dissociation are performed under aseptic conditions.NOTE: MEFs should be cultured in complete MEF medium without antibioticantimycotic after the first passage.

NOTE:
If MEFs are kept in long-term culture, i.e., for immortalization, testing the culture for mycoplasma is recommended.House male and female mice in a breeding cage overnight and check female mice for vaginal plugs the following morning.Plugged females should be separated from the male and housed until the desired stage of pregnancy is reached.Female mice can be palpated to confirm pregnancy after E11.

Materials
2. Prepare MEF harvest medium (see recipe) in the laminar flow hood and prewarm in a 37°C water bath.Prepare DPBS-/-2% FBS and DPBS-/-antibiotic/antimycotic (see recipes) in the laminar flow hood and place on ice.
These plates will be used to remove the yolk sac and placenta, head, and nonmesenchymal (red) organs.Two embryos should be processed per plate.The number of plates will depend on the number of embryos.If there are more than six embryos in the uterus, prepare additional plates after dissection of the uterine horns.To avoid crosscontamination when isolating MEFs from transgenic mouse strains and embryos with different genotypes, process one embryo per 10-cm tissue culture plate.
The two 12-well tissue culture plates are used to rinse the embryos in sterile DPBS-/-2% FBS after removing the yolk sac and placenta and before tissue dissociation (1-2 wells/embryo).
The embryos will be stored on this plate on ice until they are processed.
7. Prepare two 50-ml Falcon tubes by adding 40 ml DPBS-/-2% FBS (see recipe) per tube.These will be used to immerse the uterine horns in succession.

of 16
Current Protocols 8. Euthanize the pregnant female by cervical dislocation.
9. Place the female mouse supine on a paper bench barrier and spray generously with 70% EtOH before opening the abdominal cavity using dissection scissors (Fig. 1).
Spraying the female mouse extensively minimizes the risk of contamination by preventing the transfer of fur to the uterus during dissection.
10. Use large dissection forceps to lift the uterus, remove membranes and fat tissue, and cut the cervix using dissection scissors to isolate the uterine horns.Place the uterine horns in a 10-cm tissue culture plate containing sterile DPBS-/-2% FBS (Fig. 1).
11. Transfer the plate with uterine horns to the laminar flow hood to proceed aseptically.
14. Separate individual embryos by cutting the uterine wall between implantation sites using sterile dissection scissors.(Fig. 1).
15. Remove uterine tissue, rinse in 2 ml sterile DPBS-/-2% FBS in a 12-well tissue culture plate, and place individual embryos into a new well of a 12-well tissue culture plate containing 1 ml sterile DPBS-/-2% FBS, place on ice until all embryos are isolated (Fig. 1).
18. Decapitate embryo and remove the fetal liver and heart with two fine-point dissection forceps and rinse the embryo in 2 ml sterile DPBS-/-2% FBS in a 12-well tissue culture plate (Fig. 1).
Optional: If genotyping is required, transfer the head to a 1.5-ml tube and freeze at −20°C until further processing.If embryos are isolated from transgenic animals, and embryos with different genotypes are expected, change dissection forceps between embryos to avoid cross-contamination.
19. Transfer the embryo to a fresh 10-cm tissue culture plate containing 2 ml 0.25% trypsin-EDTA with DNase I (final concentration: 15 kU/ml) at room temperature (RT).
Depending on personal preference, 10-or 6-cm tissue culture plates can be used for tissue homogenization.Prepare an appropriate volume of 0.25% trypsin-EDTA with DNAse I (final concentration: 15 kU/ml) and add 2 ml to each 10-cm tissue culture plate.
20. Mince embryo tissue finely with a sterile blade at RT until the tissue suspension becomes smooth and gloopy in consistency (Fig. 1).
21. Tilt plate and resuspend the tissue suspension with a 1-ml filter pipette tip.
22. Place a support (i.e., a 5-or 10-ml sterile serological pipette) under the plate to keep it at an angle and place in an incubator.
Alternatively, transfer the tissue suspension to a 15-ml Falcon tube and place in a rack in the tissue culture incubator.
23. Incubate the tissue suspension at 37°C in a humidified 5% CO 2 atmosphere for 15 min.24.Add 4 ml warm MEF harvest medium to inactivate the 0.25% trypsin-EDTA with DNase I.
25. Pipette the tissue suspension up and down with a 5-ml sterile serological pipette until it is homogenous and transfer to a 15-ml Falcon tube.Keep at RT until all embryos are processed.
Optional: If large tissue aggregates are visible, the suspension can be passed through a 100-μm cell strainer into a 50-ml Falcon tube to remove undigested tissue aggregates, then transfer to a 15-ml Falcon tube.
26. Spin the tissue suspension at 500 × g and RT for 5 min using a benchtop centrifuge to pellet the cells.
27. Carefully aspirate the supernatant using a sterile aspiration pipette and vacuum trap, or use a 10-ml sterile serological pipette if a vacuum trap is unavailable.
28. Resuspend the cell pellet in 5 ml prewarmed MEF harvest medium and transfer to a T25 flask.
30.Check cell confluency the following day using an inverted microscope.If cell confluency has reached 80% to 90%, passage MEFs into a T75 flask for expansion or process for cryopreservation (see Supporting Protocol) (Fig. 2A).
If cells have not reached 80% confluency, change the MEF medium to remove debris and tissue aggregates by aspirating the medium using a vacuum trap and replacing it with fresh prewarmed (∼37°C) MEF harvest medium.

