Enhancing organoid culture: harnessing the potential of decellularized extracellular matrix hydrogels for mimicking microenvironments

Abstract

Over the past decade, organoids have emerged as a prevalent and promising research tool, mirroring the physiological architecture of the human body. However, as the field advances, the traditional use of animal or tumor-derived extracellular matrix (ECM) as scaffolds has become increasingly inadequate. This shift has led to a focus on developing synthetic scaffolds, particularly hydrogels, that more accurately mimic three-dimensional (3D) tissue structures and dynamics in vitro. The ECM-cell interaction is crucial for organoid growth, necessitating hydrogels that meet organoid-specific requirements through modifiable physical and compositional properties. Advanced composite hydrogels have been engineered to more effectively replicate in vivo conditions, offering a more accurate representation of human organs compared to traditional matrices. This review explores the evolution and current uses of decellularized ECM scaffolds, emphasizing the application of decellularized ECM hydrogels in organoid culture. It also explores the fabrication of composite hydrogels and the prospects for their future use in organoid systems.

Keywords: Decellularized Extracellular Matrix; Hydrogels; Microenvironment; Natural-based biomaterials; Organoid culture.

Fig. 1

The history of dECM scaffolds. The emergence of decellularization technology dates back to 1948 [23], primarily applied to the basic decellularization of skeletal muscle. By 2008, this technique had been applied to the study of cardiac decellularization in murine models [26]. Subsequently, the creation of decellularized liver and lung scaffolds was accomplished in 2010 [28], followed by the development of decellularized kidney scaffolds in 2013, with orthotopic transplantation subsequently performed [29]. Studies into the use of dECM for skin and pancreatic tissues were initiated in 2016 [31] and 2018 [34], respectively. Since 2014, the use of dECM has become increasingly diverse. It was first transformed into bio-ink in 2014 [37], proteomic analysis of dECM was initiated in 2016 [39], 3D constructs were created using stem-cell-supported dECM bio-ink in 2017 [33], and dECM was converted into a hydrogel for further applications in 2018 [34]. In 2019, it was verified that dECM could also be used for organoid culture [36], and in 2023, by improving the sample preparation, mass spectrometry and bioinformatics methods, more comprehensive ECM proteins group information makes in-depth analysis on composition of ECM is possible [41]

Fig. 2

The main components of ECM: collagen, proteoglycans, glycoproteins, ECM-related proteins, ECM regulators and secretory factors

Fig. 3

Transitioning from tissue to ECM requires decellularization, a process which falls into two broad categories. The first involves the selection of a reagent—either biological or chemical—followed by the application of decellularization methods such as perfusion, immersion stirring, or ultrasound. The second primarily employs physical means to disrupt the cell membrane for decellularization, with methods including freeze–thaw cycles, electroporation, and pressure

Fig. 4

A Overview of the key steps involved in endometrial hydrogel preparation from the bovine endometrium [137]. (Copyright © 2022 the Author(s). Published by PNAS). B Porcine immature testicular tissues (ITTs) were dissected in small fragments and decellularized before being lyophilized and digested in a solution of HCl/pepsin (n = 20). Drops of 5, 10, 15, 20 and 25 µL were incubated for 1 h at 34 °C to evaluate manipulability after gelation [138]. (Copyright © 2019 by the authors). C Procedure for the formation of porcine liver extracellular matrix (PLECM) gels [139]. (Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved). D Schematic illustration of the generation of gastrointestinal (GI) organoids using ECM hydrogels SEM, IEM. [Decellularized stomach derived ECM (SEM) decellularized intestine derived ECM (IEM)] [140]. (Copyright © The Author(s) 2022)

Fig. 5

A, a–d Bright-field images of mouse endometrial organoids in Matrigel and endometrial hydrogels. The organoids were cultured with hydrogels produced by tissues treated with three different detergents: 4% SDS (hereinafter referred to as P1), 1% SDS (P2) and 4% SDC (P3). A, e Shows the round-shaped mouse endometrial organoids, which was cultured in Matrigel (A, e). In comparison to circular organoids embedded in Matrigel, the organoids cultured in P1 hydrogel exhibited morphological characteristics resembling budding (A, f), tubular (A, g), and glandular structures (A, h) similar to those observed in mouse endometrial tissue. A, i The organoid forming efficiency of mouse endometrial organoids in Matrigel and P1–P3 hydrogel. A, j Percentage of formation of round, tubular and glandular mouse endometrial organoids in Matrigel and P1 hydrogel [137]. (Copyright © 2022 the Author(s). Published by PNAS). B Evaluation of Leydig cells (LC) and Sertoli cells (SC) functionality and maturation in control tissue and TOs. The authors set up three groups of experiments, with normal tissue slices and supernatant collected in vitro culture as the control group (Control). Testicular ECM hydrogels (tECM) prepared using acellular porcine immature testicular tissue (ITT) scaffolds were used as a group. There was also the Collagen hydrogel group (Collagen), which consists primarily of type I collagen but also contains small amounts of other types of collagens (II, III, V, and VI). B, a, b Testosterone and stem cell factor (SCF) was quantified in culture supernatants. B, c Maturation of SCs was monitored in control tissue and TOs by immunohistochemistry (IHC) for anti-Mullerian hormone (AMH). Maturation of SCs evaluated using a score based on AMH immunostaining demonstrated a significant decrease over time in control but not in tECM and collagen groups. B, d Representation of the scores used to determine AMH intensity staining [138]. (Copyright © 2019 by the authors). C, a Hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS) staining, immunofluorescence staining for albumin (ALB; green), and immunohistochemical staining for Ki67. The nuclei were counterstained with DAPI (blue). C, b Secretion of albumin was measured by ELISA on days 2, 4, 6, 8, 14, and 20; C, c Urea synthesis was measured on days 2, 4, 6, 8, 14, and 20. H + M + E group: hepatocytes and MSCs seeded on ECM-gel pre-coated plate; H + E: hepatocytes seeded on ECM-gel pre-coated plate; H + M: hepatocytes and MSCs seeded on ECM-gel free plate; H group: hepatocytes seeded on ECM-gel free plate; ECM: extracellular matrix [139]. (Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved). D, a–e Brightfield images of gastric organoids grown in decellularized stomach derived ECM (SEM) hydrogels and Matrigel (MAT) at day 5. D, f Comparison of expression values (log₂ [FPKM + 0.1]; FPKM, fragments per kilobase of transcript per million mapped reads) of selected genes involved in f gastric or g intestinal development and homeostasis in native gastrointestinal (GI) tissues and in GI organoids cultured in GI tissue-derived ECM hydrogels or Matrigel [140]. Core matrisome protein-encoding genes (Col4a2, Nid1, and Lama3), cytoskeleton-related genes (Flna, Gsn, and Tuba1a), intestinal epithelial gene (Tm4sf4), immune response (Procr, Mcpt2, Icam1, Cxcl10, Cxcl16, and Timp3). (Copyright © The Author(s) 2022)