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• W2126451808 abstract "Normal human keratinocytes (NHKs) are widely used in the field of basic research, and conventional monolayer cultures have been a modality for almost 30 years 1. One common trade in the standard protocols is the use of antibiotics in the culture medium to reduce infection rates. Protocols have been optimized over the last decades regarding type of medium, supplements, Ca2+ levels and pH 2, 3. In contrast, few studies have investigated the effect of the most commonly used antibiotics: gentamycin (genta) and the combination of penicillin and streptomycin (pen-strep) on keratinocyte growth and differentiation 4. Instead, focus has been directed at cytotoxicity of topically used antiseptics and antibiotics in the context of burn wounds, allograft and wound healing 5, 6. In embryonic stem cell cultures, however, it is known that genta and pen-strep have a capacity to reduce growth rate and differentiation 7. In our laboratory, we have been optimizing cell culture conditions for both conventional submerged cultures and 3D human epidermal equivalents (HEEs), and we now conclude that antibiotics should be omitted whenever possible to achieve optimal culture conditions and reproducible results. We aimed to evaluate the effects of commonly used antibiotics on keratinocyte growth and differentiation in both conventional cultures and HEE development. We herein examined the effects of genta and pen-strep on conventional NHK monolayer cultures by evaluating cell viability, cell culture confluence rates and keratinocyte gene expression levels. Epidermal morphology of HEEs was evaluated by haematoxylin and eosin staining. For detailed methods, see Method S1. To study the effect of pen-strep and genta on keratinocyte proliferation, we cultured keratinocytes and determined the NHK confluency in time. Pen-strep significantly stalled the proliferation during the first phase (days 3–6) of the culture period (Fig. 1a, open squares). Genta (closed squares) showed an intermediate inhibitory effect with significantly reduced cell confluency at day 5 of the culture period as compared to the antibiotic-free NHK cultures (closed circles). EdU incorporation into newly synthesized DNA during the S-phase of dividing cells was reduced in cultures with pen-strep or genta (Fig. S1a,b). In these proliferating cultures, we found no differences in Ki67, filaggrin (FLG), keratin 10 (KRT10) or KRT16 mRNA expression levels due to pen-strep or genta (Fig. S2a). However, expression of the terminal differentiation genes, FLG and loricrin (LOR), was nearly completely inhibited by pen-strep and genta after 24 and 48 h of differentiation. Expression of the early differentiation marker keratin 10 (KRT10) was unaffected by antibiotic use (Fig. 1b). Similarly, no statistically significant differences were found for Ki67, KRT16, involucrin (IVL), transglutaminase 1 (TGM-1) and late cornified envelope 1 (LCE1) expression (Fig. S2b). The attachment of keratinocytes after seeding, their proliferation and subsequent formation of a monolayer prior to air exposure are vital in the development of fully differentiated and stratified HEEs. Due to the profound effects on NHK growth, we postulated that the antibiotics would have similar effects in 3D skin cultures thereby leading to disturbed epidermal morphogenesis during the HEE development. Antibiotic-free HEEs showed a multilayered and fully differentiated epidermis (Fig. 2, control). Pen-strep severely impaired epidermal development as shown by the almost total absence of epidermis in donor 3 (95% reduction in epidermal thickness). Epidermal thickness was also reduced in HEEs generated with NHKs from donor 1 (65% reduction) and donor 2 (75% reduction). The absence of a stratum granulosum and stratum corneum in HEEs cultured with pen-strep indicates the disturbed epidermal differentiation as shown by the lack of protein expression of early (keratin 10) and late (filaggrin and loricrin) differentiation markers (Fig. S3). Genta also interfered with normal HEE development as indicated by the randomly distributed cells and complete lack of stratification (Fig. 2b). Notably, the addition of pen-strep during the final days of the HEE culture (from day 5 of the air–liquid interface culture onwards) did not alter the epidermal morphology (Fig. S4). In this study, we have shown a strong negative effect of commonly used antibiotics on NHK growth and differentiation in conventional NHK cultures, with an even more pronounced impact on HEE cultures. Although the conventional submerged NHK cultures do reach 100% confluency, albeit a few days later than antibiotic-free NHK cultures, and their gene expression is not altered in the proliferating phase, we strongly suggest to avoid antibiotics as the induction of epidermal differentiation in these cultures was hampered due to pen-strep and genta. As pen-strep did not alter the HEE morphology when added during the last days of culture, the profound effects on HEE development are most likely caused by growth inhibition during the first days of the culture period, where optimal cell adhesion and proliferation is a necessity for proper monolayer formation prior to lifting the HEEs to the air–liquid interface. However, as we found epidermal differentiation defects in both our submerged cultures and HEEs by pen-strep and genta, these antibiotics may also directly interfere with the epidermal differentiation programme leading to cessation of stratification. Already in 1996, a review on the prophylactic use of antibiotics in cell culture summarized the inhibition of protein biosynthesis and morphological changes by aminoglycosides (e.g. streptomycin) and other adverse effects of commonly used antibiotics on various cell cultures 8. In addition, aminoglycosides are known to affect cells from both the kidney and cochlea in vivo 9. Facing methodological problems in the laboratory when culturing keratinocytes, one should consider the use of antibiotics as a potential culprit. Influencing some of the keystone biological hallmarks of cell biology gives rise to the question whether the common use of antibiotics might do more harm than good and hence should be avoided whenever possible. This work was funded by ZonMw Top grant (91211052, Netherlands Organisation of Scientific Research (NWO)). EvdB and HN designed and supervised this study. UN, HN, GR and DR-O performed the experiments. UN, HN, EvdB and JS wrote the manuscript. The authors have declared no conflicting interests. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W2126451808 date "2015-10-06" @default.
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• W2126451808 title "Antibiotics in cell culture: friend or foe? Suppression of keratinocyte growth and differentiation in monolayer cultures and 3D skin models" @default.
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