In 2009, Sato et al. published a landmark study showing that adult intestinal stem cells could form complex 3D structures in vitro, each containing multiple cell types matching the organ source. Termed organoids, these have since become essential tools for drug discovery and development by serving as more physiologically relevant alternatives to human cell lines and animal disease models. Natural basement membrane extracts (BMEs) derived from gene-edited mouse tumor cells are widely used for organoid culture, typically yielding better results than synthetic derivatives. However, natural BMEs can suffer from batch-to-batch diversity and are often difficult to work with. Next-generation BMEs offer several advantages for organoid-based research, including improved product consistency for more reproducible data.
Although 2D cell cultures have long been used as in vitro models for drug discovery and development, the data that are generated cannot always be replicated in vivo. And, while animal models provide valuable information about drug efficacy, absorption, and potential toxicities, such findings cannot be guaranteed to translate into humans. For these reasons, the pharmaceutical industry has long required a different approach for evaluating potential drug candidates—a need compounded by recent Food and Drug Administration (FDA) guidance recommending that animal use for research be reduced.
Figure 1. BME supports organoid culture
Organoid-based disease modeling addresses these challenges to allow for more accurate assessment of novel drugs. The types of studies performed using organoids include comparing how different drugs influence cell signaling pathways in normal and diseased tissues, and monitoring the process of drug absorption, both of which are key to informing which molecules should be progressed for further optimization. To date, organoids have been derived from organs including the small intestine, heart, and liver, as well as the kidney, lungs, and brain. In addition, tumor-derived organoids have been generated to advance oncology research.
Basement membranes are thin sheets of extracellular matrix (ECM) proteins that are found at the interface between different cell types. Here, they form a scaffold for cellular growth and are involved in critical processes such as adhesion, migration, proliferation, and differentiation. Solubilized basement membrane preparations are core components of organoid culture. They are commonly extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor that is rich in ECM proteins such as laminin, collagen IV, entactin, and heparin sulfate proteoglycan.
Depending on the type of organoid being generated, the BME may be layered into the wells of a microplate prior to cell addition, used to form a sandwich around the growing culture, or be mixed with the cells before being dispensed in a dome formation. In many cases, the BME must be supplemented with various additives. For example, human colonic organoids require epidermal growth factor, Noggin, R-spondin and Wnt as growth factors, along with A83-01 and SB202190 to respectively block TGFβ and p38 signaling. Whichever method is chosen for organoid culture, the primary function of the BME is to provide the conditions needed to support cell growth and differentiation, which can subsequently be analyzed using techniques such as microscopy and single-cell RNA sequencing (scRNA-seq).
Figure 2. Human tumor organoids (A: colorectal cancer organoids, B: gastric cancer organoids, C: breast cancer organoids, D: hepatocellular carcinoma organoids) can grow well in greater than 70% Matrigengel Matrix.
A main challenge when working with BMEs is that they undergo gelation at ambient temperatures. This means that both the BME and any pipette tips used for sample handling must remain chilled to prevent early polymerization. The presence of unwanted growth factors can also be problematic. Using a reduced growth factor BME is often recommended to minimize differentiation influences from intrinsic EHS components. Further issues may arise when working with difficult-to-culture organoids such as those derived from the murine intestine or human brain, which frequently necessitate the use of optimized BME formulations.
Next-generation BMEs are designed to overcome many of the challenges associated with organoid culture. As well as offering improved batch-to-batch consistency for more reproducible results and being formulated to prevent interference from contaminating growth factors, BMEs are now available to support specified uses. Included among these, Matrigengel Matrix BMEs polymerize at 37oC for easier handling and are provided with comprehensive validation data for a broad range of organoid-based applications.
To learn more about Matrigengel Matrix and how it can benefit your research, visit ACROBiosystems.com