THESE INSTRUCTIONS APPLY TO ORDERS CONTAINING THE FOLLOWING CELL PRODUCTS

Cryopreserved Cells (Single donor)

Proliferating Cells

Proliferating Cells in Preseeded? 96-well Plates

1. Check all containers for leakage or breakage. Fibroblasts are available in 6, 12, 24 and 48 well plates, T-150 and T-225 flasks. Please call your Technical Specialist for details and also prices.

2. For cryopreserved cells - If there is dry ice left in the package, place cryopreserved cell cryovials immediately into liquid nitrogen. If no dry ice is left in the package, thaw and use them immediately. 
   
   For proliferating cells - Swab down the flask of proliferating cells with 70% ethanol or isopropanol, then place the flask in 37·C, 5% CO2, humidified incubator and allow to equilibrate for three to four hours. After cells have equilibrated, remove shipping medium from the flask following instructions on page 20. 
   

3. Store cell culture medium in a 4·C refrigerator.
   

4. If you plan to proceed within 3 days, store all growth supplements, HEPES Buffered Saline Solution (HEPES- BSS) and Trypsin Neutralizing Solution at 4·C. Trypsin/EDTA Solution has a limited shelf life or activation at 4·C. If, upon arrival, Trypsin/EDTA is thawed, immediately aliquot and refreeze at -20·C. If frozen, store at - 20·C. If you do not plan to set up the cell culture within 3 days, store all growth supplements and subculture reagents in a -20·C freezer.

Please read and follow these instructions carefully and completely. BioWhittaker is not responsible for product loss due to improper receipt and handling of its products by customers. Replacement product will be sent at the customer's expense.

*AA-1005*
AA-1005-1 Rev. 04/98

Fibroblast Cell System

1. Normal Human Dermal Fibroblasts and Normal Human Lung Fibroblasts from single donors, as either:

The proliferating cultures are shipped in flasks or plates filled with medium. The cells should be between 30 and 100% confluent upon arrival. A Certificate of Analysis is provided with each cell strain and indicates QC performance results and donor information.

The cryopreserved cultures are shipped in a screw cap cryovial containing approximately 500,000 cells. A Certificate of Analysis is provided with each cell strain and indicates date of cryopreservation, QC performance results, donor information and the number of cells contained in the cryovial.

2. Fibroblast Growth Medium (FGM?), as either:

Fibroblast Growth Medium BulletKit? (FGM? BulletKit?) (CC-3130), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)

1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
5 mg/ml Insulin (CC-4021) 0.5 ml
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml

(or)

Fibroblast Growth Medium-2 BulletKit? (FGM?-2 BulletKit? ) (CC-3132), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)

1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
5 mg/ml Insulin (CC-4021) 0.5 ml
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml
10 ml FBS (Fetal Bovine Serum) (CC-4101)

3. ReagentPackTM (CC-5034) contains one 100 ml bottle of each of the following subculture reagents:

HEPES Buffered Saline Solution (HEPES-BSS) (CC-5022)1 x 100 ml bottle
Trypsin/EDTA Solution (T/E) (CC-5012) 1 x 100 ml bottle
Trypsin Neutralizing Solution (TNS) (CC-5002) 1 x 100 ml bottle

NOTE: If you use a different Clonetics? medium, see Appendix A.

General Information

Product Applications
Clonetics? Normal Human NHDF and NHLF are:

1. FOR RESEARCH USE ONLY.

2. NOT approved for human or veterinary use, for application to humans or animals, or for in vitro diagnostic procedures.

Materials Not Provided
Fibroblast Cell Systems do not contain plasticware, glassware or other laboratory equipment used in a cell culture laboratory. Individual components are available separately.

Product Warranty 
CULTURES HAVE A FINITE LIFESPAN IN VITRO. BioWhittaker warrants its Clonetics? cells in the following manner only if Clonetics? media and reagents are used.

1. NHDF and NHLF cryopreserved cultures are assured for experimental use for fifteen population doublings.

2. NHDF and NHLF proliferating cultures are assured for experimental use for ten population doublings.

3. Additional population doublings and subcultures are possible, but growth rate, biological responsiveness and function deteriorate with subsequent passage.

4. NHDF and NHLF can become irreversibly contact-inhibited if maintained at confluence for more than two days. To avoid the loss of your cells and forfeiture of your warranty, we recommend that you subculture cells before they reach 90% confluence.

Cell Isolation 
Fibroblast cultures are established at BioWhittaker's cell culture facility from normal human tissue.

1. NHDF are isolated according to proprietary procedures, incubated until they approach 90% confluence, harvested and cryopreserved as frozen primaries.

2. NHLF are isolated according to proprietary procedures, incubated until they reach 90% confluence, subcultured once and cryopreserved as frozen secondary cultures.

3. NHDF and NHLF are cryopreserved in FGM? (or) FGM?-2 supplemented with 10% v/v fetal bovine serum and 10% v/v dimethylsulfoxide as a cryopreservation solution to improve cell viability and seeding efficiency upon thawing.

Medium Information

Quality Control 
NHDF and NHLF are cultured without antimicrobial agents and assayed to ensure the absence of microbial contamination after cryopreservation.

1. All cell strains test negative by PCR(1) for HIV-1, hepatitis-B and hepatitis-C.

2. After recovery from liquid nitrogen, cells are tested for viability, growth rate, morphology, seeding efficiency, proliferative capacity, mycoplasma, yeast, fungus and bacteria. Each culture meets Clonetics? product specifications for proliferative capacity, (i.e., 15 cumulative population doublings after thaw).

3. Additional Testing: 
   

   Cell Type	Von Willebrand Factor	Smooth Muscle Alpha Actin	Cytokeratins 18 & 19
   NHLF	Negative	Negative	Negative

4. Before shipping, all basal media, growth factors and cell culture reagents are tested for sterility and cell culture performance.

Subculture Reagent Storage
1. Subculture reagents are stored at -20·C until shipped from BioWhittaker's Distribution Centers.

2. Subculture reagents may thaw during transport. They may be refrozen once.

3. Subculture reagents can be stored at -20·C for up to one year after thawing once and refreezing.

4. To keep Trypsin/EDTA fresh and active after thawing, you should aliquot it into five 20 ml sterile centrifuge tubes and refreeze at -20·C. Trypsin/EDTA may be stored frozen up to one year.

5. We recommend that HEPES-BSS and the Trypsin Neutralizing Solution, once stored at 4·C, be used within one month.