
==== Front
STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00431-3
10.1016/j.xpro.2024.103266
103266
Protocol
Protocol for in vitro evaluation of effects of stiffness on patient-derived glioblastoma
Sohrabi Alireza 12
Seidlits Stephanie K. seidlits@utexas.edu
13∗
1 Department of Biomedical Engineering, University of Texas at Austin, Austin, TX 78712, USA
∗ Corresponding author seidlits@utexas.edu
2 Technical contact

3 Lead contact

14 8 2024
20 9 2024
14 8 2024
5 3 103266© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Summary

Glioblastoma (GBM) is the most common and lethal type of primary brain tumor. Physiologically, GBM cells experience a heterogeneous mechanical landscape. Here, we present an in vitro method to study the effects of tissue stiffness on patient-derived GBM that utilizes hyaluronic acid (HA)-based, mechanically tunable scaffolds for three-dimensional (3D) culture of patient-derived GBM spheroids. We describe steps to fabricate and characterize HA-based scaffolds, culture GBM spheroids within 3D hydrogel scaffolds, and prepare cultured cells for a variety of experimental assessments.

For complete details on the use and execution of this protocol, please refer to Sohrabi et al.1

Graphical abstract

Highlights

• Protocol for culturing patient-derived glioblastoma cells in 3D hydrogels

• Procedure for fabricating hyaluronic acid-based hydrogels

• Procedures for post-encapsulation processing of hydrogels such as cryofreezing

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Glioblastoma (GBM) is the most common and lethal type of primary brain tumor. Physiologically, GBM cells experience a heterogeneous mechanical landscape. Here, we present an in vitro method to study the effects of tissue stiffness on patient-derived GBM that utilizes hyaluronic acid (HA)-based, mechanically tunable scaffolds for three-dimensional (3D) culture of patient-derived GBM spheroids. We describe steps to fabricate and characterize HA-based scaffolds, culture GBM spheroids within 3D hydrogel scaffolds, and prepare cultured cells for a variety of experimental assessments.

Subject areas

Cancer
Material sciences
Tissue Engineering
==== Body
pmcBefore you begin

Glioblastoma (GBM) cells dynamically interact with their surrounding extracellular matrix.2 GBM cells, like cells of other solid tumors, hyper-secrete matrix, which causes progressive stiffening of their microenvironment.3 This report describes detailed methods for culture of patient-derived, primary GBM cells, forming these GBM cells into uniformly sized spheroids, culturing spheroids in 3D scaffolds with varying mechanical properties, and analyzing GBM cell phenotypes after 3D culture. This general method for 3D culture can also be applied to other cell and scaffold types. Here, we describe methods for forming hydrogel scaffolds using a UV light-initiated, thiol-ene chemistry.4,5 High molecular weight (∼750 kDa) hyaluronic acid (HA), modified with thiols, comprises the hydrogel backbone and norbornene- and thiol-polyethylene glycol (PEG) macromers are used as crosslinkers. While this protocol describes steps to generate hydrogels with varying stiffness without changing the total HA concentration, we have also published how the same platform can be used to form hydrogels in which HA concentration can be varied while maintaining a consistent soft, brain-like stiffness.6 All cell lines were collected with strict adherence to UCLA Institutional Review Board protocol 10–000655.

Preparation of hydrogel material

Timing: 2 h

Note: All material preparation and experimental steps should be done using sterile supplies and techniques, as appropriate for human cell culture.

1. Aliquot each component described below in 1.5 mL or 0.6 mL centrifuge tubes and store at −20°C under desiccation until use. Avoid multiple freeze-thaw cycles.a. Prepare 20 mM HEPES/HBSS buffer.i. Dissolve 238.3 mg of HEPES powder in 50 mL of HBSS.

ii. Adjust the pH to 7 if needed.

b. Thiol-functionalized, 4-arm polyethylene glycol (PEG-SH): Aliquot 20–30 mg of PEG-SH into each 1.5 mL microfuge tube and seal the lid with parafilm. Aliquots can be stored in −20°C up to a year.

c. Norbornene-functionalized, 8-arm polyethylene glycol (PEG-Norb):i. Dissolve PEG-Norb in 20 mM HEPES buffer in Hank’s Balanced Salt Solution (HEPES/HBSS, pH 7) at 100 mg/mL. Double-check that the pH is at 7, but no adjustments are typically needed.Note: In this protocol HEPES buffer refers to HEPES/HBSS, unless mentioned otherwise.

