Everything you ever wanted to ask about Corning® Matrigel® Matrix

The minimum protein concentration may be application dependent. You should use the lot specific protein concentration from the Certificate of Analysis to determine the optimal protein concentration range for your specific application.  In general, Matrigel matrix diluted to a concentration of 3 mg/mL will form a firm gel.  For in vivo applications do not dilute Matrigel to a final concentration below 4 mg/mL.

Store Matrigel matrix at -20°C in a non-frost-free freezer. If you aliquot Matrigel matrix after the first thaw, store at -70°C or -20°C in a non-frost-free freezer using polypropylene or other compatible tubes that can withstand the cold temperature.

Corning Matrigel matrix precoated plates are well suited in circumstances where a specific application protocol is being followed.  Test conditions necessary for optimal cell functionality, including Matrigel concentration and volume, have been empirically demonstrated and a protocol provided. Examples of such assays are angiogenesis tube formation, primary hepatocyte culture, tumor invasion and stem cell culture; all of which benefit from proven, published protocols and the reliability of precoated plates.  Manufacturing consistency, quality control testing, shelf life, stability testing and off-the-shelf use are key benefits.

For less established protocols, a drawback to precoated plates is the inability to titrate concentration and volume to drive to the functional cell response desired.

If a specific Matrigel matrix formulation is needed, Corning can work with you to provide a custom precoated Matrigel matrix solution in various formats, ranging from high-throughput for drug screening and toxicity applications to multi-well plates and flasks for cell culture. Please contact your Corning representative for details on custom Matrigel matrix custom solutions.

Phenol red-free formulations are manufactured using DMEM that does not contain phenol red, so as a result the product is colorless.  Phenol red-free formulations are recommended for assays that require color detection.  Phenol red may exhibit estrogenic effects, so we recommend using phenol red-free Matrigel matrix if estrogenic effects are an application concern.

Phenol red containing formulations are manufactured with DMEM containing phenol red.  The color variations that are observed in frozen & thawed Matrigel matrix products that contain phenol red may range from straw yellow to dark red. This is due to the interaction of carbon dioxide with the bicarbonate buffer and the phenol red.  The color variation does not affect product efficacy, and will disappear upon equilibration with 5% CO₂.

Yes, you can thaw Corning® Matrigel® matrix overnight in a refrigerator. First submerge Matrigel matrix vials in an ice bucket filled with ice at 2°C to 8°C. Place the covered ice bucket toward the back of the refrigerator where it will not be subjected to temperature change. Use an adequate amount of ice so that the Matrigel matrix vial is in ice for the entire thawing process (not in cold water). Once thawed, swirl the vials in ice to ensure the material is evenly distributed.

Not always. Corning offers hESC-qualified Corning Matrigel matrix (Corning Cat. No. 354277) which is QC tested for hESC maintenance to ensure consistency, reproducibility, and reliability in performance. This product has been qualified for use with STEMCELL Technologies’ mTeSR™1 medium. It has been shown that human embryonic stem cells grown in mTeSR1 on Corning Matrigel matrix hESC-qualified matrix-coated plates for five passages remain undifferentiated by standard morphology and surface marker expression.

In addition, Corning BioCoat™ Matrigel matrix 6-well plates (Corning Cat. No. 354671) are ready to use and offer lot-to-lot consistency for culturing human ES cells while maintaining their ability for self-renewal and pluripotency. Although non-hESC-qualified Corning Matrigel matrix may work for this application, the results may vary because these products are not qualified for use with hES cells.

Corning Matrigel hESC-qualified matrix has been used extensively as a substrate for culturing human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSCs) with numerous hPSC culture media such as mTeSR™1, TeSR™2, E8, NutriStem®, and MEF conditioned media.

Corning has published a protocol describing the use of Corning® Matrigel® hESC-qualified matrix-coated 6-well plate with mTeSR™1 media for hESC culture. This protocol may be adapted to other media as well. Results may vary depending upon the cell line used, media, and dissociation technique, etc. You should optimize conditions for your own system.

Transitioning hPSCs from mouse embryonic fibroblasts (MEFs) to Matrigel matrix does not typically require any special process steps. Cells can be plated in medium of your choice on Matrigel hESC-qualified matrix-coated vessels at the time of passage. You should optimize your passaging conditions based on the cell line used, media, and dissociation technique. You can watch a video on how to passage ES cells from MEFs to Matrigel hESC-qualified matrix on the Journal of Visual Experiments (JoVE) web site.

For further support or troubleshooting advice, feel free to reach to the Corning Scientific Support team.

There are many methods that can be used to fluorescently label and image cells in a Matrigel matrix system. Here we cover three topics: labeling cells in Matrigel matrix assays, immunofluorescence analysis for surface markers (such as those used in 3D culture and culture of pluripotent stem cells), and fixing and embedding cells in Matrigel matrix prior to sectioning.