of 16
Current Protocols Passaging and expansion of MEFs 31.Prewarm MEF culture medium and DPBS-/-in a 37°C water bath and keep 0.25% trypsin-EDTA at RT. 32.Aspirate medium with a sterile aspiration pipette using a vacuum trap or a 10-ml sterile serological pipette if a vacuum trap is unavailable.
36.Incubate the flask at 37°C in a humidified 5% CO 2 tissue culture incubator for around 5 min or until cells have detached.
Check the level of cell detachment using the inverted microscope.Tapping the side of the flask facilitates cell lifting.
38. Transfer an aliquot of the cell suspension to a Beckman Coulter Counter cup using a 300-μl filter pipette tip and perform a cell count using a Beckman Coulter Counter (according to manufacturer instructions: Beckman Coulter Z series User Manual 9914591-DA), an equivalent cell counter, or count manually using a hematocytometer (according to manufacturer instructions).
39. Transfer cells within the optimal MEF density range of ∼2to 4 × 10 4 cells/cm 2 to a fresh T75 flask and add MEF culture medium to a volume of 20 ml.
40.Incubate the T75 flask at 37°C in a humidified 5% CO 2 atmosphere in a tissue culture incubator.
41. Check cell confluency daily using an inverted microscope.Freeze (see Support Protocol 1) or passage the cells when they reach 80%-90% confluency.

CRYOPRESERVATION AND THAWING OF MEFS
MEFs can be cryopreserved at P0 if >80% confluent or after expansion at P1-5.The cells can be frozen naïve or, if isolated for use as feeder cells, after mitotic inactivation (Conner, 2001).When freezing mammalian cells, it is important to use a cryoprotective agent that prevents damage to the cell membrane by blocking ice crystal formation.Dimethyl Sulfoxide (DMSO) is most routinely used in mammalian tissue culture.Another aspect important for cryopreservation is the controlled gradual decrease in temperature at approximately −1°C per minute down to −80°C.Optimal cell recovery post cryopreservation requires rapid thawing of the cryovial and optimal cell density.To maintain high-quality cryopreserved MEFs, cryovials should be transferred to liquid nitrogen or specialized ultralow freezers (−150°C) for long-term storage.
If not treated for mitotic inactivation, MEFs should be frozen in the exponential proliferation phase and ideally at a low passage number at P0 post-harvest or P1-4 after expansion.
2. Prepare sterile MEF freeze-down medium under aseptic conditions in the laminar flow hood.
Spray all items before transferring them into the laminar flow hood with 70% EtOH to minimize the risk of contamination.
3. Aspirate medium with a sterile aspiration pipette and vacuum trap.
6. Add prewarmed 0.25% trypsin-EDTA to the tissue culture flask to detach the cells using a 5-ml sterile serological pipette.

