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Matrigel
Matrigel
Matrigel
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Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin/collagen IV-rich basement membrane extracellular environment found in many tissues and is used by cell biologists as a substrate (basement membrane matrix) for culturing cells.[1][2]

Cell culture

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A common laboratory procedure is to dispense small volumes of chilled (4 °C) liquid Matrigel onto plastic tissue culture labware. When incubated at 37 °C (body temperature) the Matrigel proteins polymerize (solidify) producing a recombinant basement membrane that covers the labware's surface.

Cells cultured on Matrigel demonstrate complex cellular behavior that is otherwise difficult to observe under laboratory conditions. For example, endothelial cells create intricate spiderweb-like networks on Matrigel-coated surfaces but not on plastic surfaces.[3] Such networks are highly suggestive of the microvascular capillary systems that suffuse living tissues with blood. Hence, Matrigel allows them to observe the process by which endothelial cells construct such networks that are of great research interest.[citation needed]

Metastasis model

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In some instances researchers may prefer to use greater volumes of Matrigel to produce thick three-dimensional gels. Thick gels induce cells to migrate from the gel's surface to its interior. This migratory behavior is studied by researchers as a model of tumor cell metastasis.[citation needed]

Cancer drug screening

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Pharmaceutical scientists use Matrigel to screen drug molecules. A typical experiment consists of adding a test molecule to Matrigel and observing cellular behavior. Test molecules that promote endothelial cell network formation are candidates for tissue regeneration therapies whereas test molecules that inhibit endothelial cell network formation are candidates for anti-cancer therapies. Likewise, test molecules that inhibit tumor cell migration may also have potential as anti-cancer drugs. Matrigel is also commonly used to prepare human tumor xenografts in rodents as part of a cancer drug discovery program.[4] Matrigel is mixed with immortalized human cancer cells and the mixture is injected subcutaneously in immunodeficient mice. A human tumor usually forms in two to four weeks. This model system allows a researcher to test an anti-cancer compound in a surrogate host.

Constituents

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The ability of Matrigel to stimulate complex cell behavior is a consequence of its heterogeneous composition. The chief components of Matrigel are structural proteins such as laminin, nidogen, collagen and heparan sulfate proteoglycans which present cultured cells with the adhesive peptide sequences that they would encounter in their natural environment.[1] Also present are growth factors like TGF-beta and EGF that prevent differentiation and promote proliferation of many cell types. A growth-factor-reduced Matrigel is also available. Matrigel contains other proteins in small amounts and its exact composition can vary from lot to lot. For this reason, Matrigel may not be appropriate for experiments that require precise knowledge of all proteins and concentrations.

Embryonic stem cells

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Matrigel is also used as an attachment substrate in embryonic stem cell culture.[5] When embryonic stem cells are grown in the absence of feeder cells, extracellular matrix components are needed to maintain the pluripotent, undifferentiated state (self-renewal). One of these matrices that can be used is diluted Matrigel. When used undiluted, Matrigel promotes stem cell growth and differentiation.

See also

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References

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Matrigel

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Background and History

Discovery and Development

Matrigel originated from research on the Engelbreth-Holm-Swarm (EHS) mouse sarcoma tumor, a transplantable tumor first described in the 1950s and studied extensively in the 1960s at the National Institute of Dental Research (NIDR) at the National Institutes of Health (NIH) for its rich extracellular matrix components. George R. Martin, a pioneering matrix biologist at NIH, initiated these tumor studies in the 1960s to investigate basement membrane collagens, leading to the identification of type IV collagen as a distinct basement membrane component in 1969. This work built on earlier findings, such as Nicholas A. Kefalides' 1966 isolation of type IV collagen from glomerular basement membranes, but the EHS tumor provided an abundant, reproducible source for further extraction and analysis. In the 1970s and early , researchers including Roslyn W. Orkin and Pamela Gehron Robey at NIH characterized the EHS tumor's properties, confirming its similarity to natural tissue matrices. Hynda K. Kleinman, who joined the Laboratory of Developmental Biology and Anomalies (LDBA) at NIDR in 1975, advanced the development of an extraction method using high-salt and chaotropic agents to isolate a soluble complex that gelled at physiological temperatures. John R. Hassell, a collaborator in the LDBA, coined the term "Matrigel" for this extract in the early , reflecting its matrix-like gel formation. The first scientific description of Matrigel appeared in a 1986 publication by Kleinman and colleagues, detailing its composition and biological activity as a reconstituted . Milestone applications followed rapidly: in 1987, Albini et al. developed the first invasion assay using Matrigel to quantify tumor cell invasive potential . By 1988, et al. demonstrated its utility in assays, where endothelial cells formed capillary-like structures on Matrigel, establishing it as a key tool for studying vascular development. NIH filed a for the extraction method in 1985 (issued as US4829000A in 1989), paving the way for commercialization. In the 1990s, Matrigel transitioned to a commercial product distributed by companies including Collaborative Biomedical Products and later Becton Dickinson, with production now handled by Corning Life Sciences, enabling widespread adoption in research.

