3D Skin Culture Research Articles

Disease-related biomarkers as experimental endpoints in 3D skin culture models.

The online version contains supplementary material available at 10.1007/s10616-023-00574-2.

Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers.

The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2β1 integrin, which is present only on basal and stem cells. The presence of β1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.

Technological advances in three-dimensional skin tissue engineering

Abstract Tissue engineering is an enabling technology that can be used to repair, replace, and regenerate different types of biological tissues and holds great potential in various biomedical applications. As the first line of defense for the human body, the skin has a complex structure. When skin is injured by trauma or disease, the skin tissues may regenerate under natural conditions, though often resulting in irreversible and aesthetically unpleasant scarring. The development of skin tissue engineering strategies was reviewed. Although the traditional approaches to skin tissue engineering have made good progress, they are still unable to effectively deal with large-area injuries or produce full-thickness grafts. In vitro three-dimensional (3D) skin constructs are good skin equivalent substitutes and they have promoted many major innovative discoveries in biology and medicine. 3D skin manufacturing technology can be divided into two categories: scaffold-free and scaffold-based. The representatives of traditional scaffold-free approaches are transwell/Boyden chamber approach and organotypic 3D skin culture. Because of its low cost and high repeatability, the scaffold-free 3D skin model is currently commonly used for cytotoxicity analysis, cell biochemical analysis, and high-throughput cell function. At present, many drug experiments use artificial skin developed by traditional approaches to replace animal models. 3D bioprinting technology is a scaffold-based approach. As a novel tissue manufacturing technology, it can quickly design and build a multi-functional human skin model. This technology offers new opportunities to build tissues and organs layer by layer, and it is now used in regenerative medicine to meet the increasing need for tissues and organs suitable for transplantation. 3D bioprinting can generate skin substitutes with improved quality and high complexity for wound healing and in vitro disease modeling. In this review, we analyze different types of conventional techniques to engineer skin and compare them with 3D bioprinting. We also summarized different types of equipment, bioinks, and scaffolds used in 3D skin engineering. In these skin culture techniques, we focus on 3D skin bioprinting technology. While 3D bioprinting technology is still maturing and improvements to the techniques and protocols are required, this technology holds great promise in skin-related applications.

Arctigenin-Enriched Burdock Seed Oil (ABSO): A New Skin Brightening Botanical Extract

The Arctium lappa L. plant, commonly known as burdock, has been used therapeutically for hundreds of years. Arctigenin (ATG) is an active ingredient in burdock, albeit at low quantities or mostly in the form of acrtiin (arctigenin-4-glucoside). ATG has been touted for its anti-inflammatory properties in many cell types and disease states; however, its role in skin and melanin production has not been extensively studied. Our aims for this study were to develop a burdock seed extract enriched for ATG that is amenable to quasi-drug development, determine in vitro brightening activity, and evaluate safety and skin brightening efficacy clinically in human subjects. Arctiin and ATG content were measured by high-performance liquid chromatography (HPLC). In vitro studies utilized EpiDermTM tissues for skin irritation test, and MelanoDermTM tissues for melanin reduction capacity. A 45 subject clinical study was performed in adult subjects ranging in age from 30 to 60 years old (mean = 41.9 ± 6.7) to test the safety and skin brightening potential of 1% ABSO lotion. We demonstrate by HPLC that ABSO is a natural extract that contains ~5-times more arctigenin than BSO. Furthermore, ABSO inhibits melanin production better than BSO and retains the same melanin-reducing capacity as synthetic ATG in vitro in MelanoDerm™ 3D skin cultures. ABSO also adheres to quasi-drug criteria according to the Japanese Standards of Quasi-Drug Ingredients as determined by infrared absorption spectrum method, unsaponifiable matter, heavy metal and arsenic content, and acid, saponification, and iodine value methods. Clinical assessment of 1% ABSO lotion shows it is well-tolerated in human skin and demonstrates improved brightness and skin tone evenness. ABSO is a natural arctigenin-enriched burdock seed extract that reduces melanin content in vitro and clinically improves skin brightness.

