Name: hydrogel arrays enable increased throughput for screening effects of matrix components and therapeutics in 3d tumor models
Uploaded: 2022-06-16T13:10:00
Description: Watch this Scientific Journal Video about Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models at JoVE.com

The authors would like to specifically acknowledge Carolyn Kim, Amelia Lao, Ryan Stoutamore, and Itay Solomon for their contributions to earlier iterations of the photogelation scheme. Cell lines GS122 and GS304 were generously provided by David Nathanson. All figures were created with BioRender.com. UCLA core facilities, the Molecular Screening Shared Resources, and the Nano and Pico Characterization Laboratory were instrumental to the work. Chen Chia-Chun was supported by the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Training Program. Grigor Varuzhanyan was supported by a Tumor Cell Biology Training Program NIH Grant (T32 CA 009056).

Patient-derived GBM cell lines (GS122 and GS304) were provided by Professor David Nathanson (our collaborator), who developed these lines under a protocol approved by the UCLA Institutional Review Board (IRB# 10-000655). Cells were provided de-identified so that the cell lines could not be linked back to the individual patients.
1. Preparation of hydrogel solution
<ol>
	<li>Prepare HEPES-buffered solution by dissolving HEPES powder at 20 mM in Hank&#39;s balanced salt soluti...

<ol>
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The current work presents methods to generate 3D, miniaturized cultures within HA-based while simultaneously altering matrix stiffness and peptides available for integrin engagement. This technique enables the systematic study of how matrix parameters affect cellular phenotypes (e.g., the viability of cancer cells exposed to chemotherapy) with increased throughput. Previous approaches, including that presented herein, have tuned hydrogel stiffness by varying the percent total polymer in the final formulation, where stiff...

