Immortalized Mouse Primary Kidney Fibroblasts

Description

Kidney-derived fibroblast studies often require a consistent cell source that can be maintained beyond the limited expansion window associated with conventional primary cultures. Immortalized Mouse Primary Kidney Fibroblasts provide a C57BL/6 mouse kidney-derived fibroblast culture with an extended lifespan for in vitro research requiring continued cell expansion.

These immortalized mouse kidney fibroblasts are isolated from the kidney tissues of pathogen-free laboratory C57BL/6 mice. Cells are grown in gelatin pre-coated tissue culture flasks with Complete Growth Medium, harvested at passages 2–3, and cryopreserved. Each vial contains 0.5 × 10⁶ cells per mL and is delivered frozen.

Cell characterization includes spindle-shaped fibroblast morphology and immunofluorescence staining with an anti-FSP1/S100A4 antibody. The cells are documented as negative for bacteria, yeast, fungi, and mycoplasma. Under the specified culture conditions, this immortalized mouse kidney fibroblast cell line can be expanded for more than 10 passages at a recommended split ratio of 1:2.

The supplier describes its immortalized primary cell range as being developed through overexpression of either simian virus 40 large T antigen (SV40 large T antigen) or human telomerase reverse transcriptase (hTERT). The supplied product documentation does not identify which of these methods was used specifically for Catalog No. C57-6016IM. Researchers whose experiments depend on the immortalization method should request confirmation before ordering.

For Research Use Only. Not authorized for human use, in vitro diagnostic use, or therapeutic procedures.

Specifications

Features

• Kidney-derived mouse fibroblasts isolated from pathogen-free laboratory C57BL/6 mice
• Primary cell-derived immortalized culture with an extended lifespan
• Harvested and cryopreserved at passages 2–3
• Supplied at 0.5 × 10⁶ cells/mL per vial
• Characterized by spindle-shaped morphology
• Characterized by anti-FSP1/S100A4 immunofluorescence staining
• Documented as negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for more than 10 passages under the specified culture conditions
• Recommended split ratio of 1:2
• Compatible with gelatin-coated adherent culture conditions described in the supplied protocol

Applications

Immortalized C57BL/6 mouse kidney fibroblasts provide a kidney-derived adherent cell culture for research workflows requiring mouse renal fibroblasts with an extended culture lifespan. Applications supported by the supplied product and culture documentation include:

• In vitro mouse kidney fibroblast research
• Kidney-derived fibroblast culture and expansion
• Fibroblast morphology assessment
• FSP1/S100A4-associated cell characterization
• Immunofluorescence staining workflows
• Flow cytometry sample preparation
• Extended-passage fibroblast culture
• Appropriately designed cell-based assays

The supplied documentation does not provide application-specific validation for renal fibrosis, chronic kidney disease, TGF-β signaling, extracellular matrix deposition, collagen production, nephrotoxicity testing, or drug screening. Researchers requiring a validated disease model should request supporting data before use.

Cell Characterization and Quality Control

The product documentation does not state an FSP1/S100A4-positive percentage, post-thaw viability specification, doubling time, or lot-specific identity test. Contact technical support if these parameters are required for study qualification.

Recommended Culture System

For culture performance consistent with the supplied protocol, the following medium and reagents are referenced in the product documentation. Confirm product availability and whether individual components are supplied separately before ordering.

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic technique to prevent microbial contamination.
• Sterile filter culture media, supplements, and reagents through a 0.2 μm filter.
• Prewarm only the amount of medium required for immediate use to 37°C.
• For cell recovery, medium changes, or splitting, use 5 mL for a T25 flask and 8–10 mL for a T75 flask.
• Repeated prewarming of complete culture medium is not recommended.

Gelatin Coating

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

Recovery from a Cryopreserved Vial

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 immediately and disinfect its exterior with 70% ethanol.
3. Transfer the cells into a sterile 15 mL conical tube containing 5 mL of prewarmed cell culture medium.
4. Rinse the vial with an additional 0.5–1 mL of medium to collect remaining cells.
5. Centrifuge the cells at 200 × g for 5 minutes to remove residual DMSO.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of prewarmed growth medium.
7. Seed the resuspended cells into a gelatin-coated T25 flask.
8. Place the flask in a humidified 37°C, 5% CO₂ incubator and leave it undisturbed for 12–16 hours.
9. Change the medium the following day and every 24 hours thereafter.
10. Check confluence and appropriate cell morphology daily under a microscope.

Expansion and Subculture

1. Remove the culture medium and wash adherent cells twice with 1X PBS without calcium and magnesium.
2. Incubate cells with prewarmed 0.25% Trypsin-EDTA solution at 37°C for 3–5 minutes.
3. Use approximately 0.5–1 mL for a T25 flask or 2–3 mL for a T75 flask, as described in the current protocol.
4. Once cells detach, add complete medium containing FBS to neutralize the trypsin and gently pipette the suspension.
5. Seed cells into fresh gelatin-coated culture vessels.
6. Return the cultures to a humidified 37°C, 5% CO₂ incubator.
7. Use the recommended split ratio of 1:2.

Preparation for Cell-Based Experiments

• Once cells reach confluence, cultures may be allowed to overgrow for up to 24 hours before cell-based assays, staining, flow cytometry analysis, or other appropriately designed experiments.
• Change the medium daily and pre-wash the cells twice with 1X PBS between medium changes.
• Before an experiment, cells may be filtered through a 40 μm cell strainer to remove cell clumps and most dead or floating large cells.

Cryopreservation

• Use freezing medium containing 10% DMSO, 50% FBS, and 40% culture medium, referenced as CB6916.
• Add 1 mL of freezing medium per vial 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 a liquid nitrogen tank for long-term storage.

Shipping, Storage, and Handling

• Cryopreserved cells are shipped frozen with dry ice overnight.
• Upon arrival, immediately transfer frozen cells to a liquid nitrogen storage tank until ready for use.
• Do not use a −20°C or −80°C freezer for routine long-term storage of the received cryopreserved cells.
• The temporary −80°C step described in the freezing protocol applies only to controlled-rate cryopreservation before transfer to liquid nitrogen.
• Repeated freezing and thawing of cells are not recommended.
• Live-cell shipment may be available upon request.
• Handle all animal cells with caution as potential biohazards because no testing procedure can guarantee the complete absence of infectious agents.

Use and Warranty Information

• This product is distributed for research purposes only.
• It is not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.
• The supplier documentation states that its cell guarantee applies when the cells are used with the specified medium and coating solution, cultured and tested according to the supplied protocol, and evaluated within 35 days of delivery.
• Review the applicable terms and conditions before ordering or beginning cell culture.

Documents

Product Datasheet – C57-6016IM

Current Cell Thawing & Culture Protocol – Cell-Culture-Protocol-CB

Archived Cell Culture Protocol – 1708462493

Cell Culture Products Safety Data Sheet – 1698246847

Frequently Asked Questions