Staining has been done with all types of Matrigel matrix. However, the use of phenol red-free Matrigel matrix will reduce autofluorescence. We also recommend the use of Falcon® culture slides (Corning Cat No. 354180) for in situ analysis such as immunofluorescence studies. The slides are a specially cleaned and triple rinsed glass with an upper polystyrene chamber.

Labeling Cells in Matrigel Matrix Assays

Labeling is easiest if you culture cells on a thin coat of Matrigel matrix or you image cells from a Matrigel matrix assay such as invasion or tube formation. Most fluorescent methods can be used directly as described by manufacturers. In addition to pre-labeling intrinsic dyes such as green fluorescent protein (GFP), Calcien AM and DiI are frequently used, depending on how long the dye is required to remain stable.

Protocol for labeling cells using Corning Calcein AM dye:

Corning Calcein AM dye is generally used at 8 μg/mL in Hanks Balanced Salt Solution (HBSS). HBSS is recommended because the use of culture medium results in auto­hydrolysis of the label, which results in unacceptably high backgrounds. Remove medium from the plates being careful to avoid disruption of the matrix by gently aspirating the medium using a Pasteur pipet. Wash the plate with HBSS and repeat the wash a second time. Label the cells by adding 8 μg/mL Calcein AM in HBSS and incubate for 30 to 40 minutes at 37°C, 5% CO₂. Gently remove the labeling solution and wash twice with HBSS. The plate is now ready for image acquisition using an automated imager or for taking pictures using a fluorescent microscope. NOTE: Once hydrolysis occurs, Calcein AM leaks out of cells resulting in a higher background. Labeled plates can be stored at 4°C for 1 to 2 hours with minimum increase in background.

Links to Corning procedures are:

Assay Methods, Protocol: Cell Invasion Assay

Assay Methods, Protocol: Endothelial Cell Tube Formation Assay

Immunofluorescence Analysis with Matrigel Matrix

Immunofluorescence analysis preparation methods using Matrigel as a 3D matrix have been widely published and are represented by this method:

Nat Cell Biol. 2001 Sep; 3(9): 785–792. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Muthuswamy, et al.

Protocol for immunofluorescence analysis:

Using the above cited method, “structures were fixed either in 2% paraformaldehyde at room temperature for 15 min. or in methanol:acetone (50:50) at -20°C for 10 min. Fixed structures were washed three times in PBS:glycine (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 100 mM glycine) for 15 min. each. The washed structures were blocked first in IF buffer (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 7.7 mM NaN₃, 0.1% BSA, 0.2% Triton™ X-100, 0.05% TWEEN® 20) plus 10% goat serum (GS) for 1–2 hr. and subsequently with 2° blocking buffer (IF buffer containing 10% GS and 20 μg ml⁻¹ goat anti-mouse F(ab′)₂) for 30–45 min. Primary antibodies were diluted in 2° blocking buffer and incubated overnight at 4°C. Unbound primary antibodies were removed by washing three times in IF buffer for 15 min. each. Anti-mouse or anti-rabbit secondary antibodies coupled with Alexa Fluor® dyes (Molecular Probes) were diluted in IF buffer containing 10% GS and incubated for 45–60 min. Unbound secondary antibodies were washed as described above. Finally, the structures were incubated for 15 min. with PBS containing 5 μM TO-PRO®-3 (Molecular Probes) and 0.5 ng ml⁻¹ DAPI (Roche) before being mounted with the anti-fade agent ProLong® (Molecular Probes). Confocal analyses were performed.”

Corning has also published numerous application notes and posters that include data on immunocytochemical detection. Examples include:

• Maintenance of Human iPS Cells in a Feeder-free Culture System
• NutriStem® hPSC XF Medium Supports Long-term Culture of Human Pluripotent Stem Cells on Corning® Matrigel® hESC-qualified Matrix
• Assay Methods Protocol: Human Embryonic Stem Cell Culture

Protocol for immunohistochemical detection of cell surface markers on human iPS cells cultured on Corning Matrigel hESC-qualified matrix:

