GFP Expressing C57BL/6 Mouse Primary Coronary Artery Smooth Muscle Cells

Description

When selecting a human dermal fibroblast model for extended in vitro culture, researchers need clear information about tissue origin, passage, characterization, expansion capacity, and culture requirements. H-6068IM Immortalized Human Primary Dermal Fibroblasts - Neonatal are isolated from human skin tissue and cultured in gelatin pre-coated tissue culture flasks with Complete Growth Medium.

These neonatal human dermal fibroblasts are harvested at Passages 2–3 and supplied as cryopreserved cells. Each vial contains 0.5 × 10⁶ cells per mL. The cells exhibit spindle-shaped fibroblast morphology and are characterized by immunofluorescence staining with anti-fibronectin antibodies.

Under the specified culture conditions, the immortalized human fibroblast cells can be expanded for more than 10 passages using a recommended split ratio of 1:2. The product datasheet reports negative results for bacteria, yeast, fungi, mycoplasma, HIV-1, hepatitis B, and hepatitis C.

Immortalized cells are derived from primary cells with an extended life span. The supplied product documentation states that the manufacturer's primary cells are immortalized through overexpression of simian virus 40 large T-antigen (SV40) or human telomerase reverse transcriptase (hTERT). The documentation does not identify which of these methods is used specifically for H-6068IM.

Repeated freezing and thawing of the cells are not recommended.

Specifications

Features

• Immortalized human primary dermal fibroblasts isolated from neonatal skin tissue
• Harvested and cryopreserved at Passages 2–3
• Supplied at a documented concentration of 0.5 × 10⁶ cells per mL
• Exhibit spindle-shaped fibroblast morphology
• Characterized by anti-fibronectin immunofluorescence staining
• Can be expanded for more than 10 passages under the specified culture conditions
• Recommended for subculture at a 1:2 split ratio
• Tested negative for bacteria, yeast, fungi, mycoplasma, HIV-1, hepatitis B, and hepatitis C
• Cultured in vessels coated with CB6950 Gelatin-Based Coating Solution
• Shipped cryopreserved on dry ice for immediate transfer to liquid nitrogen storage

Applications and Experimental Fit

H-6068IM provides a cultured human skin-derived fibroblast model for research workflows requiring immortalized neonatal human dermal fibroblasts and expansion beyond the limited number of passages typically associated with primary cell culture.

Before ordering, researchers should confirm that the documented neonatal skin origin, immortalized status, anti-fibronectin characterization, greater than 10-passage expansion capacity, and gelatin-coated culture system are appropriate for the intended experimental design.

The supplied product datasheet does not provide application-specific validation data for wound-healing assays, skin-aging studies, toxicology testing, cosmetic testing, fibrosis models, or 3D skin models. Suitability for a particular assay should therefore be determined by the investigator.

Cell Culture Requirements

Cell Culture Workflow

Culture Preparation

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

Gelatin Coating

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

Recovery of Cryopreserved Cells

1. Thaw the cryovial rapidly in a 37°C water bath for less than 1 minute or until only a few ice pieces remain.
2. Immediately remove the vial and wipe the 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 the 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 Growth Medium.
7. Seed the resuspended cells into a gelatin-coated T25 flask.
8. Place the flask in a humidified incubator at 37°C with 5% CO₂.
9. Leave the culture undisturbed for 12–16 hours.
10. Change the medium on the second day and daily thereafter according to the current culture protocol.
11. Check cell confluence and morphology daily under a microscope.

Subculture

1. Remove the culture medium and wash the cells twice with 1X phosphate-buffered saline without calcium or magnesium.
2. Incubate the cells with CB6914 0.25% Trypsin-EDTA prewarmed to 37°C 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 Growth Medium containing FBS to neutralize the trypsin.
5. Gently pipette the cell suspension up and down several times.
6. Seed the cells into fresh gelatin-coated culture vessels using the recommended 1:2 split ratio.
7. Return the culture vessels to a humidified incubator at 37°C with 5% CO₂.
8. Change the medium on the second day and daily thereafter.
9. Observe cell confluence and morphology each day.

Preparation for Experiments

• Once the cells reach confluence, the culture may be allowed to overgrow for 24 hours before cell-based assays, staining, FACS analysis, or other planned experiments.
• During this period, 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.

Freezing Cultured Cells

1. Remove the culture medium and rinse the adherent cell layer 2–3 times with sterile 1X PBS without calcium or magnesium.
2. Incubate with prewarmed CB6914 0.25% Trypsin-EDTA for 3–5 minutes.
3. Neutralize the trypsin with Complete Growth Medium containing FBS.
4. Transfer the cell suspension to a centrifuge tube and centrifuge at 200 × g for 5 minutes.
5. Carefully remove the supernatant.
6. Add 1 mL of CB6916 Freezing Medium per vial and quickly resuspend the cell pellet.
7. Place the vials in a controlled-rate freezing container filled with 100% isopropanol at -80°C for 6–12 hours.
8. Transfer the frozen vials to a liquid nitrogen tank for long-term storage.

Quality Control

Shipping, Storage, and Handling

• Cryopreserved cells are shipped frozen on dry ice overnight.
• Live cell shipment is available on request.
• Upon arrival, transfer cryopreserved cells immediately to a liquid nitrogen storage tank at -180°C until ready for use.
• Do not use a -20°C or -80°C freezer for routine storage of the supplied cryopreserved cells.
• Repeated freezing and thawing are not recommended.
• Handle the cells with caution and treat all primary-cell-derived materials as potential biohazards because no testing procedure can guarantee the complete absence of infectious agents.
• Use appropriate laboratory safety procedures when handling the cells and associated culture materials.

Product Guarantee Requirements

The supplied product datasheet states that the manufacturer's guarantee applies when the cells are purchased and used with the manufacturer's recommended medium and coating solution, cultured and tested according to the specified protocol, and evaluated within 35 days from the date of delivery.

Review the applicable terms and culture documentation before beginning cell recovery and testing.

Authorized Use

H-6068IM Immortalized Human Primary Dermal Fibroblasts - Neonatal are supplied for research use only. They are not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.

Transfer or resale of the cells or associated products to other markets, organizations, or individuals is prohibited without the manufacturer's written consent. Applicable terms and conditions must be accepted before submitting an order.

Documents

H-6068IM Product Datasheet

Cell-Culture-Protocol-CB – Current Primary Cell Culture Protocol

1708462493 – Primary Cell Culture Protocol

1698246847 – Cell Culture Products Safety Data Sheet

Frequently Asked Questions