Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Peer-reviewed article TESTING METHODS Class Rueffer In vitro methods demonstrate effects on skin self-renewal and homeostasis in 3D skin models CLAAS RÜFFER*, LISA BÖCKELMANN, WERNER VOSS *Corresponding author Dermatest GmbH Engelstrasse 37, 48143, Germany KEYWORDS: Skin homeostasis; cell renewal; proliferation; viability. ABSTRACT: In vitro 3D skin models are able to recapitulate the major events in the process of skin homeostasis very well. Two cell biological assays have been applied on Phenion®-FT-skin models and demonstrated their usefulness for describing cellular activities in the process of skin renewal and homeostasis in vitro. A cell proliferation assay was applied to study the general proliferative responsiveness of basal keratinocytes and dermal fibroblasts, whereas the MTT-viability-assay was used to analyse the overall improvement of epidermal and dermal tissue viability. For demonstration purpose, single experiments have been performed in which skin models were systematically stimulated with known cosmetic and medical actives (atRA & 13cRA), whose time and dose dependent effectiveness are well characterized (18, 21). INTRODUCTION Skin homeostasis Skin maintains its capability of self renewal throughout a whole human life. The continuum of keratinocyte birth, differentiation and desquamation comprises the coreprinciple of a physiological and physical skin barrier that holds fluids inside the body and acts as a protective shield against biological, chemical and physical environmental knoxes to the outside (3). Although skin continuously accumulates chronological and photo-induced signs of ageing, it still manages to keep up its skin barrier maintenance a life-long (6). The origin of interfollicular epidermal self regenerative capacity is located within a single, basal layer of keratinocytes, which is anchored to the papillar dermis via a range of connective structural proteins (6, 7). The first effective step in interfollicular regeneration takes place in the basal layer of epidermal keratinocytes. Undifferentiated basal keratinocytes undergo constantly acts of mitosis. Newborn daughter cells detach from the underlying basement membrane and migrate into suprabasal epidermal layers. From that point on they withdraw from cell cycle and start the highly organized sequencial program of terminal differentiation and desquamation. Terminal differentiation comprises a complex process of cornification in which granular cells transform into keratin-enriched cell remnants in order to form a multilayered interconnected protective envelope (3). Desquamation is a finely tuned and timed 70 Household and Personal Care Today - n 1/2012 sequence where outer layers of cornified keratinocytes are enzymatically detached from the skin surface. Therefore cell proliferation at the basal epidermal layer and the desquamation process of the stratum corneum are finely balanced processes which keep the constantly rejuvenating epidermis in a regulated state and the skin barrier properties permanently maintained (3, 6). Interfollicular Stem cells The homeostasis of interfollicular epidermis is characterized through a high cellular turn-over propelled by cell divisions of stem cells within the basal layer of keratinocytes. A well accepted scientific model today suggests the presence of distinct populations of stem cells that locate within the interfollicular epidermis. Epidermal stem cells have the ability to produce committed progenitor cells, also known as transient amplifying stem cell daughters (TA cells). TA cells undergo cell divisions up to 5 times before they differentiate. Therefore it is supposed that stem cells in their specific niches are rather non-dividing, mostly quiescent cells which leave most of the epidermal regenerative activity to committed and frequently activated TA cells (10, 14, 20). With ageing epidermis becomes more fragile and predisposed for trauma. A significantly reduced homeostatic activity and decline in healing rate of elder skin most probably originates from a drastic reduction of stem cell’s proliferative capacity and mobility. In general adult stem cells show the same susceptibility to chronological ageing as non-dividing cells. The chronological accumulation of intrinsic- as well as stem cell niche specific impairments is discussed to be relevant for the general and steady decline in stem cell activity (4, 11, 16, 20). In vitro skin