Matrigel Concentration Research Articles

Optimization of Matrigel concentration for salivary gland organoid culture

Background: Research on salivary gland tissue is important, both for maintaining good oral health and for understanding the properties and functions of secretory cells with similar developmental mechanisms and functions. However, research on the pathogenesis of salivary gland disease and the development of therapeutic agents is very insufficient, and no studies have yet reported effective approaches to changes that occur according to the concentration of Matrigel, which is used to mimic the interactions between cells and the extracellular matrix, in three-dimensional organoid culture systems.Methods: We aimed to identify the most suitable concentration of Matrigel for analyzing the morphology and differentiation patterns of organoids prepared using salivary submandibular gland-derived cells after inducing differentiation with or without a specific chemical, Y-27632.Results: In both low and high Matrigel concentration conditions, morphological differences were found between the control group and the Y-27632 treatment group, and the budding structure was also significantly higher in the Y-27632 treatment group. However, more distinct patterns of differentiation appeared in the high-concentration condition.Conclusion: The concentration of Matrigel in the 3-dimensional organoid culture system had a significant effect on mimicking the actual in vivo environment.

Investigation of surface endothelialization on nitinol: Effects of composite hydrogel coatings

BackgroundDue to high metal coverage and high mesh density，flow diverters (FD) are widely used for endovascular reconstruction of cerebrovascular disease. However, incomplete endothelialization, restenosis, and thrombosis hamper the long term clinical success. Thus, an effective coating to accelerate the endothelialization of the cerebrovascular FD raw materials and reduce the incidence of thrombosis and restenosis is needed. MethodsA novel composite hydrogel composed of matrigel and sodium alginate (SA) with different blending ratios, which was cross-linked with calcium chloride, was successfully prepared. The hydrophilicity of the coating surface was detected by measurement of the contact angle, and the surface morphology of the nitinol (NiTi) before and after modification was characterized by scanning electron microscopy (SEM). The elastic modulus was utilized to verify the mechanical properties and plasticity of the coating, and the release experiment was conducted to characterize the composite hydraulic coagulation. Additionally, biocompatibility was tested by in vitro cellular experiments. ResultsThe SEM observations showed that when the concentrations of matrigel and SA were 5 and 3 mg/ml, respectively, cross-linking with 40 mg/ml calcium chloride solution was resulted in formation of the uniform M/SA composite hydrogel coating. The contact angle of M/SA-NiTi was only (32.28 ± 1.55°) (P < 0.0001), indicating a strong hydrophilicity of M/SA-NiTi. The 48 h coating release rate is 61.5%. In addition, M/SA-NiTi exhibited to have better biocompatibility and plasticity than B-NiTi and M-NiTi. ConclusionsMatrigel/sodium alginate composite hydrogel coating has good biocompatibility and low thrombosis characteristics, and strong drug-carrying capacity, which is expected to accelerate the endothelialization after FD implantation. It has also a great potential application value in the surface modification of biological materials.

Extracellular Matrix Optimization for Enhanced Physiological Relevance in Hepatic Tissue-Chips.

The cellular microenvironment is influenced explicitly by the extracellular matrix (ECM), the main tissue support biomaterial, as a decisive factor for tissue growth patterns. The recent emergence of hepatic microphysiological systems (MPS) provide the basic physiological emulation of the human liver for drug screening. However, engineering microfluidic devices with standardized surface coatings of ECM may improve MPS-based organ-specific emulation for improved drug screening. The influence of surface coatings of different ECM types on tissue development needs to be optimized. Additionally, an intensity-based image processing tool and transepithelial electrical resistance (TEER) sensor may assist in the analysis of tissue formation capacity under the influence of different ECM types. The current study highlights the role of ECM coatings for improved tissue formation, implying the additional role of image processing and TEER sensors. We studied hepatic tissue formation under the influence of multiple concentrations of Matrigel, collagen, fibronectin, and poly-L-lysine. Based on experimental data, a mathematical model was developed, and ECM concentrations were validated for better tissue development. TEER sensor and image processing data were used to evaluate the development of a hepatic MPS for human liver physiology modeling. Image analysis data for tissue formation was further strengthened by metabolic quantification of albumin, urea, and cytochrome P450. Standardized ECM type for MPS may improve clinical relevance for modeling hepatic tissue microenvironment, and image processing possibly enhance the tissue analysis of the MPS.

Melt Electrowritten In Vitro Radial Device to Study Cell Growth and Migration.