Glycerol fermentation by skin bacteria generates lactic acid and upregulates the expression levels of genes associated with the skin barrier function.

Commensal bacteria play a major role in multiple skin functions by providing the first layer of defense against pathogens and maintaining the skin barrier. Staphylococcus epidermidis is one of the most common skin commensals. In this study, we showed that S. epidermidis ferments glycerol and uses it as a nutrient, while producing short-chain and organic fatty acids, with the most notable being lactic acid. Lactic acid is an alpha-hydroxy acid that inhibits the growth of pathogenic bacteria, without any negative effect on the commensal bacteria itself. Using in vivo experiments, we validated our in vitro results, showing that the skin microbiome is also capable of doing this. Finally, using 2D and 3D skin culture models, we showed that the fermentation of glycerol, mainly lactic acid, as determined by analytical methods, upregulates the expression levels of several key genes that are associated with the barrier properties of the skin. While the hydration effect of glycerol on the skin is well known, our study shows the overall benefits of glycerol on the skin microbiome, while revealing an alternate mode of action of glycerol for multiple skin benefits.

Viability Improvement of Three-Dimensional Human Skin Substitutes by Photobiomodulation during Cultivation.

Three-dimensional (3D) cultured skin containing vascular networks is a useful skin substitute that enables rapid reperfusion after transplantation. During its cultivation, however, insufficient nutrient delivery to the thick cultured tissue from the surrounding culture medium decreases the tissue viability. To solve this problem, in this study, we applied photobiomodulation (PBM), which can optically activate the electron transport chain of mitochondria, to human 3D skin cultures constructed using the layer-by-layer cell coating technique. PBM was applied once 5 days after the start of epidermal differentiation using a light-emitting diode array with a center wavelength of 440, 523, 658 or 823 nm at a constant light intensity of 15 mW cm-2 for 50 or 600 s. Two days after PBM, we assessed the viability of the tissues by a water-soluble tetrazolium-8 assay, adenosine triphosphate measurements and live/dead cell imaging, and the results showed that the PBM at 823 nm for 50 s (0.75 J cm-2 ) significantly improved the viability of the 3D-cultured skin.

Impact of diabetic (diabetes mellitus) patients immune factors on the skin cell viability in vitro

At the moment, a lot of scientific research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. A 3D skin culture system as a relevant skin model may prove valuable in investigating these mechanisms and may be a useful tool to study interactions between different cell types such as keratinocytes, fibroblasts, and immune cells. The aim of our research was to study keratinocytes and fibroblasts viability in co-culture with immune factors of patients with diabetes mellitus type 2 (DM2) and patients with diabetes and chronic foot ulcers in a 3D skin culture system. In this study, the multilayer 3D immunocompetent model of human skin comprising keratinocytes, fibroblasts, and mononuclears in an agarose-fibronectin gel was used. The human immortalized keratinocyte cell line, HaCaT, and primary fibroblast cell culture isolated from skin samples of healthy man in abdominal surgery were used for the 3D system. For the experiment 20 % serum of 9 patients with chronic diabetic foot ulcers (without active inflammation signs), 9 diabetic type 2 patients and 9 healthy people, and mononuclears of the same groups of patients were used. 9 experimental series with 3 repeats were carried out. Mononuclears of patients with DM2 and DM2 and diabetic foot syndrome (DFS) had a greater inhibitory effect on fibroblasts, significantly inhibiting their proliferation to a level of 83.78 [79.03; 89.53] % vs 70.18 [66.38; 72.10] % vs 95.40 [91.75; 99.05] %, H = 21.259, p <0.001 – DM2, DFS, and the control group, respectively. There was no significant difference in the cytoinhibitory effect of mononuclears on keratinocytes between different groups: 96.40 [92.82; 100.50] % vs 93.61 [86.80; 97.10] % vs 92.87 [85.15; 95.25] %, H = 4.459, p = 0.108 – control, DM2 and DFS group, respectively. Adding serum to the culture system influenced significantly the viability of neither keratynocytes – 99.40 [95.35; 102.05] % vs 98.60 [90.55; 100.40] % vs 94.79 [91.65; 98.16] %, H = 3.030, p = 0.220 nor of fibroblasts – 95.61 [92.39; 100.19] % vs 95.80 [88.99; 102.15] % vs 96.20 [99.69; 88.70] %, H = 0.353, p = 0.838, control, DM2 and DFS group, respectively. It was determined that the fibroblasts vialability significantly decreases after introducing mononuclears of patients with DM and patients with DM and chronic diabetic foot ulcers to the co-culture system. Adding serum of these patient groups to the culture system doesn’t influence significantly the viability of skin cells.

