Fibroblast Growth Kit-Serum-free

Your experimental design will dictate which Fibroblast Growth Kit should be used. The use of Fibroblast Growth Kit–Serum-Free creates a completely defined medium for the serum-free culture of human fibroblasts. The rate of proliferation is equal to or greater than media supplemented using FBS (at concentrations ranging from 2% to 10%) through 10 population doublings. (Proliferation rate may slow under serum-free conditions after 10 population doublings.) Under these conditions, serum-free human feeder cell layers can be established for stem cell culture, or as a model system to study wound healing, skin aging, gene delivery, toxicology and basic questions of cell biology. 

Use of the Fibroblast Growth Kit–Low Serum (ATCC PCS-100-041) will support a faster rate of proliferation compared to Fibroblast Growth Kit–Serum-Free (ATCC PCS-100-040). An upper limit for population doublings has not been established for primary fibroblasts when cultured with Fibroblast Basal Medium supplemented with Fibroblast Growth Kit–Low Serum (2% FBS). Under these conditions, primary fibroblast cultures have been maintained for 30+ population doublings, performing well when used as feeder layers to support human ES cell cultures.

1. Obtain one growth kit from the freezer; make sure that the caps of all components are tight.

2. Thaw the components of the growth kit just prior to adding to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath, and shake to dissolve any precipitates, prior to adding to the basal medium.
3. Obtain one bottle of Fibroblast Basal Medium (480 mL) 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 volume of each growth kit component, as indicated in Table 1 or 2, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. If using the Fibroblast Growth Kit–Serum-Free (ATCC^® PCS-201-040), add the indicated volume for each component in the order shown.

Component	Volume	Final Concentration
L-glutamine	18.75 mL	7.5 mM
Hydrocortisone Hemisuccinate	0.5 mL	1 µg/mL
HLL Supplement	1.25 mL	HSA 500 µg/mL Linoleic Acid 0.6 µM Lecithin 0.6 µg/mL
rh FGF basic	0.5 mL	5 ng/mL
rh EGF / TGF  β-1 Supplement	0.5 mL	5 ng/mL 30 pg/mL
rh Insulin	0.5 mL	5 µg/mL
Ascorbic acid	0.5 mL	50 µg/mL

Table 2. If using the Fibroblast Growth Kit–Low

Serum (ATCC^® PCS-201-041), add the indicated volume for each of the following components:

Component	Volume	Final Concentration
rh FGF basic	0.5 mL	5 ng/mL
L-glutamine	18.75 mL	7.5 mM
Ascorbic acid	0.5 mL	50 µg/mL
Hydrocortisone hemisuccinate	0.5 mL	1 µg/mL
rh Insulin	0.5 mL	5 µg/mL
Fetal Bovine Serum	10.0 mL	2%

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of each optional component to be added to the complete growth media is summarized in Table 3.

Table 3. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional)

Component	Volume	Final Concentration
Gentamicin-Amphotericin B Solution	0.5 mL	Gentamicin: 10 µg/mL Amphotericin B: 0.25 µg/mL
Penicillin-Streptomycin-Amphotericin B Solution	0.5 mL	Penicillin: 10 Units/mL Streptomycin: 10 µg/mL Amphotericin B: 25 ng/mL
Phenol Red	0.5 mL	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.

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.

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