Korean J Dermatol.  2005 Jun;43(6):732-742.

Effects of Keratinocyte Growth Factor (KGF), Epidermal Growth Factor (EGF), and Extracellular Calcium on the Growth of Cultured Psoriatic Keratinocytes

• Kim KH
• Kim JY
• Yoon TJ
• Kim HS
• Lee CW
• Eim JJ

Abstract

We investigated the effects of KGF and EGF, together with extracellular calcium, on the growth of cultured psoriatic keratinocytes, in order to establish a new chemically defined medium for culturing psoriatic keratinocytes using a modified form of MCDB 153 media. We compared the cell growth pattern under various culture conditions, including growth factors (KGF or EGF), and extracellular calcium concentrations, attachment and/or matrix factors (type I collagen coating or 3T3 fibroblast layering), culture durations, and types of cultured cells such as normal human epidermal keratinocytes (NHEK) and psoriatic keratinocytes. In order to achieve the above objective, semiquantitative RT-PCR for K16 mRNA, direct immunofluorescence with K8.12 as the markers of regenerating keratinocytes, and microscopic observation for cell colony formation were performed. The results are summarized as follows: 1. Psoriatic keratinocytes were grown optimally at 0.15 mM calcium, irrespective of growth factors or even in free control. They also grew well at 20 nM KGF. 2. KGF and/or EGF played an active role in cell growth, especially in the 5 days' culture. The growth stimulatory effect of EGF was suppressed by 0.5 mM calcium, but the effect of KGF was sustained at this calcium concentration. Furthermore, KGF exhibited a cell Survival effect of 18 days on type I collagen coating, and also in 12 days' culture on 3T3 fibroblast layering. 3. Cultured psoriatic keratinocytes were more vulnerable to extracellular calcium than NHEK with regard to optimal calcium concentration; they grew best at 0.15 mM, which was much lower than 1.5 mM in NHEK. 4. 3T3 fibroblasts exerted a favorable effect on cell growth and survival, especially in 12 days' culture, which may have been due to the paracrine effect of endogenous KGF from the 3T3 feeder cells, and cell reattachment/pile-up properties. To improve the culture method for psoriatic keratinocytes, the study suggests we should consider the optimal extracellular calcium concentration, introduce feeder cell layering to increase cell reattachment/pile-up, and supplement the mesenchymal paracrine growth factors such as KGF to exert a favorable effect on cell growth and survival.

Keyword

Psoriatic keratinocytes; Cell culture; KGF; EGF; Calcium; Cell growth; K16

MeSH Terms

Calcium*
Cell Culture Techniques
Cell Survival
Cells, Cultured
Collagen
Collagen Type I
Epidermal Growth Factor*
Feeder Cells
Fibroblast Growth Factor 7*
Fibroblasts
Fluorescent Antibody Technique, Direct
Humans
Intercellular Signaling Peptides and Proteins
Keratinocytes*
RNA, Messenger
Calcium
Collagen
Collagen Type I
Epidermal Growth Factor
Fibroblast Growth Factor 7
Intercellular Signaling Peptides and Proteins
RNA, Messenger