Primary Human Epidermal Keratinocytes Cell Culture System

These products are available in the US, Canada, and select European countries.

• Human Epidermal Keratinocyte Cell Culture System
• Quality Control for Primary Human Epidermal Keratinocyte Cell Culture System
• Materials for Primary Keratinocyte Culture

Human Epidermal Keratinocyte Cell Culture System

Epidermal keratinocytes make up approximately 90% of the cells of the epidermis. Keratinocytes originate in the stratum basale and migrate to the stratum corneum, the outermost barrier layer of the skin. During the gradual differentiation process, keratinocytes undergo significant morphological changes, ultimately becoming nucleus-free, flat, and highly keratinized squamous cells in the stratum corneum.

PromoCell^® Normal Human Epidermal Keratinocytes are available from single or pooled donors. The cells are isolated from juvenile foreskin or from adult normal human tissue collected from various sites that include face, breast, abdomen, and thigh. Shortly after isolation, normal Human Epidermal Keratinocytes are cryopreserved at passage 2 (P2) using proprietary serum-free freezing medium, Cryo-SFM. Each thawed cryovial contains at least 500,000 viable cells. Normal Human Epidermal Keratinocytes are also available as proliferating cell cultures generated from cryopreserved cells that have been thawed and then cultured for three days.

Cultivation Protocol for Normal Human Epidermal Keratinocytes

Keratinocyte Growth Medium 2 is a serum-free medium optimized for the in vitro cultivation of epidermal keratinocytes without feeder cells. The medium can also be used for cultivating horse, porcine, mouse, and rat keratinocytes (with appropriate calcium chloride concentration adjustment, as needed). Keratinocyte Growth Medium 2 is also suitable for rat corneal keratinocytes.

Keratinocyte Growth Medium 2 is available in two formats: Medium (ready-to-use) and Medium Kit. The Medium (ready-to-use) option includes a 500 mL bottle of Basal Medium and one vial of SupplementMix. The Medium Kit includes a 500 mL bottle of Basal Medium and the SupplementPack, which is a set of individual vials with pre-measured supplements, giving the user full control over the media formulation. Adding the SupplementMix or the SupplementPack to the Basal Media constitutes the complete Keratinocyte Growth Medium 2. The Basal Medium (with or without phenol red), SupplementMix, and SupplementPack can also be purchased separately.

Keratinocyte Growth Medium 2 is recommended for

• Normal Human Epidermal Keratinocytes (NHEK), juvenile foreskin
• Normal Human Epidermal Keratinocytes (NHEK), adult donor

Keratinocyte Growth Medium 2 Supplementation Details

Keratinocyte Growth Medium 2 includes all the components necessary for optimally growing human keratinocytes. These include bovine pituitary extract, epidermal growth factor, insulin, and transferrin (see table below for details). Keratinocyte Growth Medium 2 does not contain antibiotics or antimycotics and is formulated for use in an incubator with 5% atmospheric CO₂.

Final supplement concentrations in Keratinocyte Growth Medium (after addition)

Component	Final Concentration
Bovine Pituitary Extract	0.004 mL / mL
Epidermal Growth Factor (recombinant human)	0.125 ng / mL
Insulin (recombinant human)	5 μg / mL
Hydrocortisone	0.33 μg / mL
Epinephrine	0.39 μg / mL
Transferrin (recombinant human)	10 μg / mL
CaCl2	0.06 mM

Preparing Supplemented Media for Human Epidermal Keratinocytes

• Thaw the Keratinocyte Growth Medium 2 SupplementMix or SupplementPack at 15 – 25 °C.
• Aseptically mix the supplement solutions by gently pipetting up and down.
• Transfer the entire content of each supplement to the Keratinocyte Basal Medium.
• Close the bottle and swirl gently to form a homogenous mixture.

Storage and Stability of Primary Human Epidermal Keratinocytes and Keratinocyte Growth Media Products

• Upon arrival, immediately store the Keratinocyte Basal Medium in the dark at 4 – 8 °C; do not freeze. Store the Keratinocyte Growth Medium SupplementMix or SupplementPack at -20 °C.
• If stored appropriately, the products are stable until the expiration date stated on the label.
• Once supplements are added to the Keratinocyte Basal Medium, the shelf life of the complete medium is six weeks at 4 – 8 °C. Do not freeze the complete medium.
• To use, prewarm only the required aliquot of the complete Keratinocyte Growth Medium and keep the remaining medium refrigerated at 4 – 8 °C.
• Upon arrival, store cryopreserved cells in liquid nitrogen, or seed directly.
• Proliferating cells must be processed immediately on arrival.

Quality Control for Primary Human Epidermal Keratinocytes and Keratinocyte Growth Media

Normal Human Epidermal Keratinocytes Quality Control

The following rigid quality control tests are performed on each lot of Human Epidermal Keratinocytes:

• Cell morphology, adherence rate, and cell viability testing
• Immunohistochemical testing for the cell type-specific marker cytokeratin
• Growth performance testing through multiple passages up to 15 population doublings (PD) under culture conditions without antibiotics and antimycotics.
• Testing for the absence of HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2 and microbial contaminants (fungi, bacteria, and mycoplasma)
• A detailed certificate of analysis (CoA) for each lot is available for download.

Normal Human Epidermal Keratinocytes Specifications

Product	Plating Density	Passage after Thawing	Marker	Population Doublings
Normal Human Epidermal Keratinocytes (NHEK), juvenile foreskin, single donor	5,000 cells per cm²	P2	Cytokeratin+	> 15
Normal Human Epidermal Keratinocytes (NHEK), juvenile foreskin, pooled	5,000 cells per cm²	P2	Cytokeratin+	> 15
Normal Human Epidermal Keratinocytes (NHEK), adult, single donor	5,000 cells per cm²	P2	Cytokeratin+	> 15
Normal Human Epidermal Keratinocytes (NHEK), adult, pooled	5,000 cells per cm²	P2	Cytokeratin+	> 15

Keratinocyte Growth Medium 2 Quality Control

All lots of Keratinocyte Growth Medium 2 are subjected to comprehensive quality control testing. The tests check for growth promoting activity, adherence rate, and typical morphology of primary human keratinocytes. Approved in-house lots of media are used as a reference. Additionally, microbial testing ensures that all lots of Keratinocyte Growth Medium 2 are free of contaminants (fungi, bacteria, mycoplasma).

Intended Use of Primary Human Epidermal Keratinocytes and Keratinocyte Growth Media

Primary Human Epidermal Keratinocytes and Keratinocyte Growth Media are for in vitro use only and not for diagnostic or therapeutic procedures. For safety information, please see appropriate MSDS.

Note: PromoCell^® media have low or zero serum content and are not suitable for trypsin neutralization (for example, when subculturing the cells).  We recommend using our DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.