regeneration In vitro skin equivalents are composed of a highly differentiated, stratified epithelium which resides on top of a dermal matrix with incorporated living fibroblasts. Despite their simplified model-like character, organotypic skin equivalents are capable of a far-reaching recapitulation of the in vivo keratinocyte differentiation program (17). In contrast to real skin in vitro 3D skin models are quite short-lived. Nevertheless, for a limited time period skin models are capable of sustaining a constant pool of skin cells to keep their epidermal structure maintained (13, 17). Analogue to the in vivo skin situation, only cells within the single layer of basal keratinocytes are able to pass through the cell cycle to fuel the cellular balance. The model`s full thickness design allows possible mutual molecular dermal-epidermal crosstalk within the skin equivalent. TESTING METHODS Such cross-talk has recently been shown to favour in vitro epidermal regeneration, formation of stratum corneum and model longevity (17). Most interestingly full thickness skin models permit the analysis of dermal influences on the epidermal tissue rejuvenation and differentiation process (13, 17). A popular cell source for building 3D skin models are neonatal human foreskin-derived epidermal keratinocytes which reveal a vast proliferative capacity (8). A comparative analysis revealed that populations of human foreskin keratinocytes can be subdivided into three phenotypic and functionally distinct subgroups, which were roughly characterized as stem cell candidate-, transient amplifying- and as basal and suprabasal epidermal cells (11, 16). Experiments to test their in vitro regenerative capacity demonstrated that the stem cell fraction showed superior capabilities followed by the other two populations (11, 16). Further data revealed that besides intrinsic cellular capabilities, extrinsic or microenvironmental factors such as extracellular matrix and dermal regulatory components can act as effective restorative elements on the epidermal regenerative capacity (16). 3D Skin Models as a tool to study effects on skin renewal and epidermal homeostasis Human native skin biopsies are very close to the in vivo situation and therefore quite ideal analytical in vitro tools to evaluate endpoints of skin safety and efficacy. Because of their very limited availability and donor specific variability skin biopsies do not represent a solution for industrial high throughput applications. Therefore organotypic 3D skin models become increasingly important in medical and cosmetic skin research but also for substrate and product safety evaluations (REACH). Todays organotypic skin equivalents definitely reveal the necessary structural and physiological complexity to recapitulate important steps in the process of skin homeostasis. In this article Dermatest GmbH demonstrates two in vitro assays, which can be used to characterize the influence and effectiveness of single cosmetic and medical actives as well as whole formulations on the processes of skin renewal and revitalization. MATERIAL & METHODS In vitro 3D culture Phenion® FT skin models were Incubation at 37°C, 95 percent H2O, 5 percent CO2. Media components were purchased from Life Technologies, unless otherwise stated. Basic culture media: DMEM with Glutamax/Ham`s F12 medium (3:1) supplemented with 100 IU/ml of penicillin and 100 mg/ml of streptomycin. Supplements: DMEM 1.6 mg/ml of bovine serum albumin (Sigma, Germany), 0.4 mg/ml of hydrocortisone (Sigma, Germany), 0.12 IU/ml of insulin, 1 mM ascorbic acid 2-phosphat (Sigma, Germany). In vitro applications of atRA, 13cRA, IL-1a 10 mM stock solutions of atRA 13cRA in DMSO were prepared. 1000, 500 and 100 nM of atRA and 1000 and 100 nM 13cRA were applied to the basal medium. Pro-inflammatory cell culture conditions were generated by application of 5 pg/ml of cytokine IL-1a (R&D-Systems). Cultivation media was daily renewed. For topical application of 13cRA, Isotrex Crème 0,05 percent (Stiefel,UK) was utilized. Histological Analyisis: 3x4 mm biopsies from center of skin models were embedded in OCT at -80°C. 5 mm cryo tissue sections were mounted on Superfrost® Plus microscope slides (Menzel GmbH, Germany) and fixed in 4 percent formalin solution (Sigma, Germany). Proliferation Assay: Cell proliferation was detected for the last 24h of total incubation period by applying Click-iT® EdU Imaging Alexa 598 Kit (Life-Technologies, Germany), EdU-positive