Canine Primary Small Intestinal Fibroblasts

Description

Research involving tissue-specific primary fibroblasts requires a cell source with documented species, tissue origin, passage, characterization, and culture requirements. For laboratories working with canine small intestinal fibroblasts, these details are important when planning cell recovery, expansion, staining, and other cell-based experiments.

Canine Primary Small Intestinal Fibroblasts, Catalog No. D-6025, are primary intestinal fibroblast cells isolated from the small intestinal tissue of beagle dogs. The cells are grown in gelatin-precoated tissue culture flasks using Cell Biologics’ Complete Growth Medium.

Cells are harvested and cryopreserved at passage 2–3. Each vial contains cells at a concentration of 0.5 × 10⁶ cells per mL and is delivered frozen. Live cell shipment is also available on request in T25 or T75 tissue culture flasks.

These beagle dog small intestinal fibroblasts display spindle-shaped morphology and are characterized by immunofluorescence using anti-fibronectin antibodies. They are documented as negative for bacteria, yeast, fungi, and mycoplasma.

Under the specified primary cell culture conditions, the cells can be expanded for 3–5 passages using a recommended split ratio of 1:2. Repeated freezing and thawing are not recommended.

Canine Primary Small Intestinal Fibroblasts are supplied for research use only. They are not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.

Specifications

Features

• Primary fibroblasts isolated from beagle dog small intestinal tissue
• Cryopreserved at passage 2–3 for frozen delivery
• Supplied at a documented concentration of 0.5 × 10⁶ cells per mL
• Spindle-shaped fibroblast morphology
• Characterized by immunofluorescence using anti-fibronectin antibodies
• Documented as negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for 3–5 passages under the specified culture conditions
• Recommended 1:2 split ratio for primary fibroblast expansion
• Supported by defined gelatin-coating, thawing, culture, expansion, and freezing procedures
• Live T25 or T75 flask shipment available on request

Applications

Canine Primary Small Intestinal Fibroblasts provide a tissue-specific primary cell source for research workflows requiring fibroblasts derived from beagle dog small intestinal tissue. Application suitability should be determined by the researcher according to the experimental design and the documented characteristics of these primary cells.

• Canine intestinal fibroblast culture: Recovery, maintenance, and expansion of primary fibroblasts derived specifically from canine small intestinal tissue.
• Primary cell culture studies: Research workflows requiring adherent, spindle-shaped canine primary fibroblasts cultured under defined conditions.
• Cell-based assays: Preparation of cultured canine intestinal fibroblasts for appropriately designed cell-based experiments.
• Immunofluorescence staining: Staining and microscopy workflows using cultured primary intestinal fibroblasts.
• Flow cytometry analysis: Preparation of cultured cells for FACS analysis following appropriate filtration and experimental controls.
• Cell morphology assessment: Microscopic monitoring of fibroblast morphology and culture confluence during recovery and expansion.
• Primary fibroblast expansion: Expansion for 3–5 passages using a recommended split ratio of 1:2 under the specified culture conditions.
• Preparation for designed experiments: Generation of cultured cell material for downstream research selected and validated by the laboratory.

The supplied documents do not make a disease-specific, diagnostic, therapeutic, clinical, drug-screening, or veterinary performance claim. Researchers should independently determine suitability for their intended experiments.

Quality Control

Cell Culture Workflow

Culture Preparation

• Perform all primary cell culture procedures in a biosafety cabinet.
• Use aseptic techniques to minimize microbial contamination.
• Sterile-filter culture media, supplements, and reagents through a 0.2 μm filter.
• Prewarm only the volume of culture medium required for immediate use to 37°C.
• When recovering frozen cells, changing medium, or splitting cells, prewarm 5 mL of medium for a T25 flask or 8–10 mL for a T75 flask.
• Avoid repeatedly warming complete culture medium.

Gelatin Coating

1. Add CB6950 Gelatin-Based Coating Solution to a sterile culture flask or dish.
2. Allow the coating solution to contact the culture surface for 2 minutes.
3. Aspirate the excess coating solution before seeding the cells.