The expected cost of developing a new drug has steadily risen over the past decade, with over $1 billion in current estimates1. Part of this expense is the high failure rate of drugs entering clinical trials. Approximately 12% of drug candidates ultimately earn approval from the United States (US) Food &#38; Drug Administration (FDA) in 2019. Many drugs fail in Phase I due to unanticipated toxicity2, while others that pass safety trials may fail due to a lack of efficacy3. This attrition due to non-efficacy can partly be explained by the fact that cancer models used during drug development are notoriously non-predictive of clinical efficacy4.
Functional disparities between in vitro and in vivo models may be attributed to removing cancer cells from their native microenvironment, including non-tumor cells and the physical ECM5,6. Commonly, research groups use commercially available culture matrices, such as Matrigel (a proteinaceous basement membrane matrix derived from mouse sarcomas) to provide cultured tumor cells with a 3D matrix microenvironment. Compared to 2D culture, 3D culture in membrane matrix has improved the clinical relevance of in vitro results7,8. However, culture biomaterials from decellularized tissues, including the membrane matrix, typically exhibit batch-to-batch variability that may compromise reproducibility9. Furthermore, matrices derived from tumors with different tissue origins from those studied may not provide the appropriate physiological cues10. Finally, cancers with high degrees of intratumoral heterogeneity have microenvironmental features that vary on a submicron-size scale and which the membrane matrix cannot be tuned to recapitulate11.
Glioblastoma (GBM), a uniformly lethal brain tumor with a median survival time of approximately 15 months, is a cancer for which treatment development has been particularly difficult12,13. The current standard of care for GBM consists of primary tumor resection, followed by radiotherapy, and then chemotherapy using temozolomide (TMZ)14. Yet, more than half of clinical GBM tumors exhibit treatment resistance through various mechanisms15,16,17. Predicting the efficacy of a treatment regimen for an individual patient is extremely difficult. Standard preclinical models used to predict individual outcomes consist of patient-derived tumor cells xenografted orthotopically into immunocompromised mice. While patient-derived xenografts can recapitulate many aspects of clinical GBM tumors and are valuable for preclinical models18, they are inherently expensive, low throughput, time-consuming, and involve ethical concerns19. Cultures of patient-derived cells, on 2D plastic surfaces or as spheroids, mostly avoid these issues. While patient-derived cells preserve&#160;genetic aberrations, their cultures in 2D or as suspended spheroids have been largely poor representations of patient-derived xenografts in rodents and original patient tumors20. Previously, we, and others, have shown that GBM cells cultured in a 3D ECM that mimics the mechanical and biochemical properties of brain tissue can preserve drug resistance phenotypes10,21,22,23.
Interactions between hyaluronic acid (HA), a polysaccharide abundant in the brain ECM and overexpressed in GBM tumors, and its CD44 receptor modulate the acquisition of drug resistance in vitro21,24,25,26,27.&#160;For example, the inclusion of HA within soft, 3D cultures increased the ability of patient-derived GBM cells to acquire therapeutic resistance. This mechano-responsivity was dependent on HA binding to CD44 receptors on GBM cells21. Additionally, integrin binding to RGD-bearing peptides, incorporated into 3D culture matrices, amplified CD44-mediated chemoresistance in a stiffness-dependent manner21. Beyond HA, the expression of several ECM proteins, many containing RGD regions, vary between normal brain and GBM tumors28. For example, one study reported that 28 distinct ECM proteins were upregulated in GBM tumors29. Within this complex tumor matrix microenvironment, cancer cells integrate mechanical and biochemical cues to yield a particular resistance phenotype, which depends on relatively small differences (e.g., less than an order of magnitude) in Young&#39;s modulus or density of integrin-binding peptides28,29,30.
The present protocol characterizes how tumor cells interpret unique combinations of matrix cues and identify complex, patient-specific matrix microenvironments that promote treatment resistance (Figure 1A). A photochemical method for generating miniaturized, precisely tuned matrices for 3D culture provides a large, orthogonal variable space. A custom-built array of LEDs, run by a microcontroller, was incorporated to photocrosslink hydrogels within a 384-well plate format to increase automation and reproducibility. Exposure intensity was varied across well to alter micro-mechanical properties of resulting hydrogels, as assessed using atomic force microscopy (AFM). While this manuscript does not focus on constructing the illumination array itself, a circuit diagram (Figure 1B) and parts list (Table of Materials) are provided as aids for device reproduction.
This report demonstrates the rapid generation of an array of GBM cells cultured in unique, 3D microenvironments in which Young&#39;s modulus (four levels across a single order of magnitude) and integrin-binding peptide content (derived from four different ECM proteins) were varied orthogonally. The approach was then used to investigate the relative contributions of hydrogel mechanics and ECM-specific integrin engagement on the viability and proliferation of patient-derived GBM cells as they acquire resistance to temozolomide (TMZ) chemotherapy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.1 kOhm resistors, 6 W</td>
			<td>Digikey</td>
			<td>35601k1ft</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge tube</td>
			<td>Genesse Scientific</td>
			<td>21-108</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>365 nm LED</td>
			<td>Digikey</td>
			<td>ltpl-c034uvh365</td>
			<td></td>
		</tr>
		<tr>
			<td>384 well plate</td>
			<td>Bio Greiner One</td>