of 16
Current Protocols 7. Incubate the flask at 37°C, 5% CO 2 under humidified conditions in a tissue culture incubator for around 5 min or until cells have detached.
Check the level of cell lifting using the inverted microscope.Tap the flask on the side to facilitate cell detachment.
8. Add MEF culture medium to the flask to inactivate the 0.25% trypsin-EDTA digest and resuspend using a 5-or 10-ml sterile serological pipette to generate a homogeneous single-cell suspension.
The volume depends on tissue culture flask size: T25 flask, 4 ml; T75 flask, 8 ml 9. Transfer the cell suspension to a 15-ml Falcon tube.
10. Transfer an aliquot of the cell suspension to a Beckman Coulter Counter cup using a 300-μl filter pipette tip and perform a cell count using a Beckman Coulter Counter (according to manufacturer instructions: Beckman Coulter Z series User Manual 9914591-DA), an equivalent cell counter, or count manually using a hematocytometer (according to manufacturer instructions).
Cell viability can be estimated using the live/dead stain Trypan blue (Strober, 2015) or indirectly by measuring cell size/volume based on the Coulter principle (Graham, 2022) that detects fluctuations in the electrical resistance generated by particles in a conductive buffer that passes through the aperture.
Label the appropriate number of cryovials: cell type and origin (i.e., MEF C57BL/6), passage number, cell number per cryovial, date, and person's initials.
11. Spin the cell suspension for 5 min at 500 × g and RT using a benchtop centrifuge.
12. Carefully aspirate the supernatant without disturbing the cell pellet using a sterile aspiration pipette and vacuum trap.
13. Resuspend the pellet thoroughly to dissociate any cell aggregates in the appropriate volume of freeze-down medium to freeze 2-5 × 10 6 cells/ml per cryovial.
14. Transfer 1 ml cell suspension in MEF freeze-down medium into each labeled cryovial using a 1-ml filter pipette tip.
15. Place the cryovials in a CoolCell® Cryopreservation container to allow a gradual temperature decrease at an optimal rate of −1°C per minute to −80°C.
16. Move the CoolCell® Cryopreservation container to a −80°C freezer for overnight storage.
17. Take cryovials from the CoolCell® Cryopreservation container and transfer cryopreserved MEFs to liquid nitrogen storage or an ultralow −150°C freezer for long-term storage.
Optional: Before transferring MEF cryovials to long-term storage, defrost one vial for quality control.
Thawing of MEFs 18. Prepare and prewarm MEF culture medium in a 37°C water bath.
19. Add 9 ml prewarmed MEF culture medium to a 15-ml Falcon tube using a 10-ml sterile serological pipette.
20. Remove a MEF cryovial from liquid nitrogen or freezer and immediately place it in a 37°C water bath until defrosted.
21. Spray the MEF cryovial extensively with 70% EtOH to minimize the risk of contamination before opening in the laminar flow hood.

of 16
Current Protocols 22. Transfer the thawed cell suspension dropwise into a 15-ml Falcon tube containing 9 ml prewarmed MEF culture medium using a 1-ml filter pipette tip.
23. Spin the cell suspension for 5 min at 500 × g and RT using a benchtop centrifuge to remove the DMSO from the MEF suspension.
24. Carefully aspirate the supernatant without disturbing the cell pellet using a sterile aspiration pipette and vacuum trap.
25. Resuspend the cell pellet carefully in 5 ml prewarmed MEF culture medium and transfer to a tissue culture flask using a sterile 5-ml serological pipette.If MEFs are cultured in a T75 flask, add 15 ml MEF culture medium to the flask using a 10-ml sterile serological pipette.
27. Check viability, morphology, and percent confluency of MEFs the following day using the inverted microscope (Fig. 2B).
Optional: Replace medium to remove debris and unattached cells.

REAGENTS AND SOLUTIONS
Prepare all recipes using aseptic technique.

COMMENTARY Background Information
MEFs are a heterogenous fibroblast population of mesenchymal origin.Todaro and Green first studied and described mouse embryonic cell culture and growth characteristics in 1963.They culture-adapted mousederived embryo cells isolated from E15-17 to in vitro growth conditions through continuous passaging and generated the widely used NIH/3t3 fibroblast cell line, which has unlimited proliferative potential (Marigliani et al., 2019;Todaro & Green, 1963).The basic principle of isolating MEFs from embryos at a defined time after mating and tissue dissociation by mincing and enzymatic digestion has remained similar to the original procedure (Michalska, 2007;Qiu et al., 2016;Xu, 2005).
Over time, however, specific steps have been modified, such as the duration of tissue digestion, digestion with or without agitation, and the addition of DNase to the 0.25% trypsin-EDTA for genomic DNA degradation.The duration of tissue digestion in trypsin-EDTA varies from overnight at 4°C to 10-30 min at 37°C with or without agitation to achieve the best possible tissue