Recovery from a Cryovial

1. Thaw the cryovial rapidly in a 37°C water bath for less than 1 minute, or until only a few pieces of ice remain.
2. Immediately remove the vial and disinfect the exterior with 70% ethanol.
3. Transfer the cells into a sterile 15 mL conical tube containing 5 mL of prewarmed Cell Biologics cell culture medium.
4. Rinse the cryovial with an additional 0.5–1 mL of medium to recover remaining cells.
5. Centrifuge the cells at 200 × g for 5 minutes. Centrifugation after thawing is recommended to remove residual DMSO.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of Cell Biologics Cell Culture Growth Medium.
7. Seed the resuspended cells into a T25 flask precoated with CB6950 Gelatin-Based Coating Solution.
8. Place the flask in a humidified incubator at 37°C with 5% CO₂ and culture for 12–16 hours.
9. Change the medium on the second day and every 24 hours thereafter to remove nonadherent cells and replenish nutrients.
10. Use 5 mL of medium for a T25 flask or 10 mL for a T75 flask during daily medium changes.
11. Check cell confluence and morphology daily under a microscope.

Live Cell Arrival Handling

1. Upon receipt of live cells in a T25 or T75 flask, remove the sticker from the filter cap.
2. Place the flask, containing 6–20 mL of existing culture medium, in a 37°C incubator with 5% CO₂ for 1 hour.
3. Replace the existing medium with the desired Cell Biologics cell culture medium prewarmed to 37°C.
4. Subculture the cells at a 1:2 ratio when they reach 90% confluency.
5. Cells may be frozen for future use when they reach 95–100% confluency.

Primary Cell Expansion

1. Remove the culture medium and wash the cells twice at room temperature with calcium- and magnesium-free 1X PBS.
2. Add CB6914 0.25% Trypsin-EDTA Solution prewarmed to 37°C.
3. Use approximately 0.5–1 mL for a T25 flask or 2 mL for a T75 flask.
4. Incubate the cells with Trypsin-EDTA for 3–5 minutes and monitor cell detachment.
5. Once the cells detach, immediately add 5–10 mL of complete culture medium containing fetal bovine serum to neutralize the trypsin.
6. Gently pipette the cell suspension up and down several times.
7. Split a confluent T25 flask into two T25 flasks, using 5 mL of medium per flask.
8. Split a confluent T75 flask into two T75 flasks, using 10 mL of medium per flask.
9. Seed the cells into fresh gelatin-coated culture vessels at the recommended split ratio of 1:2.
10. Return the culture vessels to a humidified incubator at 37°C with 5% CO₂.
11. Change the medium on the second day and every 24 hours thereafter.
12. Before each medium change, wash the cells twice with calcium- and magnesium-free 1X PBS.
13. Check cell confluence and morphology daily under a microscope.

Cryopreservation

1. Remove the culture medium and rinse the adherent cell layer two or three times with sterile calcium- and magnesium-free 1X PBS.
2. Add CB6914 0.25% Trypsin-EDTA Solution prewarmed to 37°C.
3. Use approximately 0.5–1 mL for a T25 flask or 2–3 mL for a T75 flask.
4. Incubate for 3–5 minutes and monitor cell detachment.
5. Once the cells detach, immediately add 10 mL of complete culture medium containing fetal bovine serum to neutralize the trypsin.
6. Transfer the cell suspension to a centrifuge tube and centrifuge at 200 × g for 5 minutes.
7. Carefully remove the supernatant.
8. Add 1 mL of CB6916 Freezing Medium to each cryovial and quickly resuspend the cell pellet.
9. Place the cryovials in a controlled-rate freezing container filled with 100% isopropanol.
10. Freeze at −80°C for 6–12 hours as an intermediate controlled-freezing step.
11. Transfer the cryovials to a liquid nitrogen tank for long-term storage.

Recommended Freezing Ratios

• Confluent primary cells grown in one T75 flask can be frozen into two cryovials.
• Confluent primary cells grown in one T25 flask can be frozen into one cryovial.

Preparation Before Experiments

• Once cells reach confluence, they may be allowed to overgrow for 24 hours before cell-based assays, staining, FACS analysis, or other designed experiments.
• During this period, change the medium daily and wash the cells twice with calcium- and magnesium-free 1X PBS between medium changes.
• Before an experiment, filter the cells through a 40 μm cell strainer to remove cell clumps and most large dead or floating cells.