Production Methods

Matrigel is produced by isolating components from the Engelbreth-Holm-Swarm (EHS) , a tumor model that accumulates proteins. The source material consists of EHS tumors implanted subcutaneously into lathyrigenic fed a diet supplemented with β-aminopropionitrile (BAPN) to inhibit cross-linking and enhance matrix accumulation. Tumors are harvested after 3-4 weeks of growth, when they reach approximately 4 g in mass, representing about 20% of the body weight. The extraction process begins with homogenizing the harvested tumors in a saline solution to wash out soluble proteins and cellular debris. The insoluble matrix is then extracted using chaotropic agents, such as 1 M guanidine or 2 M , to disrupt non-covalent interactions while preserving protein integrity. This is followed by high-speed (typically 100,000 × g for 1 hour) to pellet the matrix components, separating them from the supernatant. Purification involves dialysis of the pelleted material against ( 7.4) at 4°C for several days to remove the chaotropic agents and restore physiological conditions. The resulting viscous, colorless solution is sterile-filtered and stored at -20°C as a liquid; upon warming to 37°C, it polymerizes into a within 30-60 minutes due to of its protein components. The final product yields approximately 60% by weight, along with other matrix proteins. Commercial production of Matrigel has been scaled up by Corning since the , following initial development at the . The process includes rigorous quality control measures, such as testing for sterility, low endotoxin levels (<1 EU/mL), and absence of mouse pathogens like LDEV (lactate dehydrogenase-elevating virus) via PCR and mouse antibody production assays. Variations include high-concentration formulations (>13 mg/mL protein) for applications and growth factor-reduced (GFR) versions, achieved through additional to lower levels of factors like TGF-β and EGF (e.g., TGF-β reduced to <0.3 ng/mL from ~2 ng/mL in standard).

Composition and Properties

Major Constituents

Matrigel is primarily composed of basement membrane proteins extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma tumor, with laminin constituting approximately 60% of its protein content. Laminin in Matrigel is predominantly the laminin-111 isoform, a heterotrimer formed by α1, β1, and γ1 chains, which plays a key role in cell adhesion and signaling. Collagen type IV accounts for about 30% of the matrix, forming intricate network structures that provide structural support and facilitate interactions with other components. , also known as entactin, comprises roughly 8% and serves as a bridging molecule that links laminin to collagen IV, enhancing the overall integrity of the matrix. Proteoglycans, particularly heparan sulfate proteoglycans such as , make up 1-2% of Matrigel's composition and contribute to its ability to bind and sequester growth factors. These proteoglycans are essential for modulating bioactive molecule presentation within the matrix. Embedded growth factors include transforming growth factor-β (TGF-β) at concentrations of 1.7-4.7 ng/mL, which promotes cell differentiation; epidermal growth factor (EGF), supporting proliferation; fibroblast growth factor-2 (FGF-2), aiding angiogenesis; and vascular endothelial growth factor (VEGF) at 5.0-7.5 ng/mL. These concentrations can vary between batches, influencing biological activity. In addition to these major elements, Matrigel contains trace amounts of other proteins such as fibronectin and vitronectin, alongside a complex mixture identified through proteomic analysis revealing nearly 2,000 unique proteins and over 14,000 peptides. Recent proteomic studies as of 2025 have confirmed over 1,800 proteins in Matrigel. This diverse proteome underscores Matrigel's role as a multifaceted extracellular matrix mimic. Overall, its composition closely resembles natural basement membranes found in tissues like the skin and kidney glomeruli, providing a supportive environment for cellular processes.

