We would like to thank the funding sources for this work: the National Institutes of Health National Cancer Institute (R37 CA222563 to J.M.), the Coulter Foundation (J.M.), and Virginia Tech ICTAS-CEH (J.M. &#38; J.H.). The GSCs used in this assay were derived by Jakub Godlewski, Ph.D. (Harvard Medical School).

This protocol was developed in compliance with the guidelines set forth by the Virginia Tech Institutional Biosafety Committee.
NOTE: Perform all procedures in a BSL-2 cell culture cabinet unless otherwise specified. Please consult the institution&#39;s biosafety committee for guidance on the use of human cells.
1. Calculations for gel preparation
<ol>
	<li>Calculate the number of conditions needed for the experiment. 
	NOTE: A &#34;condition...

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

<ol>
	<li>Ostrom, Q. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CBTRUS+statistical+report:+Primary+brain+and+other+central+nervous+system+tumors+diagnosed+in+the+United+States+in+2016-2020.">CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2016-2020.</a> Neuro Oncol. 25, 1-99 (2023).</li><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N Engl J Med. 352 (10), 987-996 (2005).</li><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+tumor-treating+fields+plus+maintenance+temozolomide+vs+maintenance+temozolomide+alone+on+survival+in+patients+with+glioblastoma:+A+randomized+clinical+trial.">Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: A randomized clinical trial.</a> JAMA. 318 (23), 2306-2316 (2017).</li><li>Lathia, J. 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The authors have no relevant conflicts of interest to disclose.

Assembling the TME model includes six fundamental steps: 1) passaging cells and separating the cells between like conditions, 2) assembling a concentrated collagen solution for all conditions, 3) combining the gel components (cells, collagen, methacrylated hyaluronic acid, and the photoinitiator) for each condition 4) plating gels, 5) crosslinking by UV exposure and heat, and 6) adding the fluid pressure head. After 18 h or more, the gels can be harvested for flow cytometry, RNA extraction, protein extraction, imaging, o...