nuclei were evaluated from 400-1000 counted DAPI-positive nuclei. Detected tissue sections were embedded in Vectorshield (Vector Lbt, USA) before microscopic analysis (Zeiss). Viability Assay: Skin models were incubated with 1 mg/ml DuraliQ stable MTT solution (Dojindo) in basic DMEM for 3h in the dark at 37°C, 95 percent H2O, 5 percent CO2. For analysis, model triplicates per endpoint were rinsed thoroughly, separated into its model components via enzymatic thermolysin activity (2.4 U/ml, 3h at 4°C) and lysed separately overnight at RT in isopropanol (epidermis: 1 ml; dermis: 5 ml, Karl Roth) on a plate shaker. ODs of lysates were measured in triplicates at 570 nm. Parameters of result acceptance are defined as follows: OD570 of formazan extracts from negative control must be greater than 0.8. For experimental exceptance, CV of identical treated tissues had to be less than 30 percent, with exception of cases with OD below 0.3. Calculation of viability: 100*(OD570 Exp/(OD570 control). RESULTS In vitro assay to evaluate in vitro cell proliferation Basal epidermal keratinocytes and dermal fibroblasts of 3D skin models undergo mitosis. Before entering the phase of active cell division or mitosis (M-Phase), cells traverse a prepreparative Interphase (I-Phase), during which pre-mitotic cells start to grow and to fully replicate their genomic DNA (S Phase). EdU® is a detectable nucleotide base analogue which is specifically inserted into replicating DNA strands during S-phase of cell cycle. By utilising the Click-iT® EdU detection kit, replicated genomic DNA in nuclei of proliferative cells could be visualized in tissue slides via immunofluorescence microscopy (Figure 1A). In vitro Modulation of cell proliferative activity The following experimental approaches revealed that the proliferative activity of basal keratinocytes and fibroblasts can be modulated by external stimulation. When grown in basal medium without growth stimuli, cells in epidermis and dermis showed a basic cell cycle activity. Explicit stimulation of cell growth could be triggered by application of growth TESTING METHODS supplements into culture media, which lead to a noticeable increase of cell proliferation activity in epidermis and dermis. Otherwise the increased proliferative activity of stimulated epidermal and dermal cells could be silenced again when an explicit pro-inflammatory milieu was created by addition of Interleukin-1 alpha (IL-1a); (12) into the culture media (Figure 1A; dermis not shown). the incubation period. Compared to basal media controls, in atRA supplemented media cultivated skin models showed an elevated proliferative activity of epidermal keratinocytes but no effect on dermis. The topically treatment of skin models with 0,05 percent 13cRA for 24 hours showed at least an increase of proliferative activity in epidermis by trend whereas the increase of proliferative activity of dermal fibroblasts was noticeable. For control the cultivation of skin models in media supplemented with ascorbic acid 2-phosphate, insulin and hydrocortisone increased epidermal and dermal proliferation likewise (Figure 1A, B, C). Figure 1. Cell proliferation. (A) 3D skin models were cultivated under basal (a, d) and under proproliferative (supplemented) media conditions (b, e). To reverse the pro-proliferative effects again, 5 pg/ml of hu-IL-1a was added to the supplemented 3D skin model cultures (c, f). (B) Effects on proliferation described in A) were quantified via Immunefluorescence (IF)-microscopy. EdU®-positive cells were counted and expressed as percentage. (C) The Click-iT®-EdU Kit was applied to tag DNA of proliferating epidermal and dermal cells during S-Phase of the cell cycle. Histological tissue slides from 3D skin models have been prepared and tagged DNA was visualized via IF analysis. Cell nuclei of proliferating cells appear red. The universal nuclei stain is blue (DAPI). The 3D skin tissue structures were visualized via DIC microscopy. (D) 3D skin models were systemically treated with 500 nM atRA or creamed with 0,05 percent 13cRA. Epidermal and dermal cell proliferation was evaluated in parallel. As expected, the data revealed that the proliferative cell activity in skin equivalents reacts dynamically according to the type of stimulus. atRA- and 13cRA treatment of in vitro 3D Models Skin models were incubated in parallel with single or combinations of different pro-proliferative actives for 4 days. Only the topical application of Isotrex® (0,05 percent of 13cRA) was time-limited to the last 24 hours of the total cultivation period in order to avoid strong irritating side effects on epidermis. 