Normal Human Epidermal Keratinocytes (NHEK)

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We use a classification system similar but not identical to the Fitzpatrick Skin Classification. The Fitzpatrick classification has six different categories (phototypes  I-VI) which correlate with the level of skin pigmentation (melanin) and sunburn following sun exposure. Fitzpatrick I corresponds with the lightest of skin complexions, while Fitzpatrick VI corresponds with the darkest skin.

• I: Pale white skin, blue/hazel eyes, blond/red hair, always burns, does not tan
• II: Fair skin, blue eyes, burns easily, tans poorly
• III: Darker white skin, tans after initial burn
• IV: Light brown skin, burns minimally, tans easily
• V: Brown skin, rarely burns, tans darkly easily
• VI: Dark brown or black skin, never burns, always tans darkly

At PromoCell, we have knowledge of the patients’ skin color (white, brown or black skin), color of eyes and hair, but we don't have any details about the burning/tanning abilities. We therefore classify our tissue donors as follows:

• Light (comprising phototypes I and II)
• Moderate (comprising phototypes III and IV)
• Dark (comprising phototypes V and VI)

Information on the phototype is available for most cell lots isolated from juvenile or adult skin.

Short protocol:

• Trypsinize the cells as usual
• Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 10⁶ cells/ml
• Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision; which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight.
• Transfer the vials into liquid nitrogen for long-term storage

• Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need Fibronectin-coating when grown in PromoCell MSC Growth Medium XF (C-28019) and when differentiated in MSC Neurogenic (C-28015); Adipogenic (C-28016); or Osteogenic (C-28013) Differentiation Media.
• Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055; C-28056).
• For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020); the TC plates should be pre-coated with collagen type I.

• Liquid phase storage provides a consistent temperature of -196°C; a longer holding time and a greater vial capacity but involves the risk of contamination issues.
• Storage in the gas phase is very safe with respect to contaminations but the holding time of the cells is shorter and the vial capacity is reduced.