The development of in vitro assays for 3D microenvironments is essential for understanding cell migration processes. A 3D-printed in vitro competitive radial device is developed to identify preferred Matrigel concentration for glioblastoma migration. Melt electrowriting (MEW) is used to fabricate the structural device with defined and intricate radial structures that are filled with Matrigel. Controlling the printing path is necessary to account for the distance lag in the molten jet, the applied electric field, and the continuous direct-writing nature of MEW. Circular printing below a diameter threshold results in substantial inward tilting of the MEW fiber wall. An eight-chamber radial device with a diameter of 9.4 mm is printed. Four different concentrations of Matrigel are dispensed into the radial chambers. Glioblastoma cells are seeded into the center and grow into all chambers within 8 days. The cell spreading area demonstrates that 6 and 8 mg mL-1 of Matrigel are preferred over 2 and 4 mg mL-1 . Furthermore, topographical cues via the MEW fiber wall are observed to promote migration even further away from the cell seeding depot. Previous studies implement MEW to fabricate cell invasive scaffolds whereas here it is applied to 3D-print in vitro tools to study cell migration.

Screening method to identify hydrogel formulations that facilitate myotube formation from encapsulated primary myoblasts.

Hydrogel‐based three‐dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of tissue specific cells embedded within a support material, such as a hydrogel. As hydrogel properties directly affect cell function, hydrogel composition is often tailored to the cell type(s) of interest and the functional objective of the model. Here, we develop a parametric analysis and screening method to identify suitable encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence‐based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.

850-P: Continued Monitoring of the Foreign-Body Response to Improve Long-Term Success of Macroencapsulation Devices for Type 1 Diabetes: A Proof-of-Concept Study

Over 463 million people are living with diabetes mellitus (DM). Type 1 DM results from an autoimmune destruction of pancreatic β-cells and accounts for 5-10% of cases. Macroencapsulation devices are a promising therapy for the treatment of T1DM as they improve insulin producing cell retention and viability. However they have had limited success due to a complex cascade of events, causing fibrous encapsulation and ultimately device failure (the foreign body response (FBR)). The FBR is a critical hurdle associated with the success of implantable devices. Here, we aim to develop a biosensing membrane that monitors the FBR in real time; ensuring steps can be taken to modulate the host’s response to the implant. A biosensing membrane was developed consisting of a conductive layer of nonmetallic, highly conductive acrylic adhesive sandwiched between two layers of 0.08 mm thermoplastic polyurethane (TPU). Thereafter the membrane was incorporated into a refillable macroencapsulation device with soft robotic capabilities. In vitro experiments were carried out using electrical impedance spectroscopy. Baseline impedance measurement were taken for porous and non-porous devices using a buffered saline (PBS). Various concentrations of Matrigel (30%, 40%, 50% and 60%) were left to solidify on the membrane, as it contains extracellular matrix proteins, making it analogous to fibrotic tissue components. A significant increase of impedance was seen across the Matrigel groups when compared to PBS alone (p&amp;lt;0.001). A significant difference was also evident between the varying concentrations of Matrigel along the spectra, an example of which is 30% vs. 50% Matrigel at 1.2649 Hz (p&amp;lt;0.001). We have developed a novel approach to measure the membrane permeability in vitro. Monitoring the FBR in vivo, will provide feedback on the functionality of implanted devices, to improve their longevity for the delivery of insulin producing cells. Disclosure R.M. Beatty: None. E.B. Dolan: None. S.T. Robinson: None. R. OConnor: None. R. Wylie: None. G.P. Duffy: None.

The use of mixed collagen-Matrigel matrices of increasing complexity recapitulates the biphasic role of cell adhesion in cancer cell migration: ECM sensing, remodeling and forces at the leading edge of cancer invasion.

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes β1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility.

Influence of matrigel on the shape and dynamics of cancer cells

The interaction between extracellular matrices and cancer cells plays an important role in regulating cancer cell behaviors. In this article, we use matrigel to mimic extracellular matrices and investigate experimentally how matrigel influences the shape and dynamics of breast cancer cells (MDA-MB-231-GFP cells). We find that matrigel facilitates cancer cells’ migration and shape deformation. The influences of the matrigel concentration are also reported.

Diversity of collective migration patterns of invasive breast cancer cells emerging during microtrack invasion.

Understanding the mechanisms underlying the diversity of tumor invasion dynamics, including single-cell migration, multicellular streaming, and the emergence of various collective migration patterns, is a long-standing problem in cancer research. Here we have designed and fabricated a series of microchips containing high-throughput microscale tracks using protein repelling coating technology, which were then covered with a thin Matrigel layer. By varying the geometrical confinement (track width) and microenvironment factors (Matrigel concentration), we have reproduced a diversity of collective migration patterns in the chips, which were also observed in vivo. We have further classified the collective patterns and quantified the emergence probability of each class of patterns as a function of microtrack width and Matrigel concentration to devise a quantitive "collective pattern diagram." To elucidate the mechanisms behind the emergence of various collective patterns, we employed cellular automaton simulations, incorporating the effects of both direct cell-cell interactions and microenvironment factors (e.g., chemical gradient and extracellular matrix degradation). Our simulations suggest that tumor cell phenotype heterogeneity, and the associated dynamic selection of a favorable phenotype via cell-microenivronment interactions, are key to the emergence of the observed collective patterns in vitro.