Remove culture media from the cultureware. Wash the hES cells twice with 2 mL of PBS. Fix the cells with 1 mL of 4% paraformaldehyde for 20 minutes at room temperature. Wash the cells twice with 2 mL of PBS for 5 minutes. Block the cells with 1 mL of 0.1% BSA, 10% normal goat serum* in PBS at room temperature for 45 minutes to 1 hour. NOTE: For Oct-3/4 staining, permeabilize in 0.1% Triton X-100, and block with 1% BSA, 10% normal rabbit serum in PBS at room temperature for 45 minutes. 7.2.5 During the blocking step, prepare the primary antibody working solution with PBS containing 1% BSA and 10% normal goat serum* to a final desired concentration. After blocking, incubate the cells with 1 mL/well of diluted primary antibody working solution overnight at 2°C to 8°C, or 1 hour at room temperature. Wash the cells three times with 2 mL of PBS containing 1% BSA for 5 minutes each. Dilute the secondary antibody (fluorescence-conjugated) in PBS containing 1% BSA. Incubate the cells with secondary antibody at 1 mL per well for 60 minutes at room temperature in the dark. NOTE: If using pre-conjugated antibody, secondary antibody will not be required. Wash the cells three times with 2 mL of PBS containing 1% BSA for 5 minutes each. Cover the cells with 4 mL of PBS and visualize using a fluorescence microscope.

Fixing and Embedding Cultures in Matrigel Matrix

Rijal and Li have recently published a method for histological studies:

Sci Adv. 2017 Sep; 3(9): e1700764. A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening, Girdhari Rijal and Weimin Li

Protocol for histology and immunostaining:

“The cell-laden scaffolds from the tissue cultures were washed twice with ice-cold 1X PBS and fixed in 10% neutral buffer formalin solution for 24–48 hours at 4°C. After rinsing with cold 1X PBS, the 3D cultures were embedded into OCT or paraffin following standard protocols and sectioned at a thickness of 10 μm using a cryostat or a microtome. For the sections produced using the paraffin fixation, a deparaffinization and rehydration process was performed, followed by antigen retrieval using the tris-EDTA buffer [10 mM tris base, 1 mM EDTA solution, and 0.05% TWEEN 20 (pH 9.0)]. The sections were washed several times with water, stained with H&E or IF antibodies (corresponding primary and Alexa fluorophore–conjugated secondary antibodies) as described previously (Circ. Res. 100, 79–87 (2007), and imaged using light or fluorescence microscopy for further analysis.”

Cell viability, immunofluorescence analysis and advanced imaging technologies are frequently used to interrogate Matrigel matrix enabled 3D cultures.

Viability can be measured via the detection of DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU). A protocol can be found below:

A chemical method for fast and sensitive detection of DNA synthesis in vivo

The labs of Mina Bissell at Lawrence Berkeley National Laboratory and Joan Brugge at Harvard Medical School have published extensively on 3D models using Matrigel matrix and have included a widely used immunofluorescence analysis preparation method. A few protocols can be found below:

ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini

Three-dimensional culture models of normal and malignant breast epithelial cells

Many labs have studied 3D architecture utilizing advanced imaging technologies. In the publication by Jorgens, et al. many of these methods were employed and are covered in the materials and methods section of the paper. Spheroid/organoid size and morphology, as well as cryogenic techniques, volume electron microscopy, and super-resolution light microscopy have been used to study phenotypic and functional attributes. A protocol can be found below:

Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture

Finally, recovery of cells from 3D Matrigel matrix cultures to be used for cell number determination, RNA isolation, and qPCR analysis can be accomplished using Corning cell recovery solution. Using the solution at low temperature (on ice) and applying mechanical disruption such as pipetting or the use of an orbital shaker will help de-polymerize the Matrigel matrix. Cell-cell interactions can be disrupted through the use of chelators and/or proteolytic enzymes such as Trypsin or Dispase.

Invasion assays for cancer cell analysis is an extensively studied and published application area where Matrigel matrix has been effectively used. To establish a reproducible assay many factors need optimization, some examples are:

• Cell seed
• Chemoattractant type and concentration
• Matrigel matrix protein concentration and volume (which relates to thickness)
• Duration of the assay
• Use (or not) of serum starvation prior to the assay
• Selection of the appropriate control

As a starting point we recommend coating an 8 micron 24-well insert (Corning Cat. No. 353097) with 0.1 mL of 200 to 300 μg/mL of Corning Matrigel matrix (Corning Cat. Nos. 354234 and 354230) per insert. Titration of the concentration and volume will help to tighten the conditions for your assay. There are many helpful documents on the Corning website, including:

• Corning Matrigel Matrix Frequently Asked Questions
• Considerations when Optimizing your Chemotaxis or Invasion Assay

Here are some examples from other literature:

• Current Opinion in Cell Biology, Vol 36, Oct 2015, 13-22. Modes of cancer cell invasion and the role of the microenvironment, G. Clark, D. Matic Vignjevic
• Cancer Res. 2016 Aug 15;76 (16):4595-7. Extracellular Matrix Invasion in Metastases and Angiogenesis: Commentary on the Matrigel "Chemoinvasion Assay", Albini A.