13-cis-retinoic acid (13cRa) is an isomeric derivative of all-trans retinoic acid (atRA) and often used for medical skin applications (18). Epidermal and dermal proliferative cell activity was marked with Click iT® EdU for the last 24 hours of 74 Household and Personal Care Today - n 1/2012 The MTT viability assay Viability is defined as the overall capacity of living. That summarizes all cellular metabolic activities which are needed for house-keeping, growth and division (1). A wellestablished method to measure the viability of tissues and cells is the MTT-assay. 3-(4,5-Dimethylthiazol-yl)2,5-diphenyl-tetrazoliumbromide or short MTT is a yellow, water-soluble tetrazolium salt, which is reduced via tetrazolium-ring cleavage and transformed into water-insoluble purple-blue formazan. The waterinsoluble, crystalline formazan precipitates into the cellular cytosol and the amount of precipitate correlates quantitatively with the degree of overall cell viability. After stopping the MTT reaction, formazan is released via chemical tissue lysis and its dissolution`s optical density is measured. The MTT transforming reaction is mediated by enzymes associated with the endoplasmatic reticulum and the mitochondria. Recent data showed that MTT reduction predominantly involves NAD(P)H-dependend enzymes of the endoplasmatic reticulum which are involved in glycolysis. Whereas in contrary to previous beliefs the mitochondrial succinate dehydrogenase activity, involved in cell respiration, adds a much smaller contribution to the overall MTT turnover in cultured human cells (1). Measuring epidermal and dermal cell viability 3D skin models offer the opportunity for a differential analysis of dermal and epidermal viability (Figure 2). For assay demonstration, skin models have been systemically incubated with concentrations of 1000 and 100 nM of atRA and 13cRA (18, 19). Recent growth response studies on epidermal keratinocytes have demonstrated that both Vitamin A substances in named concentrations have a markly positive influence on tissue viability (18). After four and seven days of atRA and 13cRA treatment, viability of skin models was tested via MTT-assay (Figure 2A, B). A subsequent analysis of epidermal and dermal equivalents revealed that in correlation with recent data both Vitamin A substances showed an positive effect on skin model`s epidermal or dermal cell viability after seven but not after four days (not shown) of incubation in a dose-dependent manner (Figure 2A). TESTING METHODS via tuned and coordinated enzymatic activity inside the native stratum corneum (3). Such functional microenvironmental conditions seem to be strongly disturbed in reconstructed skin models (2). 3D skin models as a useful tool to study cosmetic and medical actives Skin models allow the correlating analysis of substance and dose Figure2. MTT-viability: (A) 3D skin models were cultivated over 7 days in media with 1000 and 100 nM of atRA dependent epidermaland 13cRA applied. The status of organotypic epidermal and dermal tissue viability was measured in parallel. (B) Preparation of MTT-treated Phenion® FT skin models. An enzymatic separation of the 3D skin and dermal cell responses. model in its epidermal and dermal equivalents allows a differential MTT-analysis. Cell proliferation of basal epidermal keratinocytes and dermal fibroblasts DISCUSSION are important episodes in the process of skin renewal and homeostasis (3). A characteristic morphology and a clearly Participation of hair follicles in epidermal homeostasis and defined localization make basal keratinocytes and dermal regeneration fibroblasts good study objects for a microscopic analysis (13). Under physiological conditions the activity of hair follicles and The stimulation with pro-proliferative retinoic actives in different their stem cells are nearly dispensable for regular epidermal ways of application revealed a noticeable susceptibility of in processes of skin homeostasis (9). Under conditions of severe vitro skin cells for proliferative stimuli, which is adjustable through trauma and wounding, when larger amounts of lost cell the definition of medium parameter. As a result pro- as well as material have to be replaced in due time, hair follicles and anti-proliferative, agent and dose-dependent