ПРОТЕКТИВНЫЙ ЭФФЕКТ ИНТЕРЛЕЙКИНА 4 НА ФИБРОБЛАСТЫ В 3D-КЛЕТОЧНОЙ МОДЕЛИ ВЗАИМОДЕЙСТВИЯ МЕЖДУ КЛЕТКАМИ КОЖИ И ИММУННЫМИ КЛЕТКАМИ ПАЦИЕНТОВ С САХАРНЫМ ДИАБЕТОМ 2 ТИПА И САХАРНЫМ ДИАБЕТОМ 2 ТИПА С ХРОНИЧЕСКИМИ ЯЗВАМИ СТОП

Our research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. A 3D skin culture system, as a relevant skin model, has proved valuable in investigating these mechanisms and is a useful tool to study interactions between keratinocytes, fibroblasts and immune cells. The aim of this study was to evaluate the effect of IL1 and IL4 on the keratinocytes and fibroblasts viability and assess endogenous interleukin (IL1,4,6,8) production into the culture medium by the skin cells cocultured with mononuclears of patients with type 2 diabetes mellitus (T2DM) and those with T2DM and chronic foot ulcers (CFU) in a 3D culture model. The main result of this study was the protective effect of IL4 on fibroblasts – this interleukin (50 pg/ml) significantly increased the percentage of viable fibroblasts cocultured with keratinocytes, mononuclears and blood serum of T2DM patients (85.13 [80.81; 87.82]% vs. 90.34 [85.92; 94.74]%, T=-2.668, p=0.008) and those with T2DM and CFU (70.42 [68.10; 74.79]% vs. 82.10 [76.37; 84.39]%, T = -2.666, p=0.008). IL1 in high concentration potentiated the toxic effect of mononuclears in T2DM and T2DM with CFU patients on fibroblasts and also decreased keratinocytes viability in the 3D culture system. An increase in IL1 and absence of a sufficient and significant decrease in IL6 production into the culture medium during 24-hour coincubation of skin cells with T2DM patients mononuclears indicate the activation of immune mechanisms and the maintenance of persistent inflammatory response.

Application of 3D skin culture model as a tool to study the role of immune mechanisms in chronic diabetic foot ulcers pathogenesis

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection.

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

Galectin-7 is extracellularly released from epidermal keratinocytes in atopic dermatitis and serves as immunomodulator of Langerhans cells

