Immortalized Mouse Primary Embryonic Fibroblasts

Description

Maintaining a consistent mouse embryonic fibroblast culture across extended research workflows can be difficult when the limited lifespan of primary cells restricts the number of usable passages. C57-6028IM provides immortalized mouse embryonic fibroblasts derived from the embryonic tissues of pathogen-free laboratory C57BL/6 mice for researchers who require an expandable mouse fibroblast culture.

The cells are grown in gelatin-precoated tissue culture flasks with Cell Biologics' Complete Growth Medium. Cells at passages 2–3 are harvested and cryopreserved in vials at a stated concentration of 0.5 × 10⁶ cells/mL. The supplied product datasheet does not specify the vial fill volume.

These C57BL/6 immortalized MEF cells are characterized by spindle-shaped morphology and immunofluorescence staining with anti-FSP1/S100A4 antibodies. The product datasheet reports negative test results for bacteria, yeast, fungi, and mycoplasma.

Under the specified Cell Biologics culture conditions, the cells can be expanded for more than 10 passages using a recommended split ratio of 1:2. M2267 Complete Fibroblast Medium and gelatin-precoated culture vessels are recommended. Repeated freezing and thawing are not recommended.

Specifications

The supplied product datasheet states that Cell Biologics primary cells may be immortalized through overexpression of SV40 large T-antigen or hTERT, but it does not identify the specific immortalization method used for C57-6028IM.

Features

• C57BL/6 immortalized mouse embryonic fibroblasts derived from embryonic tissue
• Harvested and cryopreserved at passages 2–3
• Supplied at a stated concentration of 0.5 × 10⁶ cells/mL
• Characterized by spindle-shaped fibroblast morphology
• Characterized by anti-FSP1/S100A4 immunofluorescence staining
• Tested negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for more than 10 passages under the specified culture conditions
• Recommended 1:2 split ratio
• Compatible with the specified gelatin-coated adherent culture workflow
• Shipped frozen on dry ice for immediate transfer to liquid nitrogen

Applications

C57-6028IM provides an expandable C57BL/6-derived fibroblast culture for research workflows requiring immortalized mouse embryonic fibroblasts. The extended lifespan documented for these immortalized MEF cells can help reduce the passage limitations associated with finite primary fibroblast cultures.

The supplied general cell culture protocol includes preparation guidance for downstream cell-based assays, staining, FACS analysis, and investigator-designed experiments. Researchers should independently determine the suitability of C57-6028IM for each experimental design.

• Research workflows requiring an expandable C57BL/6 mouse embryonic fibroblast culture
• Routine culture and expansion of immortalized MEF cells
• Preparation of cultured fibroblasts for cell-based assays
• Preparation of cultured cells for staining or FACS analysis
• Investigator-designed laboratory research under validated experimental conditions

The supplied product documents do not identify C57-6028IM as irradiated, mitomycin C-treated, mitotically arrested, or validated for use as a feeder layer.

Cell Culture Requirements

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet using aseptic technique.
• Sterile-filter culture media, supplements, and reagents through a 0.2 µm filter.
• Prewarm only the amount of culture medium required for immediate use.
• Use 5 mL of medium for a T25 flask and 8–10 mL for a T75 flask when recovering cryopreserved cells, changing medium, or splitting cells.
• Avoid repeatedly warming complete culture medium.

Gelatin Coating

• Coat sterile culture vessels with CB6950 Gelatin-Based Coating Solution for 2 minutes.
• Aspirate the excess coating solution before seeding cells.

Recovery of Cryopreserved Cells

1. Thaw the vial rapidly in a 37°C water bath for less than 1 minute or until only a few ice pieces remain.
2. Remove the vial and disinfect the exterior with 70% ethanol.
3. Transfer the cells to a sterile 15 mL conical tube containing 5 mL of prewarmed culture medium.
4. Rinse the vial with an additional 0.5–1 mL of medium to recover remaining cells.
5. Centrifuge at 200 × g for 5 minutes to remove residual DMSO.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of growth medium.
7. Seed the cells into a T25 flask precoated with CB6950 Gelatin-Based Coating Solution.
8. Incubate at 37°C with 5% CO₂ and leave the culture undisturbed for 12–16 hours.
9. Change the medium the following day and every 24 hours thereafter.
10. Check cell confluence and morphology daily under a microscope.

Cell Expansion

1. Remove the culture medium and wash the adherent cells twice with 1X PBS without calcium or magnesium.
2. Incubate with prewarmed 0.25% Trypsin-EDTA for 3–5 minutes.
3. Use 0.5–1 mL of Trypsin-EDTA for a T25 flask or 2 mL for a T75 flask.
4. Once the cells detach, immediately add 5–10 mL of complete culture medium containing FBS to neutralize the trypsin.
5. Gently pipette the cell suspension several times.
6. Seed cells into fresh gelatin-precoated culture vessels.
7. Use the recommended 1:2 split ratio.
8. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.

