Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability. / NextBioForm

Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är tvådimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cellextracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDAMB- 231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3DBiosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var nedreglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. / Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triplenegative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RTqPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype.