Immortalized GFP-Expressing Mouse Primary Pulmonary Vein Fibroblasts

Description

Research that requires repeated expansion and fluorescence-based observation of tissue-specific mouse fibroblasts can be limited by the finite growth capacity of conventional primary cultures and the additional work needed to introduce a fluorescent marker. Immortalized GFP-Expressing Mouse Primary Pulmonary Vein Fibroblasts provide a cryopreserved, GFP-expressing fibroblast model derived from pulmonary vein tissue of pathogen-free laboratory C57BL/6 mice.

The cells originate from primary mouse pulmonary vein fibroblasts and are supplied as an immortalized GFP-expressing culture. According to the product datasheet, GFP-expressing cells are developed by transduction with GFP-lentiviral particles. The green fluorescent protein produces bright green fluorescence when exposed to blue light, supporting experiments requiring fluorescence-based cell visualization.

Cells are harvested and cryopreserved at passages 2–3. Each vial contains 0.5 × 10⁶ cells per mL and is delivered frozen. The cells display spindle morphology and are characterized by immunofluorescence staining with anti-FSP1/S100A4 antibodies.

Supplier testing reports negative results for bacteria, yeast, fungi, and mycoplasma. Under the specified cell culture conditions, these immortalized GFP-expressing mouse pulmonary vein fibroblasts can be expanded for more than 10 passages at a recommended split ratio of 1:2. Repeated freezing and thawing are not recommended.

Specifications

Features

• Derived from pulmonary vein tissue of pathogen-free laboratory C57BL/6 mice
• Immortalized GFP-expressing fibroblast culture derived from primary pulmonary vein fibroblasts
• GFP expression introduced using GFP-lentiviral particles
• Harvested and cryopreserved at passages 2–3
• Contains 0.5 × 10⁶ cells per mL per vial
• Displays documented spindle 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 for subculture at a split ratio of 1:2
• Cultured in gelatin-coated culture vessels using Complete Fibroblast Medium
• Shipped frozen on dry ice for immediate transfer to liquid nitrogen storage

Applications

These GFP-expressing C57BL/6 mouse pulmonary vein fibroblasts are intended for in vitro research requiring an expandable, tissue-derived fibroblast culture with a fluorescent marker. Suitability for a specific experimental endpoint should be determined by the investigator.

• Fluorescence-based visualization of GFP-expressing fibroblasts
• In vitro culture of mouse pulmonary vein-derived fibroblasts
• Fibroblast morphology observation
• Immunofluorescence staining experiments
• Flow cytometry sample preparation
• Cell-based assays performed under the specified culture conditions
• Experiments requiring an expandable, reporter-labeled mouse fibroblast model

The supplied documents do not establish this product as a validated disease model or confirm suitability for a particular diagnostic, clinical, therapeutic, drug-screening, or in vivo application.

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet using aseptic technique.
• Use culture media, supplements, and reagents sterilized by filtration through a 0.2 μm filter.
• Prewarm only the volume of medium required for immediate use to 37°C.
• Use approximately 5 mL of medium for a T25 flask and 8–10 mL for a T75 flask.
• Avoid repeatedly warming complete cell culture medium.

Gelatin Coating

• Coat sterile culture dishes or flasks with CB6950 Gelatin-Based Coating Solution for 2 minutes.
• Aspirate 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 small amount of ice remains.
2. Remove the vial promptly and disinfect the exterior with 70% ethanol.
3. Transfer the cells to 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 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 Complete Fibroblast Medium.
7. Seed the resuspended cells into a gelatin-coated T25 flask.
8. Incubate at 37°C with 5% CO₂ in a humidified incubator.
9. Leave the newly seeded culture undisturbed for 12–16 hours.
10. Change the medium the following day and then according to the applicable culture protocol.
11. Inspect cell confluence and morphology daily under a microscope.

Expansion and Subculture

1. Remove the culture medium and wash the adherent cells twice with calcium- and magnesium-free 1X PBS.
2. Incubate with prewarmed 0.25% Trypsin-EDTA solution (Catalog No. CB6914) for 3–5 minutes.
3. Use 0.5–1 mL of Trypsin-EDTA for a T25 flask or approximately 2 mL for a T75 flask, following the current culture protocol.
4. When the cells detach, add 5–10 mL of complete medium containing FBS to neutralize the trypsin.
5. Gently pipette the suspension several times to disperse the cells.
6. Seed cells into fresh gelatin-coated flasks or plates at the recommended 1:2 split ratio.
7. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.
8. Monitor confluence and spindle morphology daily.