Storage and Handling

• Cryopreserved cells are shipped overnight on dry ice.
• Immediately transfer frozen cells from the dry ice shipping container to liquid nitrogen at −180°C upon arrival.
• Do not store delivered primary cells in a −20°C or −80°C freezer.
• The −80°C step described in the freezing protocol is only an intermediate controlled-freezing step for 6–12 hours before transfer to liquid nitrogen.
• Repeated freezing and thawing are not recommended.
• Live cell shipment is available on request in T25 or T75 flasks.
• Handle animal-derived cells with caution and treat them as potential pathogens because no test procedure can completely guarantee the absence of infectious agents.
• Follow applicable institutional biosafety requirements when handling and culturing the cells.

Documents

D-6025

Cell-Culture-Protocol-CB

Frequently Asked Questions

What are Canine Primary Small Intestinal Fibroblasts?

They are primary fibroblast cells isolated from the small intestinal tissue of beagle dogs. Catalog D-6025 cells are cultured in gelatin-precoated tissue culture flasks and supplied for research use.

Are these canine intestinal fibroblasts primary cells or an immortalized cell line?

They are primary canine small intestinal fibroblasts. The supplied documents do not describe the cells as immortalized or genetically modified.

What is the tissue source of D-6025 cells?

D-6025 cells are isolated from the small intestinal tissue of beagle dogs.

At what passage are the cells cryopreserved?

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

How many cells are supplied?

The product datasheet states that each vial contains cells at a concentration of 0.5 × 10⁶ cells per mL. The document does not specify the total volume of each vial.

What morphology do the cells display?

Canine Primary Small Intestinal Fibroblasts display spindle-shaped morphology.

How are the cells characterized?

The cells are characterized by immunofluorescence using anti-fibronectin antibodies.

What contamination testing is documented?

The cells are documented as 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 3–5 passages under the specified primary cell culture conditions.

What split ratio is recommended?

The recommended split ratio for these primary fibroblasts is 1:2. A confluent T25 flask can be split into two T25 flasks, and a confluent T75 flask can be split into two T75 flasks.

What medium is recommended for these canine primary fibroblasts?

M2267 Complete Fibroblast Medium w/ Kit, 500 mL, is the suggested medium.

Do the culture vessels require gelatin coating?

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

What culture conditions are recommended?

Culture the cells in a humidified incubator at 37°C with 5% CO₂. Monitor cell morphology and confluence daily.

How should the cryovial be thawed?

Thaw the vial rapidly in a 37°C water bath for less than 1 minute, or until only a few pieces of ice remain. Immediately remove the vial and disinfect the exterior with 70% ethanol.

Should residual freezing medium be removed after thawing?

Yes. The protocol recommends centrifuging the cells at 200 × g for 5 minutes after thawing to remove residual DMSO-containing freezing medium.

How often should the culture medium be changed?

Change the medium on the second day after seeding and every 24 hours thereafter. The protocol specifies 5 mL for a T25 flask and 10 mL for a T75 flask during routine medium changes.

Are live T25 or T75 flask shipments available?

Yes. The product datasheet states that live cell shipment is available on request. The culture protocol provides arrival-handling instructions for live cells shipped in T25 or T75 flasks.

How should live cells be handled upon arrival?

Remove the sticker from the filter cap and place the flask with its existing culture medium in a 37°C, 5% CO₂ incubator for 1 hour. Then replace the existing medium with the desired Cell Biologics cell culture medium prewarmed to 37°C.

How should cryopreserved cells be stored upon arrival?

Immediately transfer the cryopreserved cells from the dry ice shipping container to a liquid nitrogen storage tank at −180°C.

Can delivered cells be stored at −80°C?

No. The product datasheet states that delivered primary cells must not be stored in a −20°C or −80°C freezer. The −80°C step in the freezing protocol is only an intermediate controlled-freezing step for 6–12 hours before transfer to liquid nitrogen.

Can the cells be repeatedly frozen and thawed?

No. Repeated freezing and thawing are not recommended.

What research applications are documented for these cells?

The protocol describes primary cell culture and expansion, cell-based assays, staining, FACS analysis, morphology monitoring, and preparation of cultured cells for designed experiments. Suitability for a specific experiment should be independently determined by the researcher.

Are D-6025 cells intended for clinical, diagnostic, or therapeutic use?

No. These canine primary small intestinal fibroblasts are for research use only and are not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.