Physical Properties and Variability

Matrigel exists as a viscous liquid when maintained at low temperatures, typically 4°C, where its protein components remain soluble, allowing for easy handling and pipetting. Upon warming to physiological temperatures around 37°C, it undergoes rapid gelation, forming a stable hydrogel through the self-assembly of its major structural proteins, such as laminin and collagen IV, which entangle to create a basement membrane-like network. This temperature-dependent phase transition is reversible below 10°C but becomes irreversible after full polymerization at 37°C, with gelation typically completing within 30-60 minutes. The resulting hydrogel exhibits pore sizes on the order of 1-5 micrometers, facilitating the embedding and three-dimensional growth of cells while permitting nutrient diffusion and cell migration. The mechanical properties of the gelled Matrigel closely mimic the softness of natural basement membranes, with an elastic modulus generally ranging from 10 to 400 Pa depending on protein concentration and measurement conditions. At standard concentrations of 8-12 mg/mL, the storage modulus (G') stabilizes around 50-250 Pa after gelation, as determined by rheological analysis using oscillatory shear testing, providing a compliant substrate that supports epithelial and stem cell morphogenesis without excessive stiffness. In its liquid form at 4°C, Matrigel has a viscosity of approximately 10-50 cP, which increases with protein concentration and necessitates specialized pipetting techniques for accurate dispensing. Batch-to-batch variability in Matrigel arises primarily from the inherent heterogeneity of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma tumors used as the source material, leading to fluctuations in protein composition and ratios, with studies reporting only about 53% similarity in identified proteins across lots. These differences can manifest as 10-30% variations in key component levels, such as laminin or entactin, affecting gelation kinetics and mechanical stiffness between preparations. To mitigate this, growth factor-reduced (GFR) formulations are produced by depleting over 90% of endogenous growth factors like TGF-β, which helps standardize functional properties for reproducible cell culture outcomes while preserving the core structural matrix. Matrigel demonstrates good long-term stability when stored undiluted at -20°C, with a typical shelf life of 1-2 years, during which it retains its ability to form functional gels upon thawing. Once gelled in culture, the matrix is susceptible to enzymatic degradation by matrix metalloproteinases (MMPs) secreted by embedded cells, leading to gradual remodeling over days to weeks, which can influence experimental timelines in 3D models. Efforts to enhance reproducibility include detailed proteomic profiling, such as a seminal 2010 study that identified over 1,193 proteins from more than 14,000 unique peptides using liquid chromatography-tandem mass spectrometry, revealing the full compositional complexity and aiding in quality control. Manufacturers like Corning implement lot-specific certification through biochemical assays and functional testing to verify protein content and gelation performance, ensuring consistency across commercial batches.

Applications

In Vitro Cell Culture

Matrigel serves as a versatile basement membrane matrix in in vitro cell culture, enabling the mimicry of extracellular matrix (ECM) environments to support cell adhesion, proliferation, and differentiation in both two-dimensional (2D) and three-dimensional (3D) formats. In 2D applications, Matrigel is applied as a thin coating to culture surfaces, typically at a dilution of 1:30 from its stock concentration (approximately 8-12 mg/mL), to enhance cell attachment and promote partial polarization of epithelial cells. This coating facilitates the formation of polarized structures, such as acini by mammary epithelial cells, by providing integrin-binding sites and biochemical cues that stabilize cell-ECM interactions. In 3D culture, cells are suspended in liquid Matrigel at 37°C, where it rapidly polymerizes into a gel, encapsulating cells to recapitulate tissue-like morphogenesis. This setup has been instrumental in studies of mammary gland branching since the 1990s, where epithelial cells embedded in Matrigel form duct-like structures in response to growth factors, highlighting the matrix's role in directing collective cell behaviors such as invasion and lumen formation. Specific applications include the endothelial tube formation assay, first described in 1988, where human endothelial cells seeded on Matrigel rapidly organize into capillary-like networks within hours, aiding the study of vascular morphogenesis. Matrigel also supports neurosphere cultures, where neural progenitor cells form floating aggregates that differentiate into neurons and glia upon matrix contact, enhancing neuronal maturation compared to standard media. In diabetes modeling, Matrigel embedding promotes the aggregation and functional maturation of pancreatic beta-cell clusters, improving insulin secretion responsiveness to glucose stimuli. These uses underscore Matrigel's ability to foster complex tissue architectures beyond simple monolayer growth. A key advantage of Matrigel in these cultures is its recapitulation of native ECM interactions, which significantly boosts cell viability—often exceeding 90% in 3D setups—compared to traditional 2D plastic substrates, where anoikis and lack of mechanical support lead to higher apoptosis rates. Standard protocols involve diluting Matrigel 1:10 to 1:100 in cold medium to achieve desired gel stiffness, followed by 24-48 hours of incubation at 37°C for matrix stabilization before cell seeding. While Matrigel promotes angiogenesis-related tube formation in endothelial assays, its broader utility lies in general morphogenesis support.