ii. Aliquot 1 mL of PEG-Norb solution in each 1.5 mL microfuge tube and seal the lid with parafilm. At −20°C aliquots are stable up to a year.

d. Dissolve RGD-SH peptide in 20 mM HEPES buffer (pH 7) to achieve a final concentration of 4 mM. Aliquot RGD-SH into 200 μL samples in 0.6 mL centrifuge tubes. Aliquots are stable in −20°C up to one month.

e. Aliquot freeze-dried, thiolated HA (HA-SH), prepared as previously described.7i. After dialysis, aliquot 5 mL of HA-SH solution in 15 mL centrifugal tubes. Freeze-dry samples before storage.

ii. Aliquots are stable in −20°C up to one month.

Preparation of GBM culture media components

Timing: 1 h

2. GBM culture media contains basal media (DMEM/F12), Gem21 Neuroplex supplement, HEF growth factor cocktail, and normocin as an antimicrobial agent.a. Store DMEM/F12 basal media at 4°C. Media can be stored at 4°C until expiration date specified by the manufacturer.

b. Prepare and store HEF (400×) aliquots at −20°C.i. Dissolve 500 μg of epidermal growth factor (EGF) in 500 μL of 20 mM HEPES (pH = 7.4) containing 0.1% (w/v) BSA (0.1% BSA-HEPES). Then, add this solution to 4.5 mL of 0.1% BSA-HEPES.

ii. Dissolve 250 μg of basic fibroblast growth factor (FGF-2) in 250 μL of 0.1% BSA-HEPES. Then, add this solution to 9.75 mL of 0.1% BSA-HEPES.

iii. Dissolve 50 mg of heparin in 10 mL 0.1% BSA-HEPES.

iv. Prepare 25 mL of HEF (heparin-EGF-FGF) solution.

v. Aliquot 100 μL of HEF into 0.7 mL centrifuge tubes and store at −20°C.

vi. HEF aliquots can be stored in −20°C up to 6 months.

c. Gem21 supplement is provided by the manufactured packaged as individual 10 mL bottles, which can be stored at −20°C until use.

d. Normocin antimicrobial agent is received in 1 × 10 mL, 5 × 1 mL, or 1 × 20 mL portions. Make aliquots of 1 mL into 1.5 mL microfuge tubes and store at −20°C. Aliquots can be stored in −20°C up to 6 months.

Preparation for GBM spheroid formation

Timing: 1 h

3. Prepare 5% (w/v) Pluronic F-127 in 1× PBS.a. Example: For 40 mL of the solution, dissolve 2 g of Pluronic F-127 in 40 mL 1× PBS in a 50 mL centrifuge tube.

b. To dissolve, set the tube on a rotator at 22°C for 1 h.

c. After complete dissolution, filter the solution using bottle-top, polyethersulfone (PES), 0.22 μm pore size filters.

d. Pluronic solution can be prepared in advance and stored at 4°C for up to 6 months.

Preparation for 4% paraformaldehyde (PFA)

Timing: 1 day

4. To prepare 1 L of 4% PFA in PBS, dissolve 40 g of PFA in 1000 mL 1× PBS.a. Dissolve 40 g of PFA in 800 mL 1× PBS.

b. Stir the mixture at 60°C in a ventilated hood.

c. Raise the pH by adding 5 M NaOH drop-by-drop until the solution is clear.

d. Adjust the volume to 1 L with 1× PBS.

e. Aliquot into 40 mL in 50 mL centrifuge tubes.

f. Frozen aliquots are stable at −20°C up to a year.

