preparation of hyaluronic acid-collagen hydrogel in a tissue culture insert for replicating the glioma tumor microenvironment

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

Glioblastoma is a devastating disease, marked by short survival1 that is extended only moderately by clinically-available treatments2,3. This impediment to effective therapy is largely driven by the highly infiltrative nature of chemoresistant glioma stem cells (GSCs) that are inaccessible to resection and seed recurrent tumors4. Concurrently, GSCs are highly plastic, responding to diverse stimuli in the tumor microenvironment (TME) in order to survive and invade5,6. Specifically, the densely populated tumor and leaky vasculature produce a steep pressure differential at the tumor border, increasing interstitial fluid flow (IFF) in distinct regions that correlate with GSC invasion7. This increased IFF influences8,9,10 and often enhances8,9 GSC invasion via molecular pathways that are amenable to pharmacological inhibition. However, this process is confounded by the influence of glia in the TME on glioma invasion; indeed, glia have been described to modulate glioma invasion in numerous contexts11, and preliminary evidence in our lab indicates a similar influence of glia on IFF-enhanced glioma invasion12. Thus, our lab has developed a tunable, 3D TME model that replicates these invasive tumor border regions by incorporating both IFF and glia-GSC interactions to quantify GSC invasion and other outcomes (i.e., surface or intracellular protein expression, RNA expression, etc.) as influenced by IFF, glia, and/or candidate therapeutics12(Figure 1).
The TME model is similar to other tissue culture insert-based interstitial fluid flow assays (i.e., static/flow assays) that have been widely published8,9,10,13,14,15,16,17,18. The main distinctions of this model are the incorporation of a nonproprietary and tunable collagen-hyaluronic acid matrix, inclusion of astrocytes and microglia into the matrix at ratios representative of patient TMEs12, and the system is entirely enclosed in a well-plate, lacking external tubing, reservoirs, or pumps. Hyaluronic acid is among the principal components of the brain extracellular matrix19, but it is not sufficient to permit fluid flow; thus, collagen I is included in the mixture. Crosslinking occurs in two steps: a free-radical-initiated chain growth polymerization whereby upon exposure to ultraviolet (UV) light, free radicals are produced from the photoinitiator and generate chemical crosslinks between methacrylated hyaluronic acid molecules20, and neutralization of acid-preserved collagen and exposure to heat to form collagen fibers21,22,23.
Various parameters that were optimized for this assay have been described in previously published work in thiol-modified hyaluronan-collagen hydrogels8,12. Specifically, the cells (primary human GSCs24, primary human cortical astrocytes, and&#160;immortalized human microglia) used for this assay are at least 60% viable for 3 days in gels composed of astrobasal media supplemented with GSC growth factors, 0.5% v/v N-2 and 1% v/v B-27 without vitamin A12. In cases where the gels must be degraded (e.g., for protein extraction or flow cytometry), collagenase and dispase are used and maintain sufficient viability (Figure 2).
To quantify invasion, GSCs must be fluorescently labeled before they are combined with glia in the matrix. This may be achieved by labeling with Hoechst 33342 (as described below), cell trackers12, genetic modification, or transduction. Regardless, the viability of cells labeled with new markers should be measured before incorporating them into the TME model. Alternatively, if invasion will not be quantified but the gels will be harvested for protein extractions, RNA extractions, or other outcomes, cells often do not need to be fluorescently labeled during the gel preparation process. In that case, gels can be either formalin-fixed for imaging, degraded and cells lysed for RNA extraction, or degraded using proteases for flow cytometry and subsequently lysed with protein lysis buffers for Western blots or other protein work.
Here, we present a protocol for incorporating human astrocytes, microglia, and patient-derived GSCs at a patient-defined ratio, 4:1:112, into a 1.2 mg/mL collagen I, 4 mg/mL hyaluronan matrix under static of flow conditions. The specific parameters used for an assay (i.e., cellular ratios, stiffness, cell types, etc.) can be altered to represent different contexts if viability is adequate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66604/66604fig01.jpg" /> 
Figure 1: Diagram of the TME model consisting of GSCs, astrocytes, and microglia in a hydrogel under a fluid pressure head. Cells are embedded in a collagen-hyaluronic acid hydrogel inside a tissue culture insert. Gravitational force drives the net downward flow. Pores in the tissue culture insert membrane allow cells invading downward to attach&#160;to the bottom surface of the tissue culture insert, and these cells can be fixed, imaged, and quantified. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/66604/66604fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66604/66604fig02.jpg" /> 
Figure 2: Cellular viability following hydrogel degradation. G34s (GSCs), astrocytes, and microglia were seeded in the TME model at 4:1:1 and incubated for 21 h at 37 &#176;C. Gels were removed and degraded using 0.3 mg/mL collagenase + 0.02 mg/mL dispase for a total of either 30, 45, or 60 min, pipetting to mix every 15 min (30 min condition) or at 30 min then every 15 min (45 min and 60 min conditions). After the gels were degraded, the cells were stained with acridine orange (all cells) and propidium iodide (dead cells) and counted using an automated cell counter. Viability is reported for all cells within the TME model. Cells maintained at least 70% viability over 30-60 min. Data is presented as mean &#177; SEM; n = 3 biological replicates. <a href="https://www.jove.com/files/ftp_upload/66604/66604fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Our work investigates how the tumor microenvironment drives invasion of tumor cells in glioblastoma, the deadliest form of brain cancer. Specifically, we're interested in how interstitial fluid flow driven by increased intratumoral pressures causes tumor cells to invade into the surrounding brain parenchyma. We currently use these 3D hyaluronic acid collagen system mass models combined with MII imaging and a computational analysis to study glioma invasion driven by interstitial fluid flow.For the individual approaches, fluid flow is generated by applying pressure on the top of the TME model, mimicking interstitial fluid flow in the brain. Providing the right physical cues to replicate the tissue microenvironment is a continual challenge. Our three-dimensional model uses hyaluronic acid and collagen, both found in the brain matrix, and sustained interstitial fluid flow.Together, these factors provide key physical signals to healthy and cancerous cells. Our recent work indicates that resident brain cells influence how glioma cells respond to interstitial flow. However, this response depends on patient-specific factors.To study the influence of these factors on glioma invasion, we developed a patient-informed model of the glioma invasive front that mimics native brain tissue. The patient tumor model allows for a highly controlled in vitro experiment that still represents tissue-level factors that influence glioma invasion. Further, this model is accessible compared to similar models of glioma invasion and interstitial flow because it does not require tubing or pumps, and it uses commercially available materials.

