Project description:ITo derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine, we developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. We have therefore developed a protocol for the preparation of cell-free extracellular matrix and characterized the material extensively using a combination of proteomics, immunohistochemsitry, and cell population assays. To prepare cell-free matrix, human cadaveric testicular tissue was exposed for 24 h or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulfate (SDS). The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. The protein composition was analysed by LC-MS/MS. The retention of testicular tissue-specific characteristics was evaluated by immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure. Proteomics analysis revelaed the presence of the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, numerous additional ECM proteins in DTM were detected, indicating its complex nature. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay showing a normal fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration.

Project description:The liver is the most common site for colorectal cancer (CRC) metastasis and new tissue culture models are needed to study hepatic metastasis from CRC as none of the current models mimic the biological, biochemical and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of cancer extracellular matrix (ECM) as decellularized scaffolds retained their tissue-specific features and biological properties. In the present study we created a 3D model of CRC and matched CRC liver metastasis (CRLM) using patient-derived decellularized ECM scaffolds seeded with HT-29 CRC cell line. Here we show increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. CRLM scaffolds also induced activation of epithelial-mesenchymal transition (EMT) with cancer cells showing loss of E-cadherin expression and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacethylation, cellular response to stress metabolic processes, response to oxygen level and to starvation. The complex 3D culture environment reduced the HT-29 cell response to anti-CRC drug 5-FU when used at standard IC50 determined in 2D cultures. In conclusion, our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of liver CRC metastasis formation and progression. Transcriptomic analysis was performed comparing the global gene expression profiles of HT29 cells grown on CRLM and HL scaffolds. The transcriptomic profile of each 3D subtype was compared with HT29 cells grown in plastic. Hierarchical clustering analysis revealed that the gene expression signature of recellularized CRLM scaffolds was more comparable to recellularized HL scaffolds, than to HT29 cells grown in 2D. Gene set enrichment analysis (GSEA) of the differently expressed genes (DEG) up-regulated in recellularized CRLM scaffolds compared to HT29 grown in 2D revealed that genes involved in demethylation, deacethylation and metabolic process belong to the most enriched biological pathways in repopulated-CRLM scaffolds. Comparing the DEG between recellularized CRLM scaffolds vs HT29 grown in 2D with a series of a-priori defined gene-set, called Hallmark, we obtained concordant results with the “HYPOXIA” and “EPITHELIAL_MESENCHYMAL_TRANSITION” pathway, enriched in recellularized CRLM, supporting the idea that 3D culture models are the ability to re-create a complex environment in terms of oxygen tension, nutrients, and metabolic gradient, similarly to the in vivo environment.

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Quantifying extracellular matrix turnover in human lung scaffold cultures

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Quantifying extracellular matrix turnover in human lung scaffold cultures.

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