g. When thawed, keep the PFA at 4°C for up to a month.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Sodium hyaluronate (MW = 700 kDa)	Lifecore Biomedical	HA700k-5	
8-arm norbornene functionalized polyethylene glycol (MW = 20 kDa)	JenKem	A10037-1	
4-arm thiol functionalized polyethylene glycol (MW = 20 kDa)	Laysan Bio	4arm-PEG-SH-20K-1g	
Lithium phenyl-2,4,6-trimethylbenzoylphosphinate, ≥95%	Sigma-Aldrich	900889-1G	
RGD-SH peptide (GCGYGRGDSPG)	GenScript	Custom order	
DMEM/F12 basal media	Thermo Fisher Scientific	11320082	
Gem21 NeuroPlex supplement	GeminiBio	400-160-010	
Normocin	InvivoGen	Ant-nr-2	
Animal-free recombinant EGF (500 μg)	PeproTech	AF-100-15	
Recombinant human FGF-basic (154 aa) (250 μg)	PeproTech	100-18B	
Heparin sodium salt from porcine intestinal mucosa, grade I-A, ≥180 USP units/mg, powder, BioReagent	Sigma-Aldrich	H3149-100KU	
Pluronic F-127	Sigma-Aldrich	P2443-250G	
TrypLE express enzyme (1×), no phenol red	Thermo Fisher Scientific	12604013	
Sucrose	Thermo Fisher Scientific	A15583.0E	
Scigen Tissue-Plus O.C.T. compound	Fisher Scientific	23-730-571	
RIPA lysis buffer	Thermo Fisher Scientific	89901	
2-methylbutane, 99%	Thermo Fisher Scientific	019387.AP	
Paraformaldehyde	Fisher Scientific	AC416785000	
Phosphate-buffered saline	Fisher Scientific	BP2944100	
HEPES	Fisher Scientific	BP310-100	
Hank’s balanced salt solution (HBSS)	Thermo Fisher Scientific	14025076	
	
Critical commercial assays	
	
AggreWell400 (24-well)	STEMCELL Technologies	34450	
RNeasy micro kit	QIAGEN	74004	
QIAshredder	QIAGEN	79656	
500 mL vacuum filter flasks	Fisher Scientific	09-740-63D	
	
Experimental models: Cell lines	
	
HK177 (patient-derived GBM cell line)	Kornblum Lab (UCLA)		
HK408 (patient-derived GBM cell line)	Kornblum Lab (UCLA)		
	
Other	
	
XX-15L bench UV lamp, 365 nm, 115 V	Thomas Scientific	1195F75	
UVA radiometer	LabGear USA	AJ 97-0015-02	
Silicone Isolators	Grace Bio-Labs	SKU: 664206	
Peel-A-Way embedding molds	Sigma	E6032-1CS	
NanoDrop spectrophotometer	Thermo Scientific	13-400-519	
Discovery Hybrid Rheometer 2 (DHR2)	TA Instruments	N/A	

Materials and equipment

Reagent to make HEF	Amount	Stock concentration (μg/mL)	Final concentration (μg/mL)	
EGF	5 mL	20	0.05	
FGF-2	8 mL	8	0.02	
Heparin	10 mL	2000	5	
BSA-HEPES	2 mL	N/A	N/A	
Total	25 mL	N/A	N/A	
Aliquot 105 μL of 400× HEF cocktail into 0.7 mL centrifuge tubes. HEF aliquots can be stored at −20°C for up to 6 months.

Reagent to make complete GBM media	Amount	
DMEM/F12	489 mL	
Gem21 Neuroplex	10 mL	
Normocin	1 mL	
Total	500 mL	
Aliquot 40 mL of media into 50 mL tubes. Media aliquots can be stored at −20°C for up to 6 months.

When a media aliquot is thawed, it can be stored at 4°C for one week.

Reagent to make HA hydrogels	Stock concentration	Amount for soft hydrogels (G’ = 100 ± 30 Pa)	Amount for stiff hydrogels (G’ = 1000 ±100 Pa)	
HA-SH	10 mg/mL	500 μL	500 μL	
PEG-SH	100 mg/mL	18.9 μL	118.9 μL	
PEG-Norb	100 mg/mL	36.3 μL	96.3 μL	
RGD-SH	4 mM	62.5 μL	62.5 μL	
LAP	5 mg/mL	50 μL	50 μL	
HEPES buffer	20 mM	338.5 μL	178.5 μL	
Total	N/A	1000 μL	1000 μL	

Note: The recipe provided above generates hydrogels with 0.5% (w/v) HA.

Note: The above recipe is formulated considering HA with 5% thiolation of repeated disaccharides, as determined by H-NMR. In case of different thiolation percentage of HA, amount of PEG-SH should be adjusted. For example, for a HA batch with 3% thiolation, PEG-SH volumes should be adjusted to 25.1 μL and 125.1 μL for soft and stiff hydrogels, respectively.

Note: G′ is reported as mean +/− standard deviation, as determined from shear rheology.

Note: Prepare solutions fresh before use.

Step-by-step method details

Note: All centrifugation steps are done 25°C unless mentioned otherwise.