Preparation of Hyaluronic Acid-Collagen Hydrogel in a Tissue Culture Insert for Replicating the Glioma Tumor Microenvironment

To begin, place collagen, sodium hydroxide, sterile ultrapure water, 10 times PBS, one micro centrifuge tube, methacrylated hyaluronic acid, and the photo initiator on ice. Then place the tissue culture inserts in the plate and label it. To prepare three milligrams per milliliter collagen solution, combine high concentration collagen, one normal sodium hydroxide, ultrapure water, and 10 times PBS.Add components to the micro centrifuge tube and keep the pipette tip submerged when mixing to prevent bubble formation. After that, centrifuge cells at 200 G for five minutes at room temperature. Then gently remove excess media from the top of each cell condition solution, leaving the volume of media required for the condition.Place these solutions on ice. Add the calculated volume of collagen solution to each cell condition solution. Next, add the calculated volume of 1%methacrylated hyaluronic acid, followed by 17 milligrams per milliliter LAP in each cell condition solution.Mix each condition tube one at a time while keeping the pipette tip submerged to avoid bubble formation and add 100 to 150 microliters to each tissue culture insert. Now turn on the 385 nanometer ultraviolet lamp at a constant current. To photo cross link each gel, expose each well to ultraviolet light for 45 seconds, one at a time.Then place the plate at 37 degrees Celsius for 30 to 45 minutes to cross link the collagen. Using an astral basal medium with 0.5%and two volume by volume and 1%volume by volume B27 without vitamin A, apply the fluid pressure head to gels. Add media to the well plate and tissue culture inserts for net zero flow.Place the plate at 37 degrees Celsius, 5%carbon dioxide for at least 18 hours or up to five days.

preparation-hyaluronic-acid-collagen-hydrogel-tissue-culture-insert

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JoVE Journal

Bioengineering

61 Views. To begin, place collagen, sodium hydroxide, sterile ultrapure water, 10 times PBS, one micro centrifuge tube, methacrylated hyaluronic acid, and the photo initiator on ice. Then place the tissue culture inserts in the plate and label it. To prepare three milligrams per milliliter collagen solution, combine high concentration collagen, one normal sodium hydroxide, ultrapure water, and 10 times PBS.Add components to the micro centrifuge tube and keep the pipette tip submerged when mixing...