Isolation and culture of human primary keratinocytes—a methods review

Ścieżyńska, Aneta; Nogowska, Aleksandra; Sikorska, Maria; Konys, Justyna; Karpińska, Anita; Komorowski, Michał; Ołdak, Monika; Malejczyk, Jacek

doi:10.1111/exd.13860

Cited by 24 publications

(29 citation statements)

References 51 publications

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“…Culturing in low-calcium media (0.03 mM) maintains keratinocytes in an undifferentiated state. Elevated levels of calcium (2.8 mM) in culture media induce terminal differentiation ( 44 ). We compared differentially expressed genes between low and high calcium (terminal differentiation) or doxorubicin treatment in low calcium (senescence).…”

Section: Resultsmentioning

confidence: 99%

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

DePianto¹,

Heiden²,

Morshead³

et al. 2021

JCI Insight

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Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis associated interstitial lung disease ILD (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells, and interrogated these signatures in a single-cell RNA-seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence, and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

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Section: Resultsmentioning

confidence: 99%

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

DePianto¹,

Heiden²,

Morshead³

et al. 2021

JCI Insight

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“…To generate a bioengineered artificial skin substitute (BASS), it is necessary to develop a dermal stroma substitute with human fibroblasts immersed and later, seed the keratinocytes in the upper layer [8,9]. Regarding the BASS dermal stroma, the use of scaffolds is necessary to mimic the extracellular matrix (ECM) in which fibroblasts are immersed.…”

Section: Introductionmentioning

confidence: 99%

Cytotoxicity and Epidermal Barrier Function Evaluation of Common Antiseptics for Clinical Use in an Artificial Autologous Skin Model

Quiñones-Vico

Fernández-González

Pérez-Castejón

et al. 2021

JCM

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Bioengineered artificial skin substitutes (BASS) are the main treatment used in addition to autografts when skin injuries involve a large body surface area. Antiseptic/antibiotic treatment is necessary to prevent infections in the BASS implant area. This study aims to evaluate the effect of antiseptics and antibiotics on cell viability, structural integrity, and epidermal barrier function in BASS based on hyaluronic acid during a 28 day follow-up period. Keratinocytes (KTs) and dermal fibroblasts (DFs) were isolated from skin samples and used to establish BASS. The following antibiotic/antiseptic treatment was applied every 48 h: colistin (1%), chlorhexidine digluconate (1%), sodium chloride (0.02%), and polyhexanide (0.1%). Cell viability (LIVE/DEAD® assay), structural integrity (histological evaluation), and epidermal barrier function (trans-epidermal water loss, (TEWL), Tewameter®) were also evaluated. Cell viability percentage of BASS treated with chlorhexidine digluconate was significantly lower (p ≤ 0.001) than the other antiseptics at day 28. Compared to other treatments, chlorhexidine digluconate and polyhexanide significantly affected the epithelium. No significant differences were found regarding epidermal barrier. These results may be useful for treatment protocols after implantation of BASS in patients and evaluating them in clinical practice. BASS represent a suitable model to test in vitro the impact of different treatments of other skin wounds.

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“…For instance, a recent genome-wide methylation study showed no correlation between methylation status and transcriptome changes during keratinocyte differentiation in a monolayer culture, 22 despite other data showing significant methylation changes during in vivo skin morphogenesis. 50,51 Likewise, normal expression patterns of terminal marker genes are significantly impaired in Ca 2+ induced differentiation of primary keratinocytes. 23 Yet, the biological relevance of monolayer cultures can be substantially increased by co-culturing two or more cell types.…”

Section: What Org Anot Ypi C Culture S C An and C An ' T Domentioning

confidence: 99%

“…Yet, 3D cultures of the same primary skin cells 49 are still far more complex than their monolayer counterparts. For instance, a recent genome‐wide methylation study showed no correlation between methylation status and transcriptome changes during keratinocyte differentiation in a monolayer culture, 22 despite other data showing significant methylation changes during in vivo skin morphogenesis 50,51 .…”

Section: What Organotypic Cultures Can and Can't Domentioning

confidence: 99%

Fostering a healthy culture: Biological relevance of in vitro and ex vivo skin models

Atwood

Plikus

2021

Experimental Dermatology

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The field of experimental dermatology research has dramatically benefited from the insights yielded by in vivo studies on animal models. Indeed, much of our understanding of the mechanisms that regulate embryonic skin development, adult skin homeostasis and physiological skin responses to stress, such as wound healing, has been educated by studies conducted in animal models, including mutant mice. These works become commonly published on the pages of Experimental Dermatology and, in fact, represent one of the core interests of the journal. Highlighting in vivo mouse model studies are recent works on hair follicle development and growth 1-5 and wound healing. 6-8 Further, studies in animal models often help to elucidate aspects of disease pathogenesis, and mouse models are used to investigate mechanisms of human skin conditions such as atopic dermatitis, 9-13 contact dermatitis, 14-16 psoriasis, 17,18 rosacea 19 and squamous cell carcinoma 20 to name a few. On the other hand, not all human skin diseases or aspects of human skin physiology can be reliably modelled in rodents. This is not surprising, considering that humans and rodents are separated by an estimated 96 million years of evolution. 21 Addressing this fact, many studies are being conducted on patient-derived primary skin cells, including human keratinocytes, 22-25 melanocytes, 26-30 fibroblasts 31-34 and cell co-cultures. 35-37 However, typical in vitro culture conditions fail to replicate and, in fact, do not come close to imitating the biomechanical and biochemical complexity of the microenvironment in which cells exist and to which they respond to in native tissues. Further, ingredients in commonly used cell culture media and the two-dimensional constraints of growth on plastic result in cells being exposed to a lot of artificial cues, to which they adapt but also prominently alter their gene expression profile and functional activities in the process. Therefore, behaviours displayed by skin cells in twodimensional cultures need to be comprehensively validated under more native-like conditions in order to be deemed biologically and physiologically relevant. Helping to bridge the gap, threedimensional (3D) organotypic cultures and organ culture techniques have long been a highly instructive tool for investigating complex, tissue-level behaviours by skin cells.

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Isolation and culture of human primary keratinocytes—a methods review

Cited by 24 publications

References 51 publications

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

Cytotoxicity and Epidermal Barrier Function Evaluation of Common Antiseptics for Clinical Use in an Artificial Autologous Skin Model

Fostering a healthy culture: Biological relevance of in vitro and ex vivo skin models

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