Stem Cell Research

Matrigel plays a crucial role in supporting the pluripotency of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) when used in diluted form. A standard protocol involves diluting hESC-qualified Matrigel at a ratio of 1:100 in DMEM/F-12 and coating culture vessels, followed by maintenance in mTeSR1 medium, which enables robust colony formation and self-renewal. This feeder-free system allows hESCs and iPSCs to be expanded for more than 20 passages while preserving a normal karyotype and pluripotency markers such as OCT4 and NANOG. In differentiation protocols, full-strength (undiluted) Matrigel is employed to embed stem cell aggregates, promoting directed lineage commitment through its rich extracellular matrix components that mimic basement membranes. For neural differentiation, embedding iPSC-derived neural progenitors in Matrigel enhances survival and neuronal maturation, with increased expression of markers like βIII-tubulin compared to 2D cultures. Similarly, cardiac differentiation benefits from Matrigel overlays or sandwiches, where it facilitates mesoderm formation and subsequent cardiomyocyte specification, yielding beating clusters with high troponin T positivity. Endodermal lineages, including hepatocyte-like cells, are induced using 2010s-era protocols where cells are plated on or within full-strength Matrigel, activating pathways like Wnt and BMP for efficient hepatic maturation. Matrigel serves as an essential scaffold for generating organoids from pluripotent stem cells, enabling three-dimensional self-organization and tissue-like architecture. In cerebral organoid protocols, iPSC-derived embryoid bodies are embedded in full-strength Matrigel droplets to promote neuroepithelial expansion and cortical layering, as demonstrated in the 2014 method that recapitulates human brain development over 2-3 months. For intestinal and kidney organoids, Matrigel's laminin-rich composition supports crypt-villus structures and nephron formation by facilitating epithelial polarization and signaling. This enhancement occurs via laminin-mediated integrin activation, which stabilizes cell-matrix interactions and drives morphogenesis. Specific applications highlight Matrigel's utility in stem cell research models. In 2016 studies, iPSC-derived neural rosettes cultured in Matrigel were used to investigate Zika virus infection, revealing preferential targeting of progenitors and disruption of radial glia-like structures. Additionally, Matrigel-supported co-cultures of iPSC-derived endothelial and neural cells have been employed to model the blood-brain barrier, achieving tight junction formation and permeability akin to in vivo conditions. These examples underscore Matrigel's role in creating physiologically relevant platforms for developmental and disease studies. Quantitative assessments demonstrate Matrigel's superior performance in differentiation efficiency.

Cancer Modeling and Angiogenesis

Matrigel plays a pivotal role in cancer modeling by providing a basement membrane-like extracellular matrix that recapitulates the tumor microenvironment, enabling the study of invasion, metastasis, and angiogenesis processes. One of the foundational applications is the chemoinvasion assay using Boyden chambers, developed by Albini et al. in 1987, where a thin layer of Matrigel serves as a barrier to quantify the invasive potential of tumor cells. In this assay, cancer cells, such as the highly invasive MDA-MB-231 breast cancer line, are placed in the upper chamber, and their ability to penetrate the Matrigel toward a chemoattractant in the lower chamber is measured, typically over 4-6 hours, providing insights into matrix degradation and migratory behavior. In vivo tumor xenograft models further leverage Matrigel to enhance tumor engraftment and growth. Cancer cells are suspended in Matrigel and injected subcutaneously into immunodeficient mice, where the matrix supports cell survival, proliferation, and vascularization, leading to the formation of palpable, vascularized tumors within 2-4 weeks. This approach, widely adopted since the early 1990s, improves take rates for low-tumorigenic cell lines and allows longitudinal monitoring of tumor progression and neovascularization. For angiogenesis studies, the Matrigel plug assay, pioneered by Passaniti et al. in 1992, offers a quantifiable in vivo model of vascular ingrowth. Liquid Matrigel, supplemented with pro-angiogenic factors such as fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF), is injected subcutaneously into mice, where it solidifies into a plug; after 7-14 days, vessel formation is assessed via hemoglobin content or histological analysis to evaluate functional neovascularization. This method has become a standard for screening angiogenic modulators due to its simplicity and reproducibility. Matrigel also facilitates the investigation of vascular mimicry, where aggressive cancer cells adopt endothelial-like properties to form perfusable, tube-like structures in three-dimensional cultures. In these assays, tumor cells seeded on or within Matrigel organize into branching networks that mimic blood vessels, independent of endothelial cells, highlighting alternative perfusion mechanisms in tumors. This phenomenon, first demonstrated in aggressive melanomas and extended to other cancers, underscores Matrigel's utility in elucidating non-traditional vascularization pathways. Recent advancements include the use of Matrigel to generate microtumor spheroids for hypoxia research, as optimized by Benton et al. in 2015, where breast cancer spheroids embedded in tumor-aligned Matrigel and medium replicate hypoxic gradients and stromal interactions observed in solid tumors. By 2022, Matrigel had been featured in over 12,000 publications, reflecting its enduring impact on understanding tumor biology and vascular dynamics.