			<td>781090</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>MTC bio</td>
			<td>C4040</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Armed thiol terminated polyethlene glycol (20 kDa)</td>
			<td>Laysan Bio</td>
			<td>4arm-PEG-SH-20K-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>6 NPN BJTs</td>
			<td>Digikey</td>
			<td>2n5550ta</td>
			<td></td>
		</tr>
		<tr>
			<td>80 Ohm resistors, 0.125 W</td>
			<td>Digikey</td>
			<td>erjj-6enf80r6v</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Armed norbornene terminated polyethylene glycol (20 kDa)</td>
			<td>Jenkem Technology</td>
			<td>A7025-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104500</td>
			<td>&#160;cell dissociation&#160; reagent</td>
		</tr>
		<tr>
			<td>AFM Probes</td>
			<td>Novascan</td>
			<td></td>
			<td>0.01 N/m Nominal spring constant, 2.5 &#181;m SiO2 particle</td>
		</tr>
		<tr>
			<td>Arduino IDE</td>
			<td>Arduino</td>
			<td>1.8.19</td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Nano</td>
			<td>Makerfire</td>
			<td>Mini Nano V3.0 ATmega328P Microcontroller Board</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>CCK8</td>
			<td>Abcam</td>
			<td>ab228554</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermoscientific</td>
			<td>sorvall legend xtr</td>
			<td></td>
		</tr>
		<tr>
			<td>CP100ST</td>
			<td>Gilson</td>
			<td>F148415</td>
			<td>Pipette tips for positive displacement pipette</td>
		</tr>
		<tr>
			<td>Cubis Semi-Micro Balance</td>
			<td>Sartorius</td>
			<td>MSA225S100DI</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - F12 (50-50)</td>
			<td>Life Technologies</td>
			<td>11330057</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS Ca (-) Mg (-)</td>
			<td>Genesse Scientific</td>
			<td>25-508</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF100-15</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>Ethanol, Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>A405P</td>
			<td>Add DI water to dilute to 70%</td>
		</tr>
		<tr>
			<td>Fisherbrand Class B Amber Glass threaded vials</td>
			<td>Fisher Scientific</td>
			<td>03-339-23C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Weighing Paper</td>
			<td>Fisher Scientific</td>
			<td>09-898-12B</td>
			<td></td>
		</tr>
		<tr>
			<td>G21 Supplement</td>
			<td>Gemini Bio</td>
			<td>400-160</td>
			<td>50x</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl, ACS, 12M</td>
			<td>Sigma Aldrich</td>
			<td>S25838A</td>
			<td>Add DI water to dilute to 1 M</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma Aldrich</td>
			<td>H3149-100Ku</td>
			<td>25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H7006-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot Air Gun</td>
			<td>Wagner</td>
			<td>HT1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin-binding sialoprotein (IBSP) peptide</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td>GCGYGGGGNGEPRGDTYRAY</td>
		</tr>
		<tr>
			<td>Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) , &#62;95%</td>
			<td>Sigma Aldrich</td>
			<td>900889-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir plate</td>
			<td>Thermo Scientific</td>
			<td>SP194715</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>Sorvall legend micro 21R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microman E single Channel Pipettor</td>
			<td>Gilson</td>
			<td>FD10004</td>
			<td>Positive displacement pipette</td>
		</tr>
		<tr>
			<td>Micropipette Tips</td>
			<td>Various Manufacturs</td>
			<td></td>
			<td>Various sizes</td>
		</tr>
		<tr>
			<td>mLine micropipette</td>
			<td>Sartorious</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl Cysteine</td>
			<td>Sigma Aldrich</td>
			<td>A7250-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanowizard 4</td>
			<td>Bruker</td>
			<td></td>
			<td>AFM microscope</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>ss255-1</td>
			<td>Add DI water to dilute to 1 M</td>
		</tr>
		<tr>
			<td>Normoicin</td>
			<td>Invivogen</td>
			<td>ant-nr-1</td>
			<td>500x</td>
		</tr>
		<tr>
			<td>Osteopontin Peptide</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td>GCGYGTVDVPDGRGDSLAYG</td>
		</tr>
		<tr>
			<td>Pipet Aid</td>
			<td>Drummond</td>
			<td>4000102</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Microscope Slides</td>
			<td>Globe Scientific</td>
			<td>1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Press-To-Seal silicone Isolator, 12-4.5mm diam x 2mm deep</td>
			<td>Grace Bio Labs</td>
			<td>664201-A</td>
			<td>Cut so that 8 individual molds are made from a single sheet</td>
		</tr>
		<tr>
			<td>Processing</td>
			<td>Processing</td>
			<td>3.5.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater M4</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater Pipette Tips</td>
			<td>Sartorious</td>
			<td>30089430</td>
			<td>1 mL sizes</td>
		</tr>
		<tr>
			<td>RGD Peptide</td>
			<td>Genscript</td>
			<td></td>
			<td>GCGYGRGDSPG</td>
		</tr>
		<tr>
			<td>Scoth Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Genesse Scientific</td>
			<td>12-102,12-104</td>
			<td>5,10 mL Pipettes</td>
		</tr>
		<tr>
			<td>Solder Paste</td>
			<td>Digikey</td>
			<td>315-NC191LT15T5-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Solder Wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straight dissecting forceps</td>
			<td>VWR Scientific</td>
			<td>82027-408</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy H1 Plate Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Cell Culture Treated Flask</td>
			<td>Genesee Scientific</td>
			<td>25-209</td>
			<td></td>
		</tr>
		<tr>
			<td>Temozolomide</td>
			<td>Sigma Aldrich</td>
			<td>T2577</td>
			<td>Typically used from 10 &#181;M to 100 &#181;M</td>
		</tr>
		<tr>
			<td>Tenascin-C Peptide</td>
			<td>Genscript</td>
			<td></td>
			<td>GCGYGRSTDLPGLKAATHYTITIR 
			GV</td>
		</tr>
		<tr>
			<td>Thiolated Hyaluronic Acid (700 kDa), 6-8% modified</td>
			<td>Lifecore Biomedical</td>
			<td>HA700K5</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Spinbar, Flea Micro</td>
			<td>VWR</td>
			<td>58948-375</td>
			<td></td>
		</tr>
	</tbody>
</table>