Patient-derived GBM cell culture

Timing: 2–3 h

Patient-derived GBM cells are enriched in GBM stem cells. This step describes steps toward preparing the appropriate culture media, and the cell culture method to maintain the stemness of GBM cells. GBM cells were previously isolated from patient samples and kindly provided by the Kornblum lab at the University of California, Los Angeles (UCLA).81. Prepare GBM culture media.a. Prepare and aliquot complete GBM media.i. Follow the recipe to prepare 500 mL of GBM media.

ii. Aliquot prepared media into sterile, 50 mL conical tubes.

Note: HEF aliquots described above are at 400×. Each aliquot is enough for one aliquot of GBM media (100 μL of a HEF aliquot for each 40 mL aliquot of GBM medium). Only add the HEF aliquot to the culture media immediately before use.

Note: Once the HEF aliquot is added, complete GBM media can be stored at 4°C for up to 1 week or at −20°C up to 6 months.

Note: Do not fill 50 mL tubes with more than 40 mL media if you are freezing the tubes.

2. Maintenance culture of GBM cells.a. Steps for starting culture from a frozen vial of patient-derived GBM cells.i. Prewarm the GBM media to 25°C.

ii. Thaw the frozen vial quickly at 37°C in a bead bath.

iii. Transfer the content from frozen vial of cells into a 15 mL centrifuge tube containing 4 mL of complete GBM media.

iv. Centrifuge at 400 × G for 4 min.

v. Carefully aspirate the supernatant.

vi. Resuspend GBM cell pellet in 1 mL of fresh, complete GBM media.

vii. Add the cell suspension into a T25 flask, prefilled with 5 mL complete GBM media.

viii. Every 2–3 days, add 2.5 mL of fresh complete GBM media to the flask.

ix. Passage GBM spheroids when the spheroid size is around 150–200 μm in diameter.Note: Add 100 μL of a HEF aliquot for each 40 mL aliquot of GBM medium to prepare the complete GBM medium.

Note: GBM cells cultured using this method form spheroids while growing.

b. Passage GBM spheroids.i. Collect GBM spheroids in a 15 or 50 mL centrifuge tube, depending on the total volume.

ii. Centrifuge at 400 × G for 4 min.

iii. Aspirate the media carefully so the GBM spheroid pellet is not disturbed.

iv. Add 1 mL pre-warmed TrypLE Express to the tube.

v. Tap or agitate the tube continuously for ∼5 min to help dissociate the GBM spheroids. Do not incubate cells for more than 5 min with TrypLE Express.

vi. Add 4 mL of complete GBM media to inactivate TrypLE Express.

vii. Flow the cell suspension through a 40 μm cell strainer.

viii. Count total cell numbers using a hemacytometer.

ix. Centrifuge at 400 × G for 4 min.

x. Aspirate the media carefully so the GBM spheroid pellet is not disturbed.

xi. Resuspend the cell pellet in 1 mL of complete GBM media by pipetting up and down ∼8 times.

xii. Add resuspended cells in a new culture flask with GBM media at a cell density of around 50,000–100,000 GBM cells/mL.

xiii. Add 5 mL of fresh complete GBM media to the flask every 2 or 3 days.Note: For example, for 1 million cells, you would use 10–20 mL of culture medium to yield 100,000 GBM cells/mL or 50,000 GBM cells/mL, respectively. Select culture flask size to match the volume of cell suspension at this density that you plan to use. For 10 mL, we recommend using a T75 flask.

Note: The frequency of media addition/feeding and passaging is highly dependent on the cell line. Check flasks frequently for growth rate.

Hyaluronic acid thiolation

Timing: 1 week

Hyaluronic acid (HA) is a major component of the brain extracellular matrix. HA is modified with thiol (-SH) functional groups to enable hydrogel formation in the presence of norbornene-modified PEG. Steps required to successfully conjugate thiol functional groups on HA chains with 5–10% modification (moles of -SH/moles of HA) are described in detail in Xiao et al.,7,9 and Ehsanipour et al.9

GBM spheroid formation

Timing: 1 day

When cultured in free-floating suspension, GBM cells form GBM spheroids with a wide range of sizes, which can potentially introduce variations in phenotype. For example, GBM cells at the center of larger spheroids experience more hypoxic conditions while GBM cells at the center of smaller spheroids do not. To eliminate any size-dependent variation, the following method can be used to generate GBM spheroids with a narrow range of sizes.Note: For this step, a 24-well AggreWell 400 plate is used. Each well of this plate has 1200 microwells.