High density gold nanostructure composites for precise electrochemical detection of human embryonic stem cells in cell mixture

Precise detection of undifferentiated human pluripotent stem cells (hPSCs) and their entire subsequent elimination are incredibly important in preventing teratoma formations after transplantation. Recently, electrochemical sensing platforms have demonstrated immense potential as a new tool to detect remaining hPSCs in label-free and non-destructive manner. Nevertheless, one of the critical huddles of this electrochemical sensing approach is its low sensitivity since even low concentrations of remaining hPSCs were reported to form teratoma once transplanted. To address this issue, in this study, we report an engineering-based approach to improve the sensitivity of electrochemical sensing platform for hPSC detection. By optimizing the density of gold nanostructure and the matrigel concentration to improve both electro-catalytic property and biocompatibility, the sensitivity of the developed platform toward hESCs detection could reach 12,500 cells/chip, which is close to the known critical concentration of hPSCs (˜10,000 cells) that induce teratoma formation in vivo. Remarkably, the electrochemical signals were not detectable from other types of stem cell-derived endothelial cells (CB–EPCs) even at high concentrations of CB–EPCs (40,000 cells/chip), proving the high selectivity of the developed platform toward hPSC detection. Hence, the developed platform could be highly useful to solve the safety issues that are related with clinical application of hPSC-derived cells.

The Three-Dimensional Culture System with Matrigel and Neurotrophic Factors Preserves the Structure and Function of Spiral Ganglion Neuron In Vitro.

Whole organ culture of the spiral ganglion region is a resourceful model system facilitating manipulation and analysis of live sprial ganglion neurons (SGNs). Three-dimensional (3D) cultures have been demonstrated to have many biomedical applications, but the effect of 3D culture in maintaining the SGNs structure and function in explant culture remains uninvestigated. In this study, we used the matrigel to encapsulate the spiral ganglion region isolated from neonatal mice. First, we optimized the matrigel concentration for the 3D culture system and found the 3D culture system protected the SGNs against apoptosis, preserved the structure of spiral ganglion region, and promoted the sprouting and outgrowth of SGNs neurites. Next, we found the 3D culture system promoted growth cone growth as evidenced by a higher average number and a longer average length of filopodia and a larger growth cone area. 3D culture system also significantly elevated the synapse density of SGNs. Last, we found that the 3D culture system combined with neurotrophic factors had accumulated effects in promoting the neurites outgrowth compared with 3D culture or NFs treatment only groups. Together, we conclude that the 3D culture system preserves the structure and function of SGN in explant culture.

Abstract 568: Formation of Cardiac Fibers in Matrigel Matrix

INTRODUCTION. BD Matrigel Matrix is a basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins. At room temperature, the matrigel polymerizes to produce a biologically active matrix material resembling the mammalian cellular basement membrane. Matrigel is widely used for cellular attachment, cell invasion assays and as a component of cell differentiation media. Here we report a new means for its use. METHODS. Matrigel is employed to create in vitro cardiac fibers from rat neonatal cardiomyocytes. A semi-solid “pillow” from concentrated matrigel is overlaid with a cardiomyocyte suspension in a diluted matrigel solution. Myocytes submerged in the top matrigel layer form multicellular structures of different sizes. The underling semi-solid matrigel creates an elastic environment which serves as a mechanical load against which myocytes contract. RESULTS. This new technique creates a net of actively contracting multicellular fibers which can be maintained in vitro for up to six weeks. Presented data illustrate the appearance and development of these fibers. Dependence of the fiber size on myocyte amount and matrigel concentration is also described. CONCLUSIONS. This new semi-3D model of cardiac muscle offers the ease of both structural and functional monitoring since fiber thickness of 50 –300 micron makes these structures suitable for live confocal microscopy (calcium and potentiometric indicators) and gives easy assess to the epitopes for immunostaining purposes.

Suppression of CFTR-Mediated Cl− Secretion by Enhanced Expression of Epithelial Na+ Channels in Mouse Endometrial Epithelium

The present study investigated the effect of enhanced expression of epithelial Na+ channels (ENaC) on the cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretion in the mouse endometrium using the short-circuit current technique. The amiloride sensitivity of the basal current of the cultured endometrial epithelia was found to vary with the magnitude of the basal current, the higher the basal current the greater its sensitivity to amiloride, indicating possible elevation of ENaC expression. However, the magnitude of the forskolin-induced Isc, previously demonstrated to be mediated by CFTR, decreased as the amiloride sensitivity of the basal current increased, suggesting a possible inhibitory effect of elevated expression of ENaC on CFTR-mediated Cl− secretion. The Matrigel concentration for culturing the endometrial epithelia was found to affect the amiloride sensitivity of the basal current as well as the forskolin-induced Isc in opposite directions. However, competitive RT-PCR demonstrated that the expression of both ENaC and CFTR was enhanced in Matrigel-treated culture, suggesting that the reduced forskolin-induced Isc with enhanced amiloride sensitivity was not due to a reduction in CFTR expression, but rather suppression of CFTR function by enhanced ENaC expression. In addition to the previously demonstrated inhibition of ENaC by activation of CFTR, the present results reveal possible regulation of CFTR by ENaC. The interaction between the two may be one of the underlying mechanisms for balancing Na+ absorption and Cl− secretion across epithelia.

