Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in mouse tumor-derived basement membrane extracts (BME), which is poorly defined, highly variable and limits the direct clinical applications of organoids in patients. Extracellular matrix (ECM)-derived hydrogels prepared from decellularized human or porcine livers are attractive alternative culture substrates. Here, the culture and expansion of human cholangiocyte organoids in liver ECM(LECM)-derived hydrogels is described. These hydrogels support proliferation of cholangiocyte organoids and maintain the cholangiocyte-like phenotype. The use of LECM hydrogels does not significantly alter the expression of selected genes or proteins, such as the cholangiocyte marker cytokeratin-7, and no species-specific effect is found between human or porcine LECM hydrogels. Proliferation rates of organoids cultured in LECM hydrogels are lower, but the differentiation capacity of the cholangiocyte organoids towards hepatocyte-like cells is not altered by the presence of tissue-specific ECM components. Moreover, human LECM extracts support the expansion of ICO in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Liver ECM hydrogels can successfully replace tumor-derived BME and can potentially unlock the full clinical potential of human cholangiocyte organoids.

Whole-organ decellularization generates scaffolds containing native extracellular matrix (ECM) components with preserved tissue microarchitecture, providing a promising advancement in tissue engineering and regenerative medicine. Decellularization retains the ECM integrity which is important for supporting cell attachment, growth, differentiation, and biological function. Although there are consensus guidelines to standardize decellularization processes and ECM characterization, no specific criteria or standards regarding matrix sterility and biosafety have been established so far. This regulatory gap in safety, sterilization, and regulation criteria has hampered the clinical translation of decellularized scaffolds. In this review, we identify essential criteria for the safe clinical use of decellularized products from both human and animal sources. These include the decellularization efficacy, levels of chemical residue, preservation of ECM composition and physical characteristics, and criteria for the aseptic processing of decellularization to assure sterility. Furthermore, we explore key considerations for advancing decellularized scaffolds into clinical practice, focusing on regulatory frameworks and safety requirements. Addressing these challenges is crucial for minimizing risks of adverse reactions or infection transmission, thereby accelerating the adoption of tissue-engineered products. This review aims to provide a foundation for establishing robust guidelines, supporting the safe and effective integration of decellularized scaffolds into regenerative medicine applications.

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

Tissue decellularization aims to obtain bioscaffolds for regenerative applications by removing all cellular components while preserving the extracellular matrix (ECM) architecture. Although decellularization removes the majority of linear nuclear DNA (nDNA), residual amounts remain detectable. However, the fate of circular mitochondrial DNA (mtDNA) after decellularization has not yet been reported. Cell death or injury can cause the release of mtDNA, which is resistant to breakdown by exonucleases. Extracellular mtDNA acts as a damage-associated molecular pattern (DAMP) that can trigger immune responses. The aim of this study is to assess the presence of residual mtDNA in the liver, bile duct, and vascular scaffolds after decellularization and whether this causes inflammatory responses in macrophages. Decellularized tissues showed a marked reduction in total DNA content well below the threshold of 50 ng/mg tissue. However, in liver and vascular scaffolds, a relative increase in the mtDNA:nDNA ratio was detected in the remnant DNA fraction. Residual mtDNA in bioscaffolds acted as DAMPs causing macrophage activation, as shown by increased cell proliferation and cytokine production. Strategies to further reduce remnant mtDNA were tested. We found that treatment with the endonuclease enzyme HpaII was effective in degrading residual mtDNA. Importantly, mtDNA removal resulted in a significantly reduced macrophage activation. In conclusion, our study shows that mtDNA is relatively resistant to the decellularization procedure and can act as a DAMP in bioscaffolds. This underscores the importance of removing mtDNA from decellularized bioscaffolds to improve the immunocompatibility for biomedical applications.

Purpose: Sleep problems are a frequent concern of colorectal cancer (CRC) survivors. Research on modifiable lifestyle factors that may mitigate sleep problems is sparse. Therefore, we investigated how various body composition parameters are longitudinally associated with sleep problems from 6 weeks up to 24 months post-treatment. Methods: In a prospective cohort of 396 stage I-III CRC survivors, home-based repeated measurements were conducted at diagnosis and at four post-treatment time points. The insomnia scale of the EORTC QLQ-C30 (range: 0–100) was used to measure sleep problems. Anthropometric measurements of adiposity (BMI, fat percentage, waist-hip ratio) and of muscle mass and muscle function (mid-upper arm muscle circumference, handgrip strength) were employed. Linear mixed models were applied to analyze overall longitudinal associations, and hybrid models were used to disentangle inter- and intra-individual components. Results: At 6 weeks post-treatment, 47.0% of participants reported sleep problems and symptom severity was at its highest; a decline was observed thereafter. In confounder-adjusted models, no statistically significant overall longitudinal associations of different body composition parameters with sleep problems were found. Intra-individual analyses revealed that increases in BMI were related to less sleep problems over time ( β per 1 kg/m ² : -2.8, 95% CI -4.4; -1.2). Conclusions: BMI increases in the first 24 months post-treatment were associated with decreased sleep problems. These findings must be interpreted with caution due to the observational design, yet might suggest a potential link between weight regain and sleep problems among CRC survivors recovering from the physical and mental impact of cancer treatment.