Note: Spheroid formation using this method requires overnight incubation. Non-GBM cell types may require changes to this timing or other method specifics.

3. Prepare the AggreWell 400 plate.a. Dispense 1 mL of 5% (w/v) Pluronic F-127 in each well.

b. Centrifuge the plate at 2000 × G for 5 min.

c. Incubate the plate at 25°C for 30 min.

4. Passage GBM cells as described in step 2.b except, at the end, do not plate the cells in a flask.

5. Seed GBM cells in the AggreWell 400 plate.a. Aspirate Pluronic-F-127 out of each well of the plate.

b. Dispense 1 mL of complete GBM media into each well.

c. Aspirate the media.

d. Pipette desired number of cells in each well of the plate.

e. Add fresh complete GBM media so that each well has a final volume of 1 mL.

f. Pipette the contents of each well up and down once or twice gently using a 1 mL micropipetter tip to distribute the cells evenly.

g. Centrifuge the plate at 300 × G for 3 min.

h. Incubate the plate in the cell culture incubator overnight.

Note: The final size of GBM spheroids is dependent on the initial seeding density in each microwell of the Aggrewell 400 plate. For this specific work, 500 GBM cells were seeded into each microwell (600,000 cells total in each well of the plate). GBM spheroids obtained from this seeding density were 100–150 μm in diameter. For different cell lines or types, optimization of seeding density to yield the desired spheroid size is required.

HA hydrogel formation and spheroid encapsulation

Timing: 3–4 h

Microenvironmental stiffness directly impacts cell phenotype. To investigate the role of stiffness on GBM cell phenotype, we fabricated HA-based hydrogels with mechanical properties approximating the perinecrotic tumor core (stiff, G’ = 1000 ± 100 Pa) or peritumoral brain tissue (soft, G’ = 100± 30 Pa).Note: Hydrogel fabrication using this method requires a UV light source to provide 4–5 mW/cm2 intensity of 365 nm light.

6. Dissolve HA in 20 mM HEPES (pH = 7.4) at 10 mg/mL.

Note: Keep HA at 25°C for 20 min to equilibrate before weighing and dissolving.

Note: Use a magnetic stir plate and a stir bar to dissolve HA thoroughly in buffer. This process will take at least 1 h.

7. After complete dissolution of HA, adjust the pH to 7–7.5 using 1 M NaOH.

Note: Add small volumes of 1 M NaOH (1–2 μL) at a time. Thiolated HA self-crosslinks through disulfide bond formation at pH > 8, so it is important not to overshoot pH 7.5.

8. Prepare hydrogel precursor solution.a. Weigh 3–5 mg of LAP photoinitiator into a 1.5 mL microcentrifuge tube and add 20 mM HEPES buffer to reach a final concentration of 5 mg/mL LAP.

b. Weigh 15–20 mg of PEG-SH and add 20 mM HEPES buffer to reach a final concentration of 100 mg/mL.

c. Mix hydrogel reagents following the recipe.

Note: Equilibrate all hydrogel components to 25°C before use.

9. Embedding GBM spheroids into hydrogels for 3D culture.a. Resuspend GBM spheroids by pipetting the contents AggreWell well up and down twice using a P1000 pipette.

b. Collect each 1 mL of GBM spheroids within medium from each well into a 1.5 mL microfuge tube.

c. Centrifuge at 200 × G for 1 min.Note: Faster or longer centrifugation will result in spheroid deformation.

d. Slowly remove supernatant from each tube using a micropipetter.

e. Add the desired amount of hydrogel precursor solution to spheroid pellets in each the microfuge tube.Note: The volume of hydrogel solution used depends on the number of hydrogel cultures to be prepared, the volume of each hydrogel needed, and the desired number of spheroids to be seeded into each hydrogel.

