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

Summary

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.

Abstract

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.

Introduction

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 & 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 not....

Protocol

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

1. Prepare HEPES-buffered solution by dissolving HEPES powder at 20 mM in Hank's balanced salt soluti.......

Discussion

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.......

Materials

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