Clonetics KGM-Gold Keratinocyte Growth Medium: A new robust and versatile serum-free medium formulation for high and low density normal human epidermal keratinocyte (NHEK) cultures

Cultured human keratinocytes provide a model to study a number of medically relevant topics such as metabolism, nutrition, toxicity, differentiation, various epidermal diseases, and the treatment of serious burns.

Serial cultivation of keratinocytes from human skin was first documented in the pioneering work of Rheinwald and Green (1). The system which these authors developed requires the use of fetal bovine serum and the co-cultivation of keratinocytes with a lethally irradiated layer of mouse 3T3 fibroblasts. Optimization studies by Peehl and Ham led to the ability to grow keratinocytes in the absence of feeder cells with reduced serum concentrations (2, 3). Serum-free cultivation of human keratinocytes has been possible since the mid 1980’s due to the development of MCDB 153 Basal Medium (4, 5).

The original formulation of MCDB 153 Medium has undergone several modifications and improvements by academic and industrial laboratories since its inception (6, 7). In a side-by-side evaluation of the performance of keratinocyte formulations from Lonza and competing companies, several inadequacies of commercially available keratinocyte growth media were revealed. Some of these available media formulations fail to support the maintenance of healthy, moderate to high-density keratinocyte cultures due to the exhaustion of essential nutrients. This characteristic limits the use of such formulations for applications requiring healthy, confluent keratinocyte monolayers. Other modifications fail to adequately support the clonal growth of keratinocytes, often resulting in low seeding efficiencies, slow doubling times and the presence of enlarged, senescent cells, even in early passages. In addition, claims of extended keratinocyte lifespan often neglect to unveil the growth rate, morphology, and health of the cells in later passages. It is, however, the lifespan over which keratinocytes are usable for experimentation that matters most to the researcher.

The need for an improved, robust and versatile serum-free medium for keratinocytes lead scientists at Lonza to develop Clonetics KGM-Gold Keratinocyte Growth Medium, which is designed to support both clonal growth and high density keratinocyte proliferation. Clonetics KGM-Gold Medium supports superior growth and morphology of keratinocytes isolated in Clonetics KGM-2 Keratinocyte Growth Medium-2 (Figure 1), Clonetics KGM Keratinocyte Growth Medium (Figure 2) and EpiLife Medium (Company I) (Figure 3). Furthermore, Quality Control at Lonza requires that keratinocytes pass strict morphological criteria specifying the minimal presence of enlarged and vacuolated cells, enabling Lonza’s Clonetics NHEK-Normal Human Epidermal Keratinocytes grown in Clonetics KGM-Gold Medium to be used by the researcher for ≥15 Population Doublings.

Clonetics NHEK cultured in KGM-Gold Keratinocyte Growth Medium proliferate well through at least 4 passages, reaching confluence in 5-7 days when seeded at 2000 cells/cm2 and remaining healthy and refractile. In contrast, keratinocytes proliferating in Clonetics KGM-2 Keratinocyte Growth Medium-2 begin to grow more slowly and adopt a poor morphology as cell density increases (Figure 1).

Cultures maintained in Clonetics KGM-Gold Medium tend to have a homogeneous phenotype, consisting mainly of small retractile cells, for ≥15 total Population Doublings of their in vitro lifespan. The ability of Clonetics KGM-Gold Medium to promote the health of keratinocytes positively influences keratinocyte lifespan: Clonetics KGM-Gold Medium outperformed Clonetics KGM Keratinocyte Growth Medium by 30-50% in side-by-side comparisons of keratinocyte longevity (Figure 2A). The morphology of cells cultured for 13-22 days in Clonetics KGM-Gold Medium vs KGM Medium is shown in Figure 2B. Keratinocytes in Clonetics KGM-Gold Medium appear healthy and continue to proliferate, while cells in Clonetics KGM Medium slow down and senesce, reaching only 14 Population Doublings.

We further show that even keratinocytes from Company I grow and look significantly better in Lonza’s Clonetics KGM-Gold Keratinocyte Growth Medium than in Company I’s EpiLife Medium (Figure 3).

In addition, Lonza’s Clonetics NHEK-Ad Adult Normal Human Epidermal Keratinocytes grown in Clonetics KGM-Gold Keratinocyte Growth Medium-Gold outperformed Company I’s adult keratinocytes grown in their own EpiLife Medium in growth rate and morphology (Figure 4) up to and even beyond 15 Population Doublings. This data shows that, although Company I claims extended lifespan of their keratinocytes in EpiLife Medium, the health and morphology of these adult keratinocytes is poor beyond 13-15 Population Doublings, rendering the cultures unusable for further experimentation.

Summary

Clonetics KGM-Gold Keratinocyte Growth Medium-Gold was created in response to deficiencies in the performance of keratinocyte media currently on the market. It is a rich medium, capable of maintaining high density keratinocyte cultures, while at the same time supporting clonal growth and the maintenance of healthy keratinocyte cell morphology.

By Therese Willstaedt and Zoe Damian, Lonza Walkersville, Inc.