Hyaluronic acid Methacryloyl (MW 400 kDa)

3D hydrogel synthetic scaffold culture embeds cells, spheroids, organoids, or tissue fragments inside a hydrated crosslinked polymer network so that cells receive matrix and cell-cell cues in three dimensions rather than from a flat plastic surface. A literature-supported model protocol is PEG-4MAL hydrogel culture, in which four-arm maleimide-terminated PEG is functionalized with cysteine-containing adhesive peptides such as RGD and crosslinked with protease-degradable peptides such as GPQ-W; this creates a defined, modular scaffold that supports human organoid generation and culture. The readouts are scaffold-supported growth, morphology, lumen formation, budding, viability, proliferation, lineage-marker expression, and matrix-dependent expansion or differentiation; reported assays include transmitted-light imaging, immunofluorescence, in situ hybridization, qRT-PCR, and rheological characterization.

Fibrosis and collagen morphometry is based on the quantitative visualization of fibrillar collagen deposition in tissue sections using histochemical stains such as Sirius Red (Picrosirius Red) or Masson's trichrome, followed by image-based or polarization-enhanced analysis to estimate collagen proportional area as a surrogate of extracellular matrix accumulation during fibrotic remodeling. Sirius Red combined with polarized light microscopy enhances detection of collagen fibers due to birefringence properties, enabling more specific visualization of collagen type I and III fibrils compared to conventional bright-field histology, while whole-section or region-restricted digital morphometry reduces field-selection bias in fibrosis assessment. Alternative quantitative approaches include second harmonic generation (SHG) and two-photon excited fluorescence microscopy, which enable label-free detection of fibrillar collagen and have been validated against histological staining and biochemica

Sirius Red or picrosirius red staining is a histochemical method for visualizing collagen-rich extracellular matrix in tissue sections, and collagen fibers are detected as red-stained structures under bright-field microscopy with enhanced birefringence under polarized light. Picrosirius red is useful for assessing total collagen organization, distribution, and fibrosis burden, but polarized color should not be interpreted as a definitive collagen type I versus type III readout because color is affected by fiber orientation, thickness, and packing.

Adult stem-cell epithelial organoid culture is a three-dimensional culture method in which adult epithelial stem cells or isolated epithelial crypts self-organize into organ-like epithelial structures that retain stem-cell activity and generate differentiated epithelial lineages (). In the intestinal model, Lgr5-positive crypt base columnar cells are adult stem cells that generate all intestinal epithelial lineages, and single Lgr5-positive cells or isolated crypts can form long-term crypt–villus organoids in extracellular matrix culture when supplied with epithelial niche signals (). Organoid growth reflects epithelial stem-cell survival, self-renewal, proliferation, and multilineage differentiation; the main readouts are organoid-forming efficiency, growth, morphology, passaging capacity, marker expression, and lineage composition ().

Patient-derived organoids (PDOs) are 3D in vitro models derived from patient tumor tissues that recapitulate the histological, genetic, and functional heterogeneity of the original tumors. These models are established by isolating tumor cells or tissue fragments and culturing them in a 3D extracellular matrix (ECM), such as Matrigel or decellularized ECM, to support self-organization, proliferation, and differentiation. The culture system preserves key features of the tumor microenvironment, including cell-cell interactions, stromal components, and ECM signaling, enabling accurate modeling of tumor biology and drug response for personalized medicine applications.

ALI organoid culture places organoid-derived epithelial cells or tissue fragments on a porous support or collagen-based matrix so that basal surfaces receive medium while the apical surface is exposed to air; in lung organoid-derived ALI cultures, this supports airway epithelial differentiation, barrier formation, mucus production, beating cilia, and pseudostratified epithelial architecture. Gastrointestinal ALI organoid systems similarly support long-term 3D epithelial growth with stromal/mesenchymal components and multilineage differentiation. The main readouts are morphology, barrier integrity, epithelial differentiation, and experimental response readouts. Lung ALI protocols used bright-field microscopy, TEER monitoring, immunofluorescence for ciliated, goblet, club, and basal-cell markers, flow cytometry, viral titration, RNA-seq, and spatial transcriptomic readouts after SARS-CoV-2 infection.

Organoid passaging by mechanical expansion transfers established 3D epithelial organoids from an extracellular matrix dome into fresh matrix after physical fragmentation. The readout is successful re-formation and expansion of organoid fragments into new organoids, reflecting survival of organoid-forming epithelial stem/progenitor cells and continued self-organization in a 3D matrix.

ECM-embedded organoid dome culture embeds epithelial stem cells, crypts, organoid fragments, or tumor-derived epithelial cells in a basement-membrane-like hydrogel such as Matrigel, allowing 3D growth, self-organization, lumen formation, budding or cystic morphogenesis, and lineage maintenance under defined niche-factor-containing medium. The primary readouts are organoid establishment efficiency, growth, morphology, passaging capacity, lineage-marker expression, and, when fluorescently labeled lines are used, microscopy- or flow-cytometry-based quantification of population behavior in 3D culture.

The Matrigel/ECM Transwell invasion assay measures the ability of cells to move toward a chemoattractant while crossing an extracellular-matrix barrier placed on a porous membrane; therefore, the readout reflects both chemotactic motility and matrix invasion rather than migration alone. Matrigel is a basement-membrane-rich matrix derived from Engelbreth-Holm-Swarm mouse sarcoma and has been used as a reconstituted basement membrane barrier in chemoinvasion assays. The assay readout is generated by quantifying cells that reach the underside of the insert membrane or lower compartment after incubation, commonly by staining and counting invaded cells or by fluorescence-based quantification.

Cerebral organoid culture is a cutting-edge in vitro modeling technique that utilizes human pluripotent stem cells-including both embryonic stem cells and induced pluripotent stem cells—to simulate the microenvironment and differentiation programs of human embryonic brain development within a three-dimensional in vitro culture system. Through directed differentiation, it generates structures characteristic of specific brain regions and comprises various functional neuronal cell types. This technology serves as a sophisticated model capable of recapitulating the processes of human brain development and the pathological features of neurological diseases, thereby finding application in mechanistic research, drug screening, and precision medicine.

Matrigel Transwell/Boyden chamber invasion assay measures the ability of cells to degrade or traverse an extracellular matrix-coated porous membrane and move from an upper chamber toward a chemoattractant in a lower chamber. Invasion is distinguished from migration by coating the membrane with Matrigel or basement membrane matrix; uncoated inserts measure migration, while coated inserts require cells to cross an ECM barrier before reaching the underside of the membrane.