Three-dimensional (3D) gastrointestinal (GI) organoids, miniature organs cultured in vitro, are invaluable tools in biomedical research. Their cultivation predominantly relies on Matrigel, a commercially available ECM extracted from Engelbreth-Holm-Swarm mouse sarcoma. However, Matrigel presents several significant drawbacks. Its tumor-derived origin raises concerns about batch-to-batch variability, potential pathogen transmission (e.g., lactate dehydrogenase-elevating virus), and safety issues for translational applications. Furthermore, the tumor ECM composition of Matrigel differs substantially from that of normal tissues, potentially hindering the accurate recapitulation of the GI microenvironment necessary for optimal organoid development and maturation. The high cost of Matrigel also limits its widespread use. These shortcomings have spurred the development of alternative matrices, including synthetic hydrogels like PEG modified with ECM peptides (e.g., RGD) and natural hydrogels (e.g., alginate, fibrin) supplemented with ECM proteins. While promising, these engineered matrices often lack the biochemical complexity of native tissue ECM. This research aims to develop a superior GI organoid culture platform using decellularized pig stomach and small intestinal tissues to create ECM hydrogels that better mimic the native GI microenvironment and overcome the limitations of Matrigel. This will be achieved by investigating whether these GI ECM hydrogels can effectively replace Matrigel, providing optimal cell-matrix signals for GI organoid development, long-term maintenance, and transplantation.

The existing literature extensively documents the use of Matrigel in organoid culture, highlighting its effectiveness while acknowledging its limitations. Several studies have explored alternative matrices for organoid culture to mitigate Matrigel's drawbacks. Synthetic hydrogels, particularly PEG-based systems, have shown promise. These often incorporate RGD peptides to promote cell adhesion and protease-degradable sequences to allow for ECM remodeling. Other studies have utilized natural hydrogels such as alginate or fibrin, incorporating purified ECM proteins, often laminin, to improve organoid growth. However, these approaches often lack the complexity and tissue specificity of the native ECM. The use of decellularized ECM from various tissues has also been explored, demonstrating that such matrices can support organoid growth and exhibit improved biocompatibility compared to Matrigel. However, limitations such as inconsistent ECM preservation and difficulties in achieving reproducible organoid growth have been reported, depending on the decellularization methods used. Thus, this current research aims to address the shortcomings of previous methodologies.

The study employed a multi-faceted approach to evaluate GI tissue-derived ECM hydrogels as Matrigel alternatives. First, optimal decellularization protocols were established for porcine stomach and small intestinal tissues. Two protocols were compared: Protocol 1, using a non-ionic detergent (Triton X-100), and Protocol 2, employing an ionic detergent (sodium deoxycholate, SDC). Protocol 1 was found to be superior, preserving major ECM components (e.g., glycosaminoglycans, GAGs) while effectively removing cellular components. The decellularized tissues (stomach-derived ECM, SEM; intestine-derived ECM, IEM) were then processed to create 3D hydrogels. The hydrogels' biocompatibility was assessed through endotoxin level determination, in vitro evaluation of inflammatory cytokine (TNF-α) secretion from RAW 264.7 macrophages, and in vivo subcutaneous injection into mice. A comprehensive proteomic analysis using mass spectrometry (MS) characterized the protein composition of the hydrogels, comparing them to Matrigel. This analysis assessed batch-to-batch variation and differences between tissues derived from various sources, using both principal component analysis (PCA) and Pearson's correlation analysis. The optimal concentrations of SEM and IEM hydrogels for supporting gastric and intestinal organoid growth were determined. Mouse gastric glands and intestinal crypts were seeded in varying concentrations of the hydrogels and Matrigel. Organoid formation efficiency, gene expression levels of stem cell markers (e.g., Lgr5, Axin2) and tissue-specific differentiation markers, and organoid functionality were evaluated. For gastric organoids, acid secretion was assessed using acridine orange staining, while for intestinal organoids, CFTR function was assessed using a forskolin-induced swelling assay. Immunofluorescence staining was employed to visualize the expression of various stemness and differentiation markers, and RNA sequencing was performed to profile global gene expression in organoids grown in different matrices. The long-term subculture potential of the hydrogels was evaluated through multiple passages. The effects of tissue type and donor age on organoid development were investigated by comparing hydrogels derived from different tissue types (stomach, intestine, skin, lymph, heart, and muscle) and pigs of different ages (2-month-old piglets and 6-month-old adults). The stability and bioactivity of stored ECM hydrogels were also examined. Finally, the feasibility of using the hydrogels for in vivo organoid transplantation was assessed in a mouse model of acute GI injury.