of 16
Current Protocols homogenization without reducing cell viability and proliferation capacity.For the same reason, DNase I digestion of gDNA further improves tissue dissociation after mincing and facilitates pipetting of the tissue suspension.The time of harvest usually varies between E11.5 and E15.5, while E13.5-15.5 is considered optimal for MEF isolation.Though not essential, some protocols utilize gelatincoated flasks to facilitate cell attachment of freshly isolated MEFs (Jozefczuk et al., 2012;Michalska, 2007;Qiu et al., 2016;Xu, 2005).
Primary MEFs are used for various applications.In the early 1980s, the first mouse ESC cultures were isolated and propagated on feeder cells, although Evans and Kaufman did not utilize primary MEFs (Evans & Kaufman, 1981).Mitotically inactivated MEFs are now one of the most commonly used feeder cell layers to maintain the undifferentiated growth of ESC and iPSC cultures.As MEF preparations can differ in quality and require frequent renewal due to their finite expansion potential, some laboratories use Sandos inbred mice (SIM) 6-thioguanineresistant, ouabain-resistant (STO) cell lines generated from SIM (Ware & Axelrad, 1972) or switched to feeder-free culture systems.While still controversial in ESC cultures, Saitoh et al. showed that MEFs are superior to permanent STO-feeders when first establishing iPSCs (Saitoh et al., 2015).However, undifferentiated mESCs can also be maintained on other embryonic fibroblastderived cell lines, including C3H 10T1/2 (Rathjen et al., 1990).
Generally speaking, MEFs are easy to obtain and culture.A major advantage of primary MEFs is their non-transformed state characterized by the absence of oncogenic features; thus, they are a popular cell model for studying "normal" biochemical processes and cellular signaling.MEFs can be a suitable model system for investigating mesodermtype cell differentiation (Dastagir et al., 2014;Saeed et al., 2012).Furthermore, MEFs are an excellent in vitro model to study gene function and the functional consequences of genetic alteration.For example, MEFs are used to validate the loss or mutation of a specific gene and its effect on pathway activation and the epigenetic landscape (Hannan et al., 2022;Moss et al., 2019).Therefore, MEFs can be isolated from genetically modified mouse strains or genetically altered in vitro as a more simplistic means to analyze gene function.Recently, MEFs have been used as a model system to assess devel-opmental toxicity (Hansen & Piorczynski, 2019).
A major disadvantage of MEFs is their limited proliferative capacity of approximately 20 cell divisions ex vivo (Karatza & Shall, 1984), equivalent to five passages.Thus, MEF stocks require frequent renewal, which is time-consuming.The non-physiological oxygen level is an environmental condition limiting the replicative life span of MEFs ex vivo (Parrinello et al., 2003).
Despite their limited proliferation potential, MEFs are easily immortalized through continuous passaging or genetic modifications that generate MEF-derived cell lines with infinite proliferative capacity.Immortalization through serial passage most likely occurs due to environmentally induced changes that cause a growth advantage and prevent apoptotic cell death and senescence.
Another disadvantage is considerable batch-to-batch variation, which leads to differences in cell viability and proliferative potential.These variations can be reduced through high technical skill and optimized protocols but have not been entirely resolved.