Corning also has some optimized prepackaged solutions available for tumor cell invasion assays on both clear and Corning FluoroBlok™ light-blocking permeable supports. Here are a few great resources to get you started using the Matrigel matrix invasion assay products:

• Protocol: Cell Invasion Assay
• Corning® BioCoat™ Tumor Cell Invasion Systems, Frequently Asked Questions

There are also posters and webinars on this topic available on www.corning.com/lifesciences that you may find valuable.

Corning® Matrigel® is poised to play an integral role in many 3D cell culture techniques, including bio-printing. Scientists have been using it to print many different tissues types. Listed below is a table that summarizes articles that have been published in this space recently that use Corning Matrigel in 3D bioprinting. Other 3D techniques that use Matrigel matrix are microfluidics and organ-on-a-chip for scaffold systems. For scaffold-free systems, Corning provides spheroid plates where the user can generate and analyze 3D spheres formed by one or more cell types. There is often a cross use of spheroid plates with Matrigel matrix if the customer is interested in generating self-assembled 3D structures.

Avoid disrupting the Matrigel matrix layer or coating by carefully aspirating and adding medium.  When aspirating the spent medium, tilt the vessel to pool the medium to one side minimizing contact with the Matrigel matrix layer. When adding medium, if possible, rest the tip along the side of the vessel and allow the liquid to slowly flow down and across the growth surface.

Cell culture medium should be changed as necessary to maintain the proper culture environment.

Below are a few resources that give guidance on general cell culture best practices:

• Introduction to Animal Cell Culture
• Corning Guide for Identifying and Correcting Common Cell Growth Problems

Corning Dispase or Corning cell recovery solution is recommended for recovering cells cultured on Corning Matrigel matrix. The Dispase enzyme will yield a single cell suspension more gently and effectively than trypsin, collagenase, or other proteolytic enzymes, as it minimizes cell damage and surface protein cleavage. Corning cell recovery solution is another option for cells/spheroids cultured in Matrigel matrix. This solution will allow non-enzymatic cell retrieval in small clumps and is frequently used in metabolic/RNA recovery experimentations. It can de-polymerize a thick Matrigel matrix layer at 4°C and facilitate cell retrieval. Cell-cell interactions can also be disrupted through the use of chelators and/or proteolytic enzymes such as Trypsin or Dispase. Using the solution at low temperature (on ice) and applying mechanical disruption such as pipetting or the use of an orbital shaker are other alternative methods to de-polymerize the Matrigel matrix.

Combining microfluidics and extracellular matrices (ECM) has shown to be a promising system to create more in vivo-like 3D environments. Some publications have shown different methods to craft such environments. For example:

• Bruzewicz, et al. (Lab Chip. 2008 May;8(5):663-71. doi: 10.1039/b719806j. Epub 2008 Mar 20) have shown that using a soft-lithographic molding gel such as Matrigel matrix or Collagen to encapsulate cells in a microfluidic channel and chambers yielded a permeable system where media could flow to feed the encapsulated cells.
• Jang, et al. (ACS Appl Mater Interfaces. 2015 Feb 4;7(4):2183-8. doi: 10.1021/am508292t. Epub 2015 Jan 21) have applied flow across the bulk gel during the gelation process to orient the ECM components with the direction of the flow, compared with randomly cross-linked Matrigel matrix.
• Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment is a recently published review article by Tsai, et al. (J R Soc Interface. 2017 Jun; 14(131): 20170137).

Understanding the biophysical cues of the 3D environment such as topography, stiffness, viscosity and porosity have shown to be important to mimic the in vivo environment. Modulating and tuning the tensile strength of the Matrigel matrix gel in a 3D environment may be beneficial to provide softer or stiffer gels to suit application need. Empirical studies may show that a stiffer gel (higher protein concentration), may reduce dilution of the gel caused by the flow in the microfluidic chip.

As we keep learning about these techniques and methods, we recommend you reach out to our global scientific support team to help you find the right solution for your work.

The choice of substrate for mesenchymal stem cells (MSC) in planar cultures is best determined in the context of the entire in vitro environment, including the media. For serum containing media, substrates such as tissue culture treated (TC) or Corning® CellBIND® surfaces are recommended.  If you are using xeno or animal free media then we recommend Corning human fibronectin, Corning PureCoat™ fibronectin mimetic or Synthemax® surfaces.

In a 3D culture environment, MSCs have been cultured, co-cultured and differentiated on Matrigel matrix. Li. et. al., have demonstrated that 3D co-culture of BM-MSCs and eccrine sweat gland cells in Matrigel matrix promotes trans-differentiation of BM-MSCs (J Mol. Histol. 2015. 46:431-8). Matrigel matrix can also be used in vitro as a thick gel where cells can be embedded or seeded on top of the matrix layer (overlay method). Furthermore, the elastic moduli of Matrigel matrix can also be tuned by providing softer or stiffer gels to suit application need (See Corning Application Note CLS-AC-AN-449).