hydrogel arrays enable increased throughput for screening effects of matrix components and therapeutics in 3d tumor models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

AFM measurements confirmed precise control of hydrogel mechanics as a function of UV irradiance (mW/cm2) during photo-crosslinking using a custom-built, Arduino-controlled LED array (Figure 2A). The hydrogel formulation used in this protocol can be found in Table 2. The spacing of the LEDs on the provided template matches the spacing for every other well of a 384-well plate, allowing for the formation of gels inside the plate (Figure 2B</stron...

Watch this Scientific Journal Video about Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models at JoVE.com

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

This method enables researchers to systematically probe how matrix cues affect the phenotype of cells in 3D culture. We demonstrate the procedure using cultures of GBM cells. The main advantage of this technique is high-throughput implementation, which allows us to screen many matrix conditions.This technique can be used to develop tissue-mimetic matrices that preserve specific functions in cell culture models, like drug resistance of tumor cells, and potentially identify new therapeutics. Demonstrating the procedure will be Mary Epperson and Kelly Tamura, undergraduate researchers from the Seidlits Lab. Start with preparing HEPES-buffered solution by dissolving HEPES powder at 20-millimolar in Hanks balanced salt solution.Adjust pH to seven following full sorbation. In the HEPES-buffered solution, dissolve thiolated HA so that 6 to 8%of carboxylic acid residues on each glucuronic acid are modified with a thiol at a concentration of 10 milligrams per milliliter in buffer solution. Stir at less than 1, 000 rotations per minute using a magnetic stir plate at room temperature until fully dissolved, typically around 45 minutes.While HA is dissolving, prepare separate solutions of 100 milligrams per milliliter of 8arm PEG norbornene, 100 milligrams per milliliter of 4arm PEG thiol, four-millimolar of cysteine or cysteine-containing peptide, and four milligrams per milliliter of LAP in microcentrifuge tubes. Prepare four-millimolar solutions of all peptides to be tethered within a single hydrogel at this point. Mix the individual solutions of HA, PEG norbornene, PEG thiol, and cysteine-thiol-containing peptides to achieve the final concentrations for the final hydrogel matrices.Stir at less than 1, 000 rotations per minute on a magnetic stair plate for at least 30 minutes to mix fully. Align the samples with the illumination device with every other LED in a single column of the silicone molds or 384-well plate. Open UV LED Parameters and enter values for intensity and illumination.Then click on Finish to begin illumination. Following illumination, when placed in one corner, move the well plate to the next corner and repeat. To illuminate wells on the other half of the plate, lift the plate out of the holder and rotate 180 degrees.To generate hydrogels with varying mechanics, start with cleaning the glass slides and silicone molds using tape to remove debris. Adhere the silicone molds to the glass slide, press down to ensure a good seal, and displace any air bubbles. Next, pipette 80 microliters of hydrogel precursor solution into each silicone mold on the glass slide.Place the glass slide onto the illumination device aligned with every other LED in a single column. Expose the hydrogel precursors to UV light for 15 seconds to photo cross-link. Once illumination has stopped, retrieve the slides and loosen the gels from the molds by tracing the inner circumference of the mold with a fine tip.Remove silicone molds with tweezers or forceps. Fill a 12-well plate with two milliliters of DPBS. Move cross-linked hydrogels into individual well plate by wetting a spatula and gently pushing them off the glass slide.Prepare desired cells as a single-cell solution. Collect glioblastoma spheroids, roughly 150 micrometers in diameter, from a T-75 flask suspension culture into a 15-milliliter conical tube. Rinse the culture flask with five milliliters of DPBS to remove any residual cells and media and add this volume to conical tube.Centrifuge the conical tube containing cells at 200 times G for five minutes at room temperature. Following centrifugation, remove the supernatant with a five milliliter serological pipette, taking care not to disturb the cell pellet and resuspend in five milliliters of DPBS. Centrifuge at 200 times G for five minutes at room temperature to wash cells.Aspirate the supernatant with a five-milliliter serological pipette, taking care not to disturb the cell pellet, and then resuspend cells in two milliliters of cell dissociation reagent. Incubate at room temperature for 10 to 15 minutes. Add three milliliters of complete medium and gently pipette three to five times to break down the spheroids to a