A number of galectins are localized in various organs, and galectin-7 is expressed in epidermal keratinocytes (KCs). Our proteome analysis of extracts from human stratum corneum revealed that galectin-7 is highly expressed in atopic dermatitis (AD) patients (Sakabe J et al. JACI 2014). Meanwhile, galectin-7 might bind to hyaluronan, and its intercellular accumulation can yield spongiosis. Thus, it should be elucidated how galectin-7 is induced in the epidermis and whether it can suppress or rather promote skin inflammation. To address these issues, we used 3D cultured KCs to investigate the ability of IL-4/IL-13 to induce galectin-7, and employed murine epidermal Langerhans cells (LCs) for its ability to maturate LCs. Immunohistochemical staining showed that, in healthy skin, galectin-7 was expressed in the cytoplasm of KCs in the prickle layer. In AD lesional skin, however, galectin-7 was positively stained in the intercellular space of KCs. In human 3D skin culture, incubation of KCs with IL-4/IL-13 promoted its expression in the intercellular space, suggesting that these Th2 cytokines stimulate KCs to produce galectin-7 and to extracellularly release it. These findings urged us to further investigate its effect on LCs. Epidermal suspensions were freshly prepared from C57BL/6 mice, and cells were cultured for 24 h with or without 100 ng of mouse recombinant galectin-7. The expression levels of langerin, EpCAM, CD54, CD80, CD86, and MHC class II were examined by flow cytometry. LCs, gated as langerin+ and EpCAM+ cells, expressed increased levels of CD54 and CD86, when epidermal cells were incubated with galectin-7. These results suggest that galectin-7 is extracellularly secreted by epidermal KCs in AD and may play an immunomodulatory effect on LCs.

Comprehensive analysis of long non-coding RNAs in keratinization

Long non-coding RNAs (lncRNAs) play key roles in a variety of processes necessary for homeostasis. However, their effects on keratinization have not been fully elucidated. In this study, we aimed to identify specific lncRNAs that regulate keratinization. First, we performed transcriptome sequencing of keratinocytes sampled from palmar and forearm skin and compared transcriptome profiles between them, since palmar skin is much more keratinized compared to forearm skin. These analyses led us to identify 125 candidate lncRNAs that may be involved in keratinization. Next, we knocked down the candidate lncRNAs in keratinocytes. Histological and transcriptional analysis using reconstructed 3D skin culture demonstrated that knockdown of several lncRNAs leads to disruption of keratinization. These data suggest that these lncRNAs could be required for keratinization.

Histopathological and Immunohistochemical Studies of Cowpox Virus Replication in a Three-Dimensional Skin Model

Human cowpox virus (CPXV) infections are rare, but can result in severe and sometimes fatal outcomes. The majority of recent cases were traced back to contacts with infected domestic cats or pet rats. The aim of the present study was to evaluate a three-dimensional (3D) skin model as a possible replacement for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with two CPXV strains of different pathogenic potential: a recent pet rat isolate (RatPox09) and the reference Brighton red strain. Infected 3D skin cultures exhibited histological alterations that were similar to those of mammal skin infections, but there were no differences in gene expression patterns and tissue damage between the two CPXV strains in the model system. In conclusion, 3D skin cultures reflect the development of pox lesions in the skin very well, but seem not to allow differentiation between more or less virulent virus strains, a distinction that is made possible by experimental infection in suitable animal models.

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Protein kinase C δ and η differently regulate the expression of loricrin and Jun family proteins in human keratinocytes

Barrier function of the epidermis is maintained by precise expression of keratinocyte-specific structural proteins to form the cornified cell envelope (CE). Loricrin, a major component of the CE, is expressed at the late stage of keratinocyte differentiation. In this study, we reveal the isoform-specific function of protein kinase C (PKC) in the regulation of loricrin expression. Both PKCδ and PKCη have been recognized as differentiation-promoting isoforms. However, loricrin expression was inversely controlled by PKCδ and PKCη in cultured keratinocytes and 3D skin culture; i.e. loricrin expression was decreased by PKCδ and increased by PKCη. To clarify the mechanisms that PKCδ and PKCη oppositely regulate the loricrin expression, we examined the expression of activator protein-1 (AP-1) family proteins, which modulate the transcription of loricrin and are downstream molecules of PKC. PKCδ decreased c-Jun expression, whereas PKCη increased JunD, which are positive regulators of loricrin transcription. These findings suggest that inverse effects of PKCδ and PKCη on loricrin expression attributes to the expression of c-Jun and JunD.