[These primary cells are not known to harbor an agent recognized to cause disease in healthy adult humans. Handle as a potentially biohazardous material under at least Biosafety Level 1 containment. Cells derived from primate lymphoid tissue may fall under the regulations of 29 CFR 1910.1030 Bloodborne Pathogens.  

ATCC recommends that appropriate safety procedures be used when handling all primary cells and cell lines, especially those derived from human or other primate material. Detailed discussions of laboratory safety procedures are provided in Laboratory Safety: Principles and Practice, 2nd ed. (ASM Press, Washington, DC) (Fleming et al., 1995) and Caputo, J.L. Biosafety procedures in cell culture. (1988) J. Tissue Culture Methods 11:223.

Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.]

All tissues used for isolation are obtained under informed consent and conform to HIPAA standards to protect the privacy of the donor’s personal health information. It is best to use caution when handling any human cells. We recommend that all human cells be accorded the same level of biosafety consideration as cells known to carry HIV. With infectious virus assays or viral antigen assays, even a negative test result may leave open the possible existence of a latent viral genome.

1. Obtain one Keratinocyte 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 them 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 Dermal Cell Basal Medium (485 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 indicated volume of each growth kit component, as indicated in Table 1, to the bottle of basal medium using a separate sterile pipette for each transfer.

       Table 1. Keratinocyte Growth Kit Components 

Component	Volume	Final Concentration
Bovine Pituitary Extract (BPE)	2.0 mL	0.4%
rh TGF-a	0.5 mL	0.5 ng/mL
L-Glutamine	15.0 mL	6 mM
Hydrocortisone Hemisuccinate	0.5 mL	100 ng/mL
rh Insulin	0.5 mL	5 mg/ml
Epinephrine	0.5 mL	1.0 mM
Apo-Transferrin	0.5 mL	5 mg/ml

 

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components (GA solution or PSA solution) to be added to the complete growth media is summarized in Table 2.

 

Table 2. 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.

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