Matrigel remains the most widely used scaffold in organoid and 3D culture despite its murine origin, variability, and translational limitations. This review discusses the barriers to replacing Matrigel with xeno-free alternatives and introduces practical tools, including a matrix selection checklist and scaffold assessment criteria, to guide the development and selection of tissue- and model-specific matrices. Abstract Matrigel is a crucial tool in cell biology, particularly for organoid research. It forms a stable scaffold with components like basement membrane proteins and growth factors, resembling the extracellular matrix and mimicking the cellular microenvironment. Its complex composition is both an asset and a drawback, as it is undefined and can vary from batch to batch. Another issue is the murine origin of Matrigel, raising ethical and scientific concerns. Interspecies variation hinders the successful translation of research findings from experimental models to clinical application in humans. Despite these well-known and often-discussed disadvantages, Matrigel is often the first choice of matrix. This review explores why Matrigel remains the gold standard for many human 3D culture systems despite its murine origin and well-known limitations. Therefore, challenges are identified that prevent researchers from transitioning to matrix alternatives and, eventually, developing completely xeno-free human model systems. To tackle these challenges, it is suggested to move beyond a one-for-all approach by pursuing a tissue- and model-specific focus when designing new matrices or selecting an alternative from already available options. To facilitate the implementation of Matrigel substitutes, we provide a matrix selection checklist and a scaffold assessment tool, and quantitative assessment criteria to evaluate matrix-model compatibility are suggested.

Epithelial organoids are grown without Matrigel using a single-chain antibody.

Epithelial organoids are grown without Matrigel using a single-chain antibody.

Primary astrocyte 2D cultures often exhibit a phenotype that differs significantly from astrocytes in the brain. Here, we present a detailed protocol for culturing primary murine astrocytes within a 3D Matrigel environment, which better mimics the in vivo conditions. We describe a step-by-step guide for the isolation and preparation of primary striatal astrocytes, their embedding and cultivation in Matrigel, and the functional characterization of astrocytes grown in this 3D system.

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A recent study by Technion reveals CollPlant's rhCollagen-based bioink, Collink.3D™, outmatching Matrigel® in tissue formation. Promoting eco-friendly practices in engineering.

Using phenotypic and scRNA-seq comparative analyses, Dolgos et al. show that prostate cancer patient-derived organoids (PDOs) cultured in a Matrigel-free culture system exhibit cellular heterogeneity and preserve PCa patient-specific cell populations. An integrated prostate PDO single-cell atlas (PPScA) captures a spectrum of cellular identities and reveals pathways altered in vitro.

Organoids are vital for studying cancer mechanisms and treatment resistance, but their growth usually depends on costly, animal-derived Matrigel. Here, we present a protocol for expanding and maintaining lung-derived organoids without the need for Matrigel. We describe steps for establishing lung cancer organoids, staining organoids for immunohistochemistry, and cultivating them with ClinoStar. By eliminating dependence on Matrigel and its associated costs, this protocol enhances accessibility a...