Primary Gingival Fibroblast; Normal, Human, Adult (HGF)

Human gingival fibroblasts (hGF) could potentially be an alternative source of mesenchymal stem cells (MSC) for regenerative medicine studies as they share similar morphology, CD markers, and differentiation lineage.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Obtain one vial of Primary Normal Gingival Fibroblast Cells (ATCC PCS-201-018) from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit (ATCC PCS-201-041) just prior to adding them to the basal medium (ATCC PCS-201-030).
3. Obtain one bottle of Fibroblast Basal Medium (485 mL; ATCC PCS-201-030) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component, as indicated in "A" below, to the bottle of basal medium using a separate sterile pipette for each transfer.

1. If using the Fibroblast Growth Kit-Low Serum, add the indicated volume for each of the following components
   • rh FGF b, 0.5 mL (Final concentration 5 ng/mL)
   • L-glutamine, 18.75 mL (Final concentration 7.5 mM)
   • Ascorbic acid, 0.5 mL (Final concentration 50 µg/mL)
   • Hydrocortisone Hemisuccinate, 0.5 mL (Final concentration 1 µg/mL)
   • rh Insulin, 0.5 mL (Final concentration 5 µg/mL)
   • Fetal Bovine Serum, 10.0 mL (Final concentration 2%)

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components to be added to the complete growth media is summarized below.


2. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional):
   • Penicillin-Streptomycin-Amphotericin B Solution, 0.5 mL (Final concentration Penicillin: 10 Units/mL, Streptomycin: 10 µg/mL, Amphotericin B: 25 ng/mL).
   • Phenol Red, 0.5 mL (Final concentration 33 µM)

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
4. Discard supernatant and resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
5. Refer to the batch-specific information provided on the Certificate of Analysis for the total number of viable cells recovered from this lot of ATCC PCS-201-018.
6. Using the total number of viable cells reported, determine how much surface area can be inoculated to achieve an initial seeding density of 2,500 to 5,000 cells per cm².
7. Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard culture medium.
2. Rinse the cell layer with DPBS solution for 2 minutes to remove all traces of serum that contains trypsin inhibitor.
3. Add 5.0 to 7.0 mL of Trypsin-EDTA solution to the flask and incubate at 37°C .Observe cells under an inverted microscope until cell layer is dispersed (usually within 4 to 6 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Add 5.0 to 7.0 mL of Trypsin Neutralizing Solution (ATCC^® PCS-999-004™) Centrifuge at 125 x g; 10 ± 2 minutes. Discard supernatant and resuspend the cell pellet with 8 mL of complete growth media. Gently break cell pellet by pipetting repeatedly.
4. Count cells. Seed 2,500 to 5,000 viable cells per cm². Add appropriate volume of the cell suspension to new culture vessels.
5. Incubate cultures at 37°C.

The flask was seeded with cells (see specific batch information) grown and completely filled with medium at ATCC to prevent loss of cells during shipping.

1. Upon receipt visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination.  Also check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable).
2. If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium.  The shipping medium can be saved for reuse.  Incubate the cells at 37°C in a 5% CO₂   in air atmosphere until they are ready to be subcultured.
3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 x g for 5 to 10 minutes.  Remove shipping medium and save.  Resuspend the pelleted cells in 10 mL of this medium and add to 25 cm² flask.  Incubate at 37°C in a 5% CO₂  in air atmosphere until cells are ready to be subcultured.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.