effects could their stem cells especially assist in the acute phase of rebe studied likewise. Proliferation assays reveal a high sensitivity epithelialization to accelerate the start of wound closure (5, 9). in a quite short time of incubation (Figure1). Even with relative Recent research data revealed that optimal wound healing low doses of actives and relative short periods of stimulation involves an epidermal and dermal response and definitely clear effects should be detectable especially on retinoid depends on intact hair follicles, whereas the final wound sensitive basal keratinocytes (18). Another option for studying closure is accomplished by the regenerative capacity of the effects on skin homeostasis offers the MTT-assay, which allows interfollicular epidermis (9). the measurement of whole tissue viability (1, Figure 2). Besides quantification of direct effects on skin renewal it also offers the Skin models recapitulate the key processes of terminal possibility of studying substances and formulations which act differentiation but not of desquamation over a longer time period indirectly on cell viability via DNA3D skin models can be characterized as reconstructs of protective or anti-inflammatory activities, which support tissue interfollicular skin which are able to recapitulate structural longevity by enhancing cell survivability instead of increasing and molecular processes of skin homeostasis (13, 17). In cellular turnover (1). comparison to in vivo skin in vitro constructs are quite shortlived but reveal nevertheless a good regenerative potential of the basal kerationocytes for a limited time period (13, 17). REFERENCES AND NOTES The decline in tissue regenerative potential in vitro is related to a quite high degree of replicative ageing of the proliferative 1. M.V. Berridge, et al., Biochemica, 4, pp. 14-19 (1996). 2. J.A. Bouwstra et al., J Inv Derm., 128, pp. 378-388 (2008). basal keratinocytes, especially when cultivated under basal 3. E. Candi, et al., Nature molcellbio., 6, pp. 328-340 (2005). conditions (Phenion®-Kit, 13). The absence of desquamation in 4. A. Charruyer et al., J Inv Derm., 129, pp. 2574-2583 (2009). reconstructed stratum corneum is one of the striking differences 5. C.M. Chuong, Nature, 447, pp. 265-266 (2007). in contrast to native skin (2, 3). Although the organotypic stratum 6. C. Blanpain, E. Fuchs, Nature molcellbio., 10, pp. 207-217 (2009). corneum reveals several similarities to its native counterpart, 7. E. Fuchs, JCB, 180(2), pp. 273-284 (2008). like a comparable structure and composition of lipids and 8. P. Gangatirka, et al., Nature Protocols., 2(1), pp. 178-186 (2007). proteins, the key factor desquamation is completely impaired. 9. M. Ito, C. Cotsarelis, Nature, 128, pp. 1059-1061 (2008). This circumstance leads to a continuous accumulation of 10. P. Kaur, J Inv Derm., 126, pp. 1450-1458 (2006). the cornified layers over time (2, 3). Together with certain 11. L. Ling, T.A. Rando, JCB, 193(2), pp. 257-266 (2011). intracellular lipids mainly paracellular cell to cell contacts called 12. Y. Morinanga et al., J. of Immunol., 143(11), pp. 3538-3542 (1989). 13. K. Mewes et al., Skin Pharmacol Physiol., 20, pp. 85-95 (2007). corneodesmosoms are responsible for the interconnection of 14. K.A. Moore et al., Science, 311, pp. 1880-1885 (2006). single corneocytes inside the cornified layers (2, 3). The continuous 15. K. Pankaj et al., Nature molcellbio., 12, pp. 198-202 (2011). degradation of corneodesmosoms subsequent to the terminal 16. N.E. Sharpless, R.A. DePintho, Nature molcellbio., 8, pp. 703-713 differentiation process of keratinocytes is indispensable for (2007). desquamation and precisely organized in time and space. The 17. H.J. Stark et al., J Inv Derm., 11, pp. 93-105 (2006). lack of corneodesmosom degradation in organotypic stratum 18. M. Schroeder, C.C. Zouboulis, Horm Metab Res, 39, pp. 136-140 corneum is discussed to be due to the absence of trypic, (2007). chymotryptic and proteolytic enzyme activities, which ideally 19. J.Varani, et al., Arch Dermatol. Res. 295, pp. 255-262 (2003). require finely balanced microenvironmental conditions. Local 20. C.C. Zouboulis et al, J.Exger., 43, pp. 986-997 (2008). pH, Ca2+ and water levels and the presence of cholesterol sulfate 21. C.C. Zouboulis et al., Skin Pharmacol Appl Physiol., 14, pp. 303-315 determine a precise regulation of corneosome degradation (2001). Household and Personal Care Today - n 1/2012 75