Cross-linked Collagen Hydrogel Matrix Resisting Contraction To Facilitate Full-Thickness Skin Equivalents.

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.

Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer.

PURPOSE: Pancreatic cancer 3D in vitro models including multicellular tumor spheroid (MCTS), single cell-derived tumor spheroid (SCTS), tissue-derived tumor spheroid, and organotypic models provided powerful platforms to mimic in vivo tumor. Recent work supports that circulating tumor cell (CTC) clusters are more efficient in metastasis seeding than single CTCs. The purpose of this study is to establish 3D culture models which can mimic single CTC, monoclonal CTC clusters, and the expansion of macrometastases. METHODS: Seven pancreatic ductal adenocarcinoma cell lines were used to establish MCTS and SCTS using hanging drop and ultra-low attachment plates. Spheroid immunofluorescence staining, spheroid formation assay, immunoblotting, and literature review were performed to investigate molecular biomarkers and the morphological characteristics of pancreatic tumor spheroids. RESULTS: Single cells experienced different growth patterns to form SCTS, like signet ring-like cells, blastula-like structures, and solid core spheroids. However, golf ball-like hollow spheroids could also be detected, especially when DanG and Capan-1 cells were cultivated with fibroblast-conditioned medium (p < 0.05). The size of golf ball-like hollow spheroids hardly grew after getting matured. Only DanG and Capan-1 could establish SCTS- and MCTS-derived hollow spheroids using hanging drop plates and ultra-low attachment plates. Other PDA cell lines could also establish tumor spheroid with hanging drop plates by adding methylated cellulose. Tumor spheroids derived from pancreatic cancer cell line DanG possessed asymmetrically distributed proliferation center, immune-checkpoint properties. ß-catenin, Ki-67, and F-actin were active surrounding the crater-like structure distributing on the inner layer of viable rim cover of the spheroids, which was relevant to well-differentiated tumor cells. CONCLUSIONS: It is possible to establish 3D CTC cluster models from homogenous PDA cell lines using hanging drop and ultra-low attachment plates. PDA cell line displays its own intrinsic properties or heterogeneity. The mechanism of formation of the crater-like structure as well as golf ball-like structure needs further exploration.