Critical Parameters
We recommend accounting for variability in mating success (Table 1), as not all matings produce pregnancies.Mating success can be increased by housing stud mice individually 1-2 weeks before mating and by selecting females that are in the proestrus or oestrus state (Byers et al., 2012) (https:// www.jax.org/news-and-insights/ jaxblog/ 2014/ september/ six-steps-for-settingup-timed-pregnant-mice).Timed matings are routinely set up as 1 male with 2 females (8-10 weeks of age) overnight.The following morning, the female mice are examined for the presence of a vaginal plug.
The mouse strain utilized to generate MEFs depends on the downstream application.Certain mouse strains have smaller litter sizes that need to be accounted for-the number of female mice set up for timed mating may need to be increased.The difference in litter sizes may be due to wild-type inbred strain-specific differences.For example, C57BL/6 litter sizes range from 5 to 8 pups, while FVB/NJ mice have litter sizes of 8 to 10 pups, and differences can be the phenotypic consequence of gene ablation or modification (Ferreira et al., 2007).Thus, researchers must consider the average litter size when setting up the required number of timed matings.When using genetically modified mouse strains, it is important  The size and state of the embryo will determine the proliferative capacity and overall quality of the derived MEFs.For example, suppose MEFs are not used for phenotypic characterization of specific genotypes but to generate feeder cells.In that case, we do not recommend generating MEFs from embryos that appear markedly smaller (∼25% smaller than embryos from the same female) or morphologically abnormal.In principle, MEFs can be isolated from any genetically modified mouse strain; however, if the genetic alteration is embryonic lethal before harvest (E13.5, for example), MEFs can be purified from embryos as early as E8.5.
Cell viability, cell number, and proliferative capacity are impacted by the efficiency and duration of the enzymatic tissue digestion (Table 1); thus, it is inadvisable to exceed the recommended digestion time or trypsin-EDTA concentration, and it is critical to only use tissue culture-grade reagents before their expiration date.High cell confluency (>70%) and low level of debris 24 hr post-harvest is indicative of a high-quality preparation.Ex vivo MEFs are cultured under oxidative conditions since physiological oxygen levels are around 3%, and MEFs grown in culture are exposed to 21% oxygen, which facilitates/accelerates replicative senescence (Parrinello et al., 2003).
Contamination of MEF cultures most likely occurs at the time of harvest or due to poor aseptic technique (Table 1).To minimize the risk of contamination during isolation, spray the euthanized female mouse extensively with 70% EtOH before dissecting the uterine horns.Once isolated, transfer the uterine horns in a 10-cm tissue culture plate to a laminar flow hood, rinse twice in sterile DPBS-/-, and perform all subsequent steps under aseptic conditions.To avoid contamination of media, reagents, and other consumables used for MEF harvest and culture, prepare all materials under strict aseptic conditions and purchase reagents, media, and single-use consumables MEFs should only be frozen in the exponential proliferation phase at 80% to 90% confluence, and the number of cells per cryovial should not be less than 1 × 10 6 cells/vial nor exceed 5 × 10 6 cells/vial (Table 1).To ensure optimal cell recovery after thawing, remove DMSO and transfer to an appropriately sized tissue culture flask depending on the number of cells in the cryovial.Low cell numbers can affect cell recovery as MEFs require cell-cell interactions for optimal growth.During cryopreservation, a gradual decrease in temperature is essential to prevent cell damage, and rapid thawing minimizes the cytotoxic effects of the cryoprotectant.

Understanding Results
MEF preparations from wild-type embryos are expected to reach 80% to 90% confluence in a T25 flask within 1-2 days after purification.Depending on the genetic alteration, MEFs isolated from genetically modified animals may exhibit different proliferation rates.Many dead cells and debris are expected within the first 24 hr post-preparation and should decrease significantly after the first medium exchange.The average doubling time of P1-P4 wild-type MEFs can vary between 28 hr and 48 hr and decreases with further passages due to the limited proliferation potential.Between passages 0 and 4/5, MEF morphology remains mesenchymal fibroblastic, while MEFs passaged beyond P5/6 display an increased percentage of senescent cells with an enlarged, flat, and frequently multinucleated morphology (Chen et al., 2020;Dirac & Bernards, 2003;Manning & Kumar, 2010).Cell viability of high-quality cryopreserved MEFs after thawing is expected to be around 80%.

Time Considerations
The time required to purify MEFs includes the timed mating, which ranges from E12.5 to 15.5.At the time of harvest, the time required to isolate MEFs depends on the number of pregnant females and embryos per female.The preparation of reagents (FBS and other media supplements should be defrosted overnight at 4°C) and set-up of the harvest area and laminar flow hood takes around 30 min.Euthanasia and dissection of the uterine horns take around 5 min per mouse.Embryo isolation, tissue removal, mincing, and tissue digestion require approximately 30 min per em-bryo.These steps can be staggered so that the next embryo can be processed during tissue digestion for the previous embryo.Centrifugation, aspiration of the supernatant, resuspension of the tissue pellet, and transfer to a prelabeled T25 flask will take another 10-20 min, depending on the number of samples.Overall, 2-2.5 hr should be allowed for the MEF purification from one pregnant female with a litter size of 8-9 embryos.

Figure 1
Figure 1 Workflow of MEF preparation with the corresponding steps in the Basic Protocol; pregnant female mouse before and after opening the abdominal cavity (step 9); dissected uterine horns (step 10); separated embryo (step 14); embryo after removal of the uterine tissue, leaving the embryo in the yolk sac intact (step 15); embryo (left) after removal of yolk sac and placenta (right) (step 17); embryo body (left), head (top), and removed fetal liver and heart (right) (step 18); embryonic tissue after mincing in 0.25% trypsin-EDTA with DNase I (step 20).
. All reusable dissection equipment must be sterilized/autoclaved.

Table 1
Troubleshooting Guide for MEF Purification