Example: In this work, we fabricated 30 μL hydrogels with 6 hydrogels per each condition and approximately 200 GBM spheroids per hydrogel. To do so, GBM spheroids acquired from each well of the AggreWell plate, were resuspended in 200 μL of hydrogel precursor solution.

f. For 30 μL hydrogels, add 500 μL of complete GBM media to each well of a 24-well culture plate.Note: Plates with smaller or large well volumes may be used to accommodate different hydrogel sizes, which depends on the specific experimental application.

g. Cast hydrogel precursor solution into appropriately sized silicone rubber molds, then irradiate with UV light (365 nm at 4–5 mW/cm2) for exactly 15 s to crosslink.

h. Transfer hydrogels to well plates prefilled with complete GBM media (step f) before transferring plates to the cell culture incubator.

i. Refresh half of culture media every 2–3 days by carefully pipetting 250 μL of media out from each well and adding 250 μL of fresh complete GBM media.Note: Frequency of media changes depends on the cell line. Observe cultures frequently.

HA hydrogel mechanical properties: Rheology

Timing: 2–3 h

Note: The protocol described here is based on using a DHR2 rheometer (TA instruments). While general parameters are universal, users need to optimize this part of the protocol for the available rheometer.

Note: Hydrogels were swollen in 1 mL of 1× PBS overnight before testing.

Note: Measurements shown in this report were acquired at 37°C.

10. Turn the rheometer on and make sure it’s connected to the computer.

11. Open the software (Trios).

12. Calibrate the rheometer.a. First, while the geometry is not attached, calibrate the instrument inertia.

b. Attach the proper geometry. In our experiments, we used a cross-hatched, 8 mm, parallel-plate geometry.

c. Go to the geometry calibration and calibrate inertia, friction, and rotational mapping.

Note: In any step of calibration, if the new values are more than 1% different than the previous values, contact the manufacturer to troubleshoot.

13. Setup the experiment in experiment tab.a. For each sample add the proper name.

b. Choose the destination for the data to be stored.

c. In the geometry tab, set the gap to 800 μm, loading gap to 45,000 μm, and trim gap to 50 μm.

d. In the procedure tab, set the experiment to oscillation frequency, strain to 1%, and log sweep of 0.1 Hz–1 Hz.

14. Prepare the samples.a. Transfer the sample to the bottom plate of the parallel plate geometry.

b. Using the software control, move to the trim gap.

c. Physically trim excess sample (overhanging the parallel plate geometry) with a plastic object.CRITICAL: Do not use any metal object for this step, metal object will damage the rheometer geometry.

d. Using the software control, move to the geometry gap.

e. Start the measurement in the software to acquire data.

15. Process the data.a. Export each data file to csv format.

b. For each sample, in storage modulus vs. frequency graphs, find the average of the flat portion of storage modulus (G′) graph to calculate the storage modulus of the sample.

Processing of GBM spheroids cultured in 3D hydrogels: Fixation and sample preparation for cryosectioning

Timing: 2 days

To visualize the expression of specific proteins, immunofluorescence staining of fixed cryosections of hydrogel cultures can be used. Step-by-step procedures to prepare the hydrogels for cryosectioning are described here.

Day 116. Prepare sucrose solutions.a. Solution A: 5% (w/v) sucrose buffer.i. Dissolve 2 g of sucrose in 40 mL of 1× PBS.

b. Solution B: 20% (w/v) sucrose buffer.i. Dissolve 8 g of sucrose in 40 mL of 1× PBS.

c. Solution C: 20% (w/v) sucrose-OCT.i. Dissolve 8 g of sucrose in 40 mL of OCT.

Note: Solution C requires mixing on a tube rotator for at least 24 h at 25°C to achieve full dissolution.

17. Fix 3D hydrogel cultures and prepare for OCT embedding.a. Move hydrogels to a new, sterile 24-well plate, add 1 mL of 4% (w/v) paraformaldehyde (PFA) solution, incubate at 25°C for 1 h.

b. Remove PFA add 1 mL of solution A (step 16.a) to each well with a hydrogel culture. Incubate for 1 h at 25°C on a shaker or rocker plate.Note: PFA is hazardous and need to be disposed with adherence to the institutional guidelines.

c. Carefully remove solution A, add 1 mL of solution B (step 16.b), and incubate at 25°C for 30 min on a shaker or rocker plate.

d. Carefully remove solution B, add 1 mL of fresh solution B, and incubate at 25°C for 30 min on a shaker or rocker plate. Repeat this step one more time.

e. Store the samples in fresh solution B at 4°C overnight.