Drug Discovery and Screening

Matrigel has become a cornerstone in drug discovery and screening, particularly for evaluating anticancer and angiogenic compounds in physiologically relevant three-dimensional (3D) environments that mimic the extracellular matrix of tumors. Its ability to support complex cellular interactions, such as endothelial tube formation and tumor cell invasion, enables high-throughput assays to identify inhibitors of pathological angiogenesis and tumor progression. These applications leverage Matrigel's gel-forming properties to create stable scaffolds for co-culturing cells with candidate drugs, facilitating the assessment of therapeutic efficacy before advancing to in vivo models. In angiogenesis inhibitor screening, Matrigel is widely used in tube formation assays where endothelial cells, such as human umbilical vein endothelial cells (HUVECs), are seeded on the matrix to form capillary-like structures in the presence of pro-angiogenic factors like vascular endothelial growth factor (VEGF). Candidate inhibitors disrupt this network, providing a quantifiable readout of anti-angiogenic potential. This assay's simplicity and reproducibility have made it a standard for screening compounds targeting VEGF signaling pathways. Tumor growth assays often involve embedding xenograft-derived tumor cells in Matrigel to form subcutaneous plugs or implants in animal models, allowing co-culture with test drugs to evaluate effects on proliferation and survival. Viability is typically measured using colorimetric assays like MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for metabolic activity or bioluminescence imaging with luciferase-expressing cells to track real-time tumor burden non-invasively. These Matrigel-embedded xenografts enhance tumor take rates and mimic stromal interactions, improving the reliability of drug response predictions in preclinical settings. Organotypic 3D models using better recapitulate tumor heterogeneity and drug penetration barriers compared to two-dimensional (2D) cultures, leading to more accurate predictions of clinical efficacy. In studies from 2015, Matrigel-supported organotypic assays revealed doxorubicin resistance patterns in breast cancer cells that aligned closely with patient outcomes, unlike 2D monolayers which overestimated sensitivity due to altered drug diffusion and cell-matrix interactions. This enhanced predictive power stems from Matrigel's promotion of multicellular spheroids and extracellular matrix remodeling, which influence chemotherapeutic resistance mechanisms like efflux pump expression. High-throughput adaptations of Matrigel-based assays utilize 96-well plates where small domes of the matrix are formed for embedding cells or organoids, enabling automated imaging and analysis of drug effects on morphology, viability, and invasion. By 2020, these formats had been applied in numerous screens, supporting the evaluation of thousands of compounds across cancer types through scalable protocols that integrate fluorescence microscopy for endpoints like organoid size reduction or apoptotic markers. This approach accelerates lead identification while maintaining 3D fidelity. Notable examples include the testing of bevacizumab, a monoclonal VEGF antibody, in Matrigel plug assays where it significantly reduces vascularization and hemoglobin content in implanted gels, confirming its anti-angiogenic mechanism in vivo. More recently, integration of patient-derived organoids (PDOs) in Matrigel has advanced personalized medicine; 2024 studies established PDOs from gastric cancer patients embedded in Matrigel for drug screening, achieving over 75% success rates in modeling histological features and predicting individual responses to chemotherapeutics like 5-fluorouracil. These PDO-Matrigel systems enable tailored therapy selection by correlating ex vivo sensitivities with clinical outcomes.