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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 Well Tissue Culture Plate, Sterile</td>
			<td>Celltreat Scientific Products&#160;</td>
			<td>229112</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Filter&#160; System, PES Filter Material, 0.22 &#181;m, 50 mm, Sterile</td>
			<td>DOT Scientific</td>
			<td>667706</td>
			<td></td>
		</tr>
		<tr>
			<td>385 nm, 1650 mW (Min) Mounted LED, 1700 mA&#160;</td>
			<td>Thorlabs&#160;</td>
			<td>M385LP1-C1</td>
			<td></td>
		</tr>
		<tr>
			<td>75cm2 Tissue Culture Flask - Vent Cap, Sterile</td>
			<td>Celltreat Scientific Products</td>
			<td>229341</td>
			<td></td>
		</tr>
		<tr>
			<td>8.0 &#956;m Cell Culture Plate Insert 12 mm Diameter</td>
			<td>Millicell</td>
			<td>PI8P01250</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol, 200 proof, Molecular Biology Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>T038181000CS</td>
			<td>Ethanol for flow cytometry dead cell control.</td>
		</tr>
		<tr>
			<td>Astrocyte Medium (Astrofull)</td>
			<td>ScienCell Research Laboratories</td>
			<td>1801</td>
			<td>Contains astrobasal, FBS, and penicillin/streptomycin.&#160;</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x), minus vitamin A (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (MACS)</td>
			<td>Miltenyi Biotec&#160;</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>CD71 antibody (eBioscience, Invitrogen)</td>
			<td>Fisher Scientific&#160;</td>
			<td>25-0719-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Chambered Slides</td>
			<td>Nexcelom Bioscience</td>
			<td>CHT4-PD100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scrapers</td>
			<td>Biologix USA&#160;</td>
			<td>70-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2 Fluorescent Cell Counter (Nexelcom Bioscience)</td>
			<td>VWR&#160;</td>
			<td>NEXCCMK2-SK150-FCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge - Low-Speed</td>
			<td>Eppendorf&#160;</td>
			<td>5702 R</td>
			<td>Centrifuge for cell culture.</td>
		</tr>
		<tr>
			<td>Clear Polystyrene 96-Well Microplates, Corning</td>
			<td>Fisher Scientific</td>
			<td>07-200-108</td>
			<td>V-bottom plates for flow cytometry staining.</td>
		</tr>
		<tr>
			<td>CO2&#160;Incubator, 150L, Heracell 150i (Thermo Scientific)</td>
			<td>Thermo Fisher Scientific</td>
			<td>50116047</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, High Concentration, Rat Tail</td>
			<td>Corning</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, Rat Tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>&#34;Low&#34; concentration for coating adherent flasks.</td>
		</tr>
		<tr>
			<td>Collagenase (CAS# 9001-12-1)</td>
			<td>United States Biological</td>
			<td>C7511-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimation Adapter for Olympus BX &#38; IX, AR Coating: 350 - 700 nm</td>
			<td>Thorlabs</td>
			<td>COP1-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Swabs, Q-tips Precision Tips&#160;</td>
			<td>Amazon</td>
			<td>B01KCJB3R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase (CAS# 9001-92-7)&#160;</td>
			<td>United States Biological</td>
			<td>D3760</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL</td>
			<td>Invitrogen</td>
			<td>AMF4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td>For microglia culture.</td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10% (Sigma-Aldrich)&#160;</td>
			<td>Millipore Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, Invitrogen)</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-5523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glioma stem cells</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Can be patient derived or commercial glioma stem cell lines.&#160;</td>
		</tr>
		<tr>
			<td>Guava easyCyte HT System</td>
			<td>Millipore Sigma</td>
			<td>0500-4008</td>
			<td>Flow cytometer.</td>
		</tr>
		<tr>
			<td>HBSS (Sigma-Aldrich)&#160;</td>
			<td>Millipore Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M) (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Power 1-Channel LED Driver with Pulse Modulation, 10.0 A Max, 50.0 V Max&#160;</td>
			<td>Thorlabs</td>
			<td>DC2200</td>
			<td>Interface for UV Lamp.</td>
		</tr>
		<tr>
			<td>Hoechst 33342 Solution (20 mM) (Thermo Scientific)</td>
			<td>Thermo Fisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Astrocytes</td>
			<td>ScienCell Research Laboratories</td>
			<td>1800</td>
			<td>Primary astrocytes derived from the cerebral cortex.</td>
		</tr>
		<tr>
			<td>Human EGF Recombinant Protein (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Immortalized Human Microglia - hTERT</td>
			<td>Applied Biological Materials&#160;</td>
			<td>T0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Incu-mixer MP Heated Microplate Vortexer, 2 position</td>
			<td>Benchmark Scientific&#160;</td>
			<td>H6002</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67 REAfinity, PerCP-Vio 700 antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-120-420</td>
			<td></td>
		</tr>
		<tr>
			<td>LED UV Curing Meter</td>
			<td>Gigahertz-Optik</td>
			<td>X1-RCH-116</td>
			<td>Optometer to measure UV light intensity.</td>
		</tr>
		<tr>
			<td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)&#160;</td>
			<td>Advanced Biomatrix</td>
			<td>5269-100MG&#160;</td>
			<td>Photoinitiator.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR (Invitrogen)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>L24975</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylated hyaluronic acid (photoHA)</td>
			<td>Advanced Biomatrix</td>
			<td>5212-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge, Sorvall ST8R (Thermo Scientific)</td>
			<td>Fisher Scientific</td>
			<td>75-997-203</td>
			<td>Centrifuge for flow cytometry staining.</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X) (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4 without Ca &#38; Mg</td>
			<td>Quality Biological</td>
			<td>119-069-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, pellets ACS (CAS# 1310-73-2)</td>
			<td>VWR&#160;</td>
			<td>97064-476</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy Ultrapure Water Purification System (MilliporeSigma)</td>
			<td>Fisher Scientific&#160;</td>
			<td>SYNS0HFUS</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaStain AOPI Staining Solution</td>
			<td>Nexcelom Bioscience</td>
			<td>CS2-0106</td>
			<td></td>
		</tr>
	</tbody>
</table>

a photopolymerizable hyaluronic acid-collagen model of the invasive glioma microenvironment with interstitial flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

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.

Representative data for invasion (Figure 3), viability, and the expression of Ki67 and CD71 via flow cytometry (Figure 4) are provided for GSC lines as previously published for a thio-modified hyaluronan-collagen hydrogel12. The presence of astrocytes and microglia within the TME model has a differential effect on GSC invasion dependent on the cell line (Figure 4). Specifically, the GSCs G44 and G62<sup class...

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

a-photopolymerizable-hyaluronic-acid-collagen-model-invasive-glioma

Without Section

Research

JoVE Journal

Bioengineering

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.

278 Views.  Fralin Biomedical Research Institute at Virginia Tech Carilion.  We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed. 

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

Summary