Day 218. Embedding fixed hydrogel cultures in OCT for cryosectioning.a. Carefully remove solution B from all wells, add 1 mL of solution C (step 16.c) to each well, and incubate at 4°C for 3 h.Note: Avoid storing hydrogels in solution C for longer than 3 h.

b. Transfer hydrogels to cryofreezing molds.

c. Fill the mold with more OCT until the hydrogel is fully immersed.

d. Flash freeze hydrogel cultures by dipping (using tongs) the hydrogels within cryofreezing molds into 2-methylbutane chilled on dry ice.Note: Liquid nitrogen can be used instead of 2-methylbutane.

Note: Avoid completely submerging the molds in the freezing liquid.

Processing of GBM spheroids cultured in 3D hydrogels: RNA-extraction

Timing: 2–3 h

Bulk RNA-sequencing can be utilized to understand the broad effects of microenvironmental stiffness on the transcriptional activities of GBM cells. This section describes the steps necessary to extract RNA from GBM cells cultures in 3D hydrogels in preparation for downstream RNA sequencing.Note: This protocol is adapted from QIAGEN RNeasy Microkit protocol with minor changes.

19. Transfer hydrogels from culture wells to 1.5 mL microfuge tubes.

Note: In our experiments, after around 1 week in culture we combined 5, 30 μL hydrogels—each with initial spheroid density of 200 GBM spheroids, formed from 500 cells per spheroid—per microfuge tube.

20. Add 350 μL of RLT buffer to each tube.

21. Disintegrate hydrogels and cells using mechanical forces.

Note: Tissue homogenizers are preferred. If tissue homogenizer is not available, use a 1 mL syringe, equipped with a 20G needle, and homogenize samples by 30 cycles of suction/expulsion.

22. Transfer the solution to Qiashredder columns and centrifuge at 14.8 × G for 2 min.

23. Transfer the Qiashredder column flowthrough to 2 mL microfuge tubes.

Note: Avoid pipetting the hydrogel culture precipitate at this point.

24. Add 350 μL of 100% ethanol to each microfuge tube. Mix and wait 5 min.

Note: While the QIAGEN protocol suggests using 70% ethanol for this step, in our experience, 70% ethanol will elute RNA from the column prematurely, but 100% ethanol will not.

25. Transfer the solution to Qiagen RNeasy Microkit and follow the standard protocol provided by Qiagen.

26. Measure RNA concentration using a NanoDrop microvolume spectrophotometer.

27. Store RNA samples at −80°C until ready to perform bulk RNA sequencing.

Processing of GBM spheroids cultured in 3D hydrogels: Protein extraction

Timing: 2–3 h

In this section, we describe a step-by-step method to extract proteins from hydrogel cultures for downstream applications such as Western blotting.28. Pool cultured hydrogel of the same experimental condition into a single well of a 6-well plate.

Note: Typically, after around 1 week in culture we combined 4 hydrogels—each with initial spheroid density of 200 spheres, formed from 500 cells per spheroid, per 30 μL hydrogel—per well to achieve workable level of proteins.

29. Wash hydrogels with 1× PBS for 15 min. Repeat this step 3 times.

30. Cool a microcentrifuge to 4°C.

31. Collect washed and pooled hydrogels (4 hydrogels) into 1.5 mL centrifuge tubes.

32. Add 300 μL of RIPA buffer.

Note: Other lysis buffers can be substituted for RIPA buffer, if needed.

33. Using mechanical forces, dissociate hydrogels and lyse cells.

Note: Tissue homogenizers are preferred. If tissue homogenizer is not available, use a 1 mL syringe, equipped with a 20G needle, and homogenize samples by 30 cycles of suction/expulsion as described in step 15 above.

34. Incubate samples on ice for 15 min, quickly pulse vortexing samples every 5 min during this period.

35. Centrifuge microtubes at 14.8 × G for 30 min at 4°C.

36. Collect the supernatant in a pre-chilled, fresh microfuge tube and store in −80°C until analysis.

Expected outcomes

To assemble GBM cells into GBM spheroids with a uniform size distribution, Aggrewell technology was used. GBM spheroid size can be controlled by changing the average number of cells seeded in each μ-well (Figure 1). By changing the average density from 100 cells per μ-well to 1000 cells per μ-well, we were able to achieve spheroid diameter of 75 ± 20 μm to 180 ± 35 μm. HA-based hydrogels were formed using a UV-activated thiol-ene chemistry. To achieve a wide range of mechanical properties, while keeping the final concentration at 0.5 wt %, concentration of PEG-SH (co-crosslinker) and PEG-Norb (cross-linker) were varied (Figure 2). Hydrogel formulation, in this study are shown in red (soft) and blue (stiff).Figure 1 GBM spheroids with uniform size was produced using Aggrewell

(A–D) GBM spheroids with a variety of sphere sizes were from in Aggrewell plates. Seeding densities of (A) 100, (B) 250, (C) 500, and (D) 750 cell per μ-well.