single-cell suspension.Centrifuge the single-cell suspension at 400 times G for five minutes to pellet cells at room temperature. Aspirate the supernatant with a five-milliliter serological pipette, taking care not to disturb the cell pellet. Resuspend cells in one milliliter of complete medium.Remove a portion of the cells for counting using a hemocytometer. Dilute this portion twofold with trypan blue, which permeates cells with compromised viability. Count only the live, colorless cells.Determine the number of cells necessary for encapsulation. Transfer a volume of media containing the total number of cells needed into a sterile 1.7-milliliter microcentrifuge tube. Spin down at 400 times G for five minutes at room temperature.Aspirate the supernatant with a micropipette, taking care not to disturb the cell pellet. Resuspend the cell pellet in the hydrogel precursor solution, mixing well by pipetting up and down with a 1, 000 microliter micropipette four to five times. Load the cells into a repeat pipetter set to dispense 10 microliters.To avoid bubbles and uneven dispensing, prime the repeat pipetter by dispensing an additional one to two times into a waste container. In each well of a 384-well plate, dispense 10 microliters of cells suspended in hydrogel solution from the repeat pipetter. Using the LED array, illuminate each well containing cell for 15 seconds to achieve the desired mechanical properties.Add 40 microliters of complete media to each well containing the cells. Add 50 microliters of DPBS to non-experimental dry wells surrounding the gels to minimize losses due to evaporation. For glioblastoma cells, add 40 microliters of the media-containing drug to achieve the final desired concentration, starting three days after encapsulation.Add 10 microliters of CCK8 reagent to each well containing the cells. Incubate for one to four hours, according to the manufacturer's instructions. Read absorbances at 450 nanometers for all wells following incubation.AFM interrogation of micron-scale regions at the surfaces of single hydrogels showed that hydrogels with softer average Young's moduli also had smaller ranges of moduli than stiffer hydrogels. 3D cultures of GS122 cells seeded at densities of 2, 500, 000 cells per milliliter exhibited substantially higher viabilities when assessed after seven days in culture compared to those seeded at densities of 500, 000 cells per milliliter. GS122 cells showed survival gains in the eight kilopascal condition when osteopontin-derived peptides were included in the matrix, while the incorporation of integrin-binding sialoprotein or tenascin C-derived peptides provided minimal survival benefits, such as culture and matrices with the ginger RGD peptide.In contrast, no peptides conferred survival gains in the 0.8 kilopascal culture condition. Both GS122 and GS304 cells spread when cultured in soft or stiff hydrogel matrices, including RGD-containing peptides. GS122 cells show a similar lack of spreading in both 0.8 and 8 kilopascals with osteopontin.However, GS122 only showed enhanced resistance to temozolomide in the eight kilopascals condition. Finally, this miniaturized 3D culture platform can be used to culture human cells from other tumor types, including viable organoids of terminally differentiated neuroendocrine prostate cancer cells. Following this procedure, further techniques such as single-cell RNA sequencing can be used to further characterize cell phenotypes.

hydrogel-arrays-enable-increased-throughput-for-screening-effects

Research

JoVE Journal

Bioengineering

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation in situ. Unlike conventional bulk hydrogels, interconnected microscale pores in GHS facilitate degradation-independent cell infiltration as well as oxygen, nutrient, and cellular byproduct transfer. Methacryloyl-modified gelatin (GelMA), a (photo)chemically crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties, has widely been used as a cell-responsive/instructive biomaterial. Converting bulk GelMA to GHS&#160;may open a plethora of opportunities for tissue engineering and regeneration. In this article, we demonstrate the procedures of high-throughput GelMA microgel fabrication, conversion to resuspendable dry microgels (micro-aerogels), GHS formation via the chemical assembly of microgels, and granular bioink fabrication for extrusion bioprinting. We show how a sequential physicochemical treatment via cooling and photocrosslinking enables the formation of mechanically robust GHS.&#160;When light is inaccessible (e.g., during deep tissue injection), individually crosslinked GelMA HMPs may be bioorthogonally assembled via enzymatic crosslinking using transglutaminases. Finally, three-dimensional (3D) bioprinting of microporous GHS&#160;at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.

This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting.

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...