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>
	<li>Scannell, J. W., Blanckley, A., Boldon, H., Warrington, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+the+decline+in+pharmaceutical+R&amp;D+efficiency.">Diagnosing the decline in pharmaceutical R&amp;D efficiency.</a> Nature Reviews Drug Discovery. 11 (3), 191-200 (2012).</li><li>Waring, M. J., 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=An+analysis+of+the+attrition+of+drug+candidates+from+four+major+pharmaceutical+companies.">An analysis of the attrition of drug candidates from four major pharmaceutical companies.</a> Nature Reviews Drug Discovery. 14 (7), 475-486 (2015).</li><li>Khozin, S., Liu, K., Jarow, J. 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Market indicators.</a> Nature Reviews Drug Discovery. 2 (8), 609-610 (2003).</li><li>Da Ros, M., 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=Glioblastoma+chemoresistance:+The+double+play+by+microenvironment+and+blood-brain+barrier.">Glioblastoma chemoresistance: The double play by microenvironment and blood-brain barrier.</a> International Journal of Molecular Sciences. 19 (10), 2879 (2018).</li><li>Broekman, M. L., 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=Multidimensional+communication+in+the+microenvirons+of+glioblastoma.">Multidimensional communication in the microenvirons of glioblastoma.</a> Nature Reviews Neurology. 14 (8), 482-495 (2018).</li><li>Grundy, T. 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The authors have nothing to disclose.

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.

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Bioengineering

2.4K Views.  University of California Los Angeles. 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 therapeut...