Limitations and Ethical Considerations

Challenges in Use

One major challenge in using Matrigel stems from its batch-to-batch variability, arising from the natural heterogeneity in the Engelbreth-Holm-Swarm (EHS) mouse tumor extracts used for its production. This variability manifests in differences in protein composition, growth factor concentrations, and gelation properties, leading to inconsistent cell responses across experiments. For instance, studies have reported substantial discrepancies in cell invasion assays due to altered matrix stiffness and biochemical cues. Such inconsistencies undermine reproducibility in high-throughput screening and long-term cultures, prompting calls for standardized quality controls. Biologically, Matrigel's tumor-derived origin introduces undefined factors that can confound results and pose risks. As a complex mixture extracted from murine sarcomas, it contains latent bioactive components, including angiogenic growth factors and potential contaminants like the lactate dehydrogenase-elevating virus (LDHV), which has been detected in multiple batches and may alter immune responses or cell behavior in assays. Furthermore, its composition, derived from rapidly growing tumors in young mice, poorly mimics the extracellular matrix of aged or diseased human tissues, limiting its utility in modeling age-related pathologies or chronic conditions where matrix remodeling differs significantly. Ethical concerns arise from Matrigel's reliance on animal sourcing, requiring the sacrifice of mice to harvest EHS tumors; estimates indicate that producing a single 10 mL vial necessitates euthanizing at least two animals bred specifically for tumor induction. This xenogenic material, being murine in origin, also restricts clinical translation, as residual animal proteins could elicit immune reactions in human therapies or introduce species-specific artifacts in preclinical testing. Controversies surrounding Matrigel include its potential to confound toxicity screens through nonspecific drug adsorption. The matrix's high protein content, particularly laminin and collagen IV, binds lipophilic small molecules with affinities correlating to their hydrophobicity (e.g., partition coefficients up to 483 M⁻¹ for certain benzene derivatives), reducing effective drug concentrations and masking true cytotoxic effects. In the 2010s, critiques in the organoid field highlighted over-reliance on Matrigel for its undefined nature, arguing it perpetuates variability and hampers scalable, reproducible models for disease research. Practically, Matrigel's high cost—approximately $50–$100 per mL for standard formulations, as of 2025—and inconsistent degradation in long-term cultures (>4 weeks) pose additional barriers. The matrix often undergoes premature breakdown due to enzymatic activity from embedded cells, leading to structural instability and loss of 3D architecture in extended experiments, such as maintenance beyond one month. These issues, compounded by storage requirements and limited scalability, increase experimental expenses and complexity.

Alternatives to Matrigel

Synthetic options, such as (PEG) hydrogels, provide defined, tunable matrices for culture as animal-free substitutes to Matrigel. These hydrogels can be functionalized with peptides like RGD to promote and are engineered to mimic mechanics, typically with values ranging from 200 to 500 Pa. For instance, RGD-functionalized PEG-4MAL hydrogels support the generation and expansion of human intestinal organoids from pluripotent stem cell-derived spheroids, enabling robust growth and transplantation for colonic wound repair. gels represent another synthetic alternative, valued for their highly tunable mechanical properties that allow precise control over matrix to study mechanobiology in differentiation and tissue . Natural alternatives include gels, which form through the of fibrinogen with to create biocompatible 3D networks. Typically prepared at 7.5 mg/mL fibrinogen and 0.1 U/mL , these gels support vascular differentiation from induced pluripotent stem cells (iPSCs), yielding structures with comparable size, , and endothelial composition to those cultured in Matrigel. Decellularized extracellular matrix (ECM) hydrogels, derived from tissues like the , offer species-specific matrices that retain native bioactive components such as collagens and proteoglycans. Gastrointestinal-derived hydrogels, solubilized at concentrations of 2-5 mg/mL, facilitate the culture of gastric and intestinal with morphology, proliferation, and profiles equivalent to Matrigel, while exhibiting low batch-to-batch variability and endotoxin levels below 0.5 EU/mL. Recombinant proteins, such as , serve as xeno-free coatings for 2D iPSC maintenance and as bases for 3D models. This human-derived supports feeder-free iPSC culture, preserving pluripotency markers like OCT3/4 and Nanog at levels indistinguishable from Matrigel, with over 95% of cells maintaining undifferentiated states across multiple passages. In blood-brain barrier models, Vitronectin XF enables the differentiation of iPSC-derived vascular organoids with similar barrier integrity and to Matrigel-based systems. These alternatives offer key advantages, including xeno-free composition to minimize risks and enhanced in experimental outcomes. For example, synthetic PEG hydrogels significantly reduce variability in size and viability compared to Matrigel, supporting standardized applications in drug screening and . They are particularly useful in , where defined matrices enable scalable production of organoids for transplantation. Despite these benefits, challenges persist, such as initially higher production costs for recombinant and synthetic materials compared to Matrigel. Additionally, some options like gels exhibit reduced bioactivity for certain differentiation pathways, often requiring supplemental growth factors or peptides to achieve full maturation of structures equivalent to Matrigel.

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  60. https://pmc.ncbi.nlm.nih.gov/articles/PMC5664213/
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