Preparation for Downstream Experiments

• Once cells reach confluence, the culture protocol allows cells to overgrow for up to 24 hours before cell-based assays, staining, FACS analysis, or designed experiments.
• Change the medium daily and wash the cells twice with 1X PBS between medium changes.
• Before an experiment, the protocol recommends filtering the cells through a 40 µm cell strainer to remove cell clumps and most dead or floating large cells.

Cryopreservation

• Use 1 mL of CB6916 Freezing Medium per cryovial and quickly resuspend the cell pellet.
• Place the vials in a controlled-rate freezing container filled with 100% isopropanol at −80°C for 6–12 hours.
• Transfer the vials to liquid nitrogen for long-term storage.

The −80°C step described above is part of the controlled freezing procedure for newly prepared vials. Received cryopreserved cells should be transferred directly from the dry ice shipping container to liquid nitrogen.

Quality Control

Shipping, Storage and Handling

• Cryopreserved immortalized MEF cells are shipped frozen on dry ice overnight.
• Upon arrival, transfer the frozen vial immediately to liquid nitrogen until ready for use.
• Do not keep received cryopreserved cells in a −20°C or −80°C freezer for routine storage.
• Live-cell shipment is available on request.
• Repeated freezing and thawing are not recommended.
• Handle all animal cells with caution and treat them as potential pathogens.

Warranty and Authorized Use

Cell Biologics' product warranty applies only when the cells are used with the recommended Cell Biologics medium and coating solution and are cultured and tested according to the supplied protocols. The product datasheet limits this warranty to 35 days from the date of delivery.

C57-6028IM is distributed for research purposes only. It is not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures. Transfer or resale of Cell Biologics cells or products requires the company's written consent.

Documents

C57-6028IM Product Datasheet

Current Primary Cell Culture Protocol

Archived Primary Cell Culture Protocol

Cell Culture Products Material Safety Data Sheet

Frequently Asked Questions

What are C57BL/6 immortalized mouse embryonic fibroblasts?

C57-6028IM consists of immortalized fibroblasts derived from the embryonic tissues of pathogen-free laboratory C57BL/6 mice. The product datasheet describes the cells as primary-cell-derived fibroblasts with an extended lifespan.

Are C57-6028IM cells primary or immortalized MEF cells?

They are immortalized mouse embryonic fibroblasts derived from primary cells. The cells are not presented as finite-lifespan primary MEFs; the product datasheet states that they have an extended lifespan and can be expanded for more than 10 passages under the specified culture conditions.

Which immortalization method was used for C57-6028IM?

The supplied product datasheet states that Cell Biologics primary cells may be immortalized through overexpression of SV40 large T-antigen or hTERT. However, it does not identify which method was used specifically for C57-6028IM. Contact technical support if the immortalization method is critical to your study.

What concentration of immortalized MEF cells is supplied?

The product datasheet states a supplied concentration of 0.5 × 10⁶ cells/mL. The vial fill volume and total number of cells per vial are not specified in the supplied datasheet.

At what passage are the cells cryopreserved?

The cells are harvested and cryopreserved at passages 2–3.

How are the C57BL/6 mouse embryonic fibroblasts characterized?

The cells are characterized by spindle-shaped morphology and immunofluorescence staining with anti-FSP1/S100A4 antibodies.

Are the immortalized MEF cells tested for mycoplasma and microbial contamination?

Yes. The product datasheet reports negative test results for bacteria, yeast, fungi, and mycoplasma.

How many passages can C57-6028IM cells be expanded?

The product datasheet states that the cells can be expanded for more than 10 passages under the specified Cell Biologics culture conditions using a recommended split ratio of 1:2.

What medium and coating solution are recommended?

M2267 Complete Fibroblast Medium w/ Kit is the suggested medium. CB6950 Gelatin-Based Coating Solution is recommended for coating culture vessels before cell seeding.

What are the recommended immortalized MEF culture conditions?

The supplied culture protocol specifies adherent culture in gelatin-precoated vessels at 37°C with 5% CO₂ in a humidified incubator. A 1:2 split ratio is recommended.

How should the cryopreserved cells be stored after delivery?

Transfer the frozen vial directly from the dry ice shipping container to liquid nitrogen immediately upon arrival. Do not keep the received vial in a −20°C or −80°C freezer for routine storage.

Can C57-6028IM be used as an ESC or iPSC feeder layer?

The supplied product documents do not identify C57-6028IM as irradiated, mitomycin C-treated, mitotically arrested, or validated as a feeder layer. Researchers should confirm suitability with technical support before considering any feeder-cell application.

Are these cells intended for clinical or diagnostic use?

No. C57-6028IM is for research use only and is not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.