Matrigel increases the rate of split wound healing and promotes keratinocyte ;take' in deep wounds in rats.

The influence of matrigel, a mixture of the components of thebasement membrane, on the wound healing was studied in a modelof experimental wounds in rats. Matrigel was found to increasethe rate of epithelization of split-thickness wounds. The modelof deep wound was developed in which the host animal could notprovide enough migrating and proliferating keratinocytes tocover the wound area. The model is relevant to severe burns andinjuries in humans. When rat keratinocyte suspension wastransplanted into deep wounds, cell retention in the wound bedwas only observed if matrigel was added together with the cells.Increasing matrigel concentration in the wound was seen toenhance the rate of wound area coverage by the cells. Althoughthe process of healing seemed macroscopically normal, afterhistological screening of the biopsies cell in the wouldappeared as amorphous aggregates and tubules rather thenstratified epidermis.

Human neutrophils do not degrade major basement membrane components during chemotactic migration

At sites of inflammation, circulating neutrophils (PMNs) migrate through microvessel walls into the subendothelial interstitium. While endothelial passage is mediated by adhesion proteins, including those of the integrin, selectin and immunoglobulin superfamily classes, the mechanisms used to cross the subendothelial basement membrane (BM) are unclear. Studies examining tumour cell invasion and lymphocyte extravasation suggest several possible mechanisms, including proteolysis. Different cells, however, may use different mechanisms to effect passage. To examine neutrophil-basement membrane interactions in more detail, human PMNs were embedded within reconstituted BM (Matrigel) and used in migration assays. The integrity of the gel following migration was assessed by assaying for the release of incorporated radiolabelled products and by immunoblotting for specific matrix molecule epitopes. PMNs migrated through Matrigel in response to the chemotactic peptide FMLP. Degradation products of laminin, heparan sulphate proteoglycan or of gelatin, however, were not detected. In contrast, phorbol ester, which triggers activation without migration, released ≈40% of incorporated HSPG, 30% of gelatin and 20% of laminin as intact molecules or degraded fragments. Electron microscopy of migrating cells demonstrated pseudopodia associated with channels within the Matrigel. Although the serine proteinase inhibitor DFP, plasma and a specific anti-neutrophil elastase IgG blocked degradation, these agents failed to inhibit migration. Migration was inhibited, however, when the Matrigel concentration was increased to 10 mg/ml. Thus, although PMNs will degrade matrix components they do not do so during migration, and proteolytic remodelling of the BM is not a pre-requisite for neutrophil passage.

Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology

Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary-spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.

Cell‐substratum adhesion strength as a determinant of hepatocyte aggregate morphology

Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary—spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415–426, 1997.

Development of anin vitro extracellular matrix assay for studies of brain tumor cell invasion

Invasion of brain by tumor cells is an inherent feature of the malignant phenotype. Assays to quantitate invasiveness should provide a powerful tool to investigate this phenomenon. We have developed a modified in vitro assay to measure tumor cell invasion, attachment, and chemotaxis using a barrier of the complex basement membrane Matrigel on gelatin-coated filters. Within 5 hours, 7.8% of U251MGp and 2.6% of SF126 human malignant glioma cells invaded the Matrigel and filter, compared with 0.8% of normal human leptomeningeal cells. The extent of invasion was directly proportional to incubation time and filter pore size and inversely proportional to the Matrigel concentration. Cells from exponentially growing U251MGp cultures invaded more readily (10.9%) than cells from plateau-phase cultures (2.3%); however, labeling studies with bromodeoxyuridine showed that quiescent cells and rapidly dividing cells were equally capable of invading. This suggests that the mechanisms underlying invasion by malignant glioma cells are distinct from those underlying proliferation and indicates the need for therapy aimed specifically at invasive behavior. In a practical application of this assay to test a potential anti-invasive strategy, monoclonal antibodies to the beta subunit of an integrin receptor mediating attachment to the extracellular matrix inhibited invasion by U251MGp cells in a dose-dependent manner. This assay should allow evaluation of the cellular and molecular basis of brain tumor progression and perhaps aid the development of rationally designed drugs that limit tumor invasion. It may also allow prediction of the clinical behavior of neoplasms in individual patients.