Preparation for Experiments

• Once cultures reach confluence, the current protocol permits up to 24 hours of additional growth before cell-based assays, staining, FACS analysis, or other designed experiments.
• Change the medium daily during this period and wash twice with calcium- and magnesium-free 1X PBS between medium changes.
• Before an experiment, cells may be passed through a 40 μm cell strainer to remove clumps and large dead or floating cells.

Cryopreservation

• Use freezing medium containing 10% DMSO, 50% FBS, and 40% culture medium, or Cell Biologics Freezing Buffer CB6916.
• Add 1 mL of freezing medium per cryovial and quickly resuspend the cell pellet.
• Place cryovials in a controlled-rate freezing container containing 100% isopropanol at −80°C for 6–12 hours.
• Transfer cryovials to a liquid nitrogen storage tank for long-term storage.

The temporary −80°C step described above is part of the controlled cryopreservation procedure. Received cryopreserved product should be transferred directly from the dry-ice shipping container to liquid nitrogen and should not be routinely stored at −80°C.

Quality Control

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice overnight.
• Upon arrival, transfer the frozen vial immediately to a liquid nitrogen storage system until ready for use.
• The product datasheet specifies liquid nitrogen storage at approximately −180°C.
• Do not use a −20°C or −80°C freezer for routine storage of the received cryopreserved vial.
• Live-cell shipment is available on request according to the product datasheet.
• Repeated freezing and thawing are not recommended.
• Handle all animal cells with caution as potential biohazardous material.
• Follow appropriate institutional biosafety practices and the supplied safety documentation.

Recommended Culture Products

Documents

C57-6078IM.GFP Product Datasheet

Primary Cell Thawing & Culture Protocol

Archived Primary Cell Culture Protocol

Cell Culture Products Safety Data Sheet

Frequently Asked Questions

What are Immortalized GFP-Expressing Mouse Primary Pulmonary Vein Fibroblasts?

They are immortalized GFP-expressing fibroblasts derived from primary pulmonary vein fibroblasts isolated from pathogen-free laboratory C57BL/6 mice. The cells are supplied as a cryopreserved vial for research use.

Are these primary cells or immortalized cells?

The cells originate from primary mouse pulmonary vein fibroblasts but are supplied as an immortalized GFP-expressing culture. In the product name, “primary” describes the original tissue-derived cell source rather than a finite-lifespan primary-cell preparation.

How is GFP introduced into the cells?

The product datasheet states that GFP-expressing cells are developed by transduction with GFP-lentiviral particles.

Which immortalization method is used for C57-6078IM.GFP?

The supplier documentation states that immortalized GFP-expressing cells may be produced through overexpression of SV40 large T antigen or human telomerase reverse transcriptase (hTERT). The available datasheet does not identify which of these methods applies specifically to C57-6078IM.GFP. Contact technical support if confirmation is required before ordering.

What mouse strain and tissue are the cells derived from?

The fibroblasts are derived from pulmonary vein tissue of pathogen-free laboratory C57BL/6 mice.

At what passage are the cells cryopreserved?

Cells are harvested and cryopreserved at passages 2–3.

How many cells are supplied per vial?

According to the product datasheet, each cryopreserved vial contains 0.5 × 10⁶ cells per mL.

How are the mouse pulmonary vein fibroblasts characterized?

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

What quality control testing is documented?

The supplier reports that the cells test negative for bacteria, yeast, fungi, and mycoplasma.

How many passages can the cells be expanded?

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

What medium and coating solution are recommended?

The suggested medium is M2267 Complete Fibroblast Medium w/ Kit – 500 mL. CB6950 Gelatin-Based Coating Solution is recommended for coating culture vessels before cell seeding.

What culture conditions are recommended?

The supplied protocol specifies culture at 37°C in a humidified incubator with 5% CO₂. Cells should be seeded into gelatin-coated culture vessels and monitored daily for confluence and appropriate morphology.

Can the cells be stored at −80°C after arrival?

No. Transfer the frozen vial from the dry-ice shipping container to liquid nitrogen immediately upon arrival. A −80°C freezer is used only as a temporary controlled-freezing step when newly cryopreserving cultured cells according to the supplied protocol.

Can the cells be repeatedly frozen and thawed?

No. Repeated freezing and thawing are not recommended.

What research applications are supported by the available documentation?

The documentation supports in vitro fibroblast culture, GFP fluorescence-based cell visualization, morphology observation, immunofluorescence staining, flow cytometry sample preparation, and cell-based assays conducted under the specified culture conditions. Suitability for a particular experimental endpoint should be determined by the investigator.

Are these cells authorized for clinical or diagnostic use?

No. The cells are for research use only and are not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.