The study revealed several key findings. First, a non-ionic detergent-based decellularization protocol (Protocol 1) proved superior to the ionic detergent protocol (Protocol 2) in preserving ECM components while effectively removing cells. SEM and IEM hydrogels exhibited a nanofibrous ultrastructure and demonstrated excellent biocompatibility. Proteomic analysis showed distinct ECM profiles for SEM and IEM, rich in collagens and proteoglycans, significantly differing from Matrigel's glycoprotein-rich profile. SEM and IEM hydrogels exhibited low batch-to-batch variation. GI organoids grown in SEM and IEM hydrogels demonstrated comparable or superior development, differentiation, and functionality compared to Matrigel controls. Optimized concentrations of SEM (5 mg/ml) and IEM (2 mg/ml) supported optimal organoid growth. Gastric organoids in SEM hydrogel showed comparable acid secretion to Matrigel organoids. Intestinal organoids in IEM hydrogel exhibited similar CFTR function to Matrigel organoids. The hydrogels supported long-term subculture and maintained the self-renewal ability of organoids. Tissue origin significantly impacted organoid development, with GI-derived ECMs supporting superior growth compared to ECMs from other tissues (e.g., skin). Age-related differences in ECM composition also affected organoid development, with piglet-derived ECMs showing enhanced stem cell marker expression compared to adult pig ECMs. Specifically, fibrillin 2, tenascin C, and fibronectin were identified as key proteins contributing to the improved organoid growth potential observed in piglet-derived ECMs. The hydrogels enabled cryopreservation of organoids with improved viability compared to Matrigel. Finally, in vivo transplantation studies demonstrated the efficacy of SEM and IEM hydrogels as carriers for organoid engraftment in injured GI tissues.

The findings directly address the limitations of Matrigel for GI organoid culture. The development of GI tissue-specific ECM hydrogels provides a superior alternative by overcoming the issues of batch-to-batch variation, safety concerns related to tumor-derived material, and high cost. The comprehensive proteomic analysis highlights the importance of tissue-specific ECM composition for optimal organoid development, revealing the abundance of collagens and proteoglycans, and the presence of tissue-specific non-matrisome proteins, which are notably absent in Matrigel. The demonstrated capacity for long-term subculture and cryopreservation significantly enhances the practicality and accessibility of GI organoid research. The successful in vivo transplantation studies show the therapeutic potential of these hydrogels as organoid carriers for GI tissue regeneration, paving the way for future studies in addressing various GI disorders. The data regarding the age-related ECM changes also offer crucial insights into the impact of the aging process on tissue regeneration and the development of advanced culture systems.

This study successfully demonstrates the use of decellularized GI tissue-derived ECM hydrogels as effective, biocompatible, and cost-effective alternatives to Matrigel for culturing GI organoids. These hydrogels support superior organoid development, long-term culture, cryopreservation, and in vivo transplantation. Future research should focus on further refining the hydrogel composition and exploring the therapeutic potential of these hydrogels in treating GI diseases. The identification of specific ECM components contributing to enhanced organoid growth, such as fibronectin, tenascin C, and fibrillin 2, may inform the development of chemically defined hydrogels, ultimately reducing reliance on animal-derived materials.

While this study presents compelling evidence for the advantages of GI tissue-derived ECM hydrogels, some limitations exist. The study primarily used porcine tissue; further validation using human-derived ECMs is necessary to ensure the applicability to clinical translation. Although the study showed promising results with long-term subculture and cryopreservation, further investigations are needed to fully explore the long-term effects on organoid function and genetic stability. The in vivo transplantation studies were relatively short-term; longer-term studies are required to assess the long-term regenerative capacity and potential side effects. The in vivo study used a model of acute injury, and further studies are warranted to investigate the applicability to chronic GI conditions.