(E and F) Representative images of GBM spheroids in expansion. GBM cells cultured in media grow into spheroids with a wide range of size (Scale bar = 100 μm).

(G) By changing the seeding density of each μ-well a variety of uniformed spheroid sizes were formed (∗∗∗∗: p < 0.0001, n = 100 spheroids, N = 2 biological repeats).

Figure 2 Hyaluronic acid hydrogels with variety of stiffness were fabricated using a UV-initiated thiol-ene chemistry

Storage modulus (G′) of hydrogels were measured using a rheometer. By altering the total thiol (Sh) concentration in hydrogel precursor solutions, we were able to fabricate hydrogels with a wide range of stiffness (∗∗∗∗: p < 0.0001, n = 5 hydrogels per experiments, N = 3 biological repeats).

For detailed outcomes please refer to Sohrabi et al.1

Limitations

Stiff hydrogels formed using this protocol will have more polymer crowding than soft hydrogels, which could have unknown effects on cells beyond those determined by stiffness alone. In addition, hydrogels formed using these methods are primarily elastic, rather than viscoelastic like native brain tissue. Finally, this method may not work to create hydrogels along a much stiffer modulus range without altering pore size or diffusion between soft and stiff hydrogels.

Troubleshooting

Problem 1: HA hydrogels not forming or too soft, steps 6–9

Hydrogel formulations described in above result in hydrogels with storage modulus of 100 Pa and 1000 Pa, for soft and stiff hydrogels, respectively. If hydrogels are too soft or not forming.

Potential solutions

• Make sure hydrogel components are stored at −20°C, expect LAP which should be stored at 4°C.

• Avoid freeze-thawing HA-SH and PEG-SH aliquots.

• Check the UV lamp intensity. UV lamp with intensity 4–5 mW/cm2 is necessary.

Problem 2: GBM spheroids not forming in Aggrewell, steps 3–5

GBM spheroids formed in Aggrewell using this protocol forms in 24 h of incubation. If spheroids do not form:• Pluronic solution contains wrong amount of Pluronic or is expired.

• GBM cells are in high passages (above 30).

• Inappropriate centrifugation speeds are used.

Potential solutions

• Make fresh Pluronic solutions and use correct concentrations.

• Make sure using younger cell passages.

• Pay attention to different centrifugation speeds and timing. Make sure the centrifuge is balanced.

Problem 3: RNA concentration too low for sequencing, steps 19–27

Low RNA concentration can be due to multiple reasons such as:• Low number of cells in cultures.

• Wrong dilution of ethanol used. Refer to potential solutions.

• Expired RNeasy columns.

Potential solutions

• Increase number of cells, encapsulated in each hydrogel, or combine more hydrogels for RNA extraction.

• As mentioned in the protocol, Qiagen protocol asks for mixing the cell lysates with 70% ethanol. In our experience, the water content in 70% ethanol inhibits efficient binding of RNA to the RNeasy column. Therefore, we substituted this step with 100% ethanol.

• Check the expiry date of the RNeasy columns. In addition, make sure RNeasy kit components have been stored at the correct temperature.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Stephanie Seidlits (Seidlits@utexas.edu).

Technical contact

Further information and technical questions should be directed to the technical contact, Alireza sohrabi (alireza.sohrabi@austin.utexas.edu).

Materials availability

This study did not generate new materials.

Data and code availability

This study did not generate any code or data sets.

Acknowledgments

We would like to thank the Department of Bioengineering at UCLA for their support. We would like to thank Kornblum and Nathanson labs at UCLA for providing patient-derived GBM cell lines as well as consultation. We would like to thank NIH 10.13039/100000054 NCI for funding this project (R01CA241927-01A1 ). Images depicted in the graphical abstract were designed using Biorender.com.

Author contributions

A.S. wrote the manuscript, composed and implemented the techniques described here, and performed experimental parts corresponding to this protocol. S.K.S. supervised the study. Both authors commented on and edited the manuscript.

Declaration of interests

The authors declare no competing interests.
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