Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Summary

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Abstract

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 μm. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Introduction

Tumor spheroids have emerged as useful in vitro tools in studying cancer etiology, pathology, and drug responsiveness¹. Traditionally, spheroids have been cultured in conditions such as low adhesion plates or bioreactors, where cell-cell adhesion is favored over cell-surface adhesion². However, it is now recognized that to recapitulate the tumor microenvironment more faithfully, in vitro spheroid models should capture both cell-cell and cell-matrix interactions. This has prompted multiple groups to design scaffolds, such as hydrogels, where spheroids can be encapsulated^3,

Protocol

1. Solutions preparation

1. Preparation of polydimethylsiloxane (PDMS) precursor solution
   1. Prepare the negative PDMS precursor solution (also used for the glue precursor solution). Scoop the elastomer into a weigh boat using a spatula and weigh it. Add the curing agent to the elastomer base at a 1:10 ratio. Mix the PDMS and curing agent gently and thoroughly using the spatula in the plastic weigh boat.
      NOTE: This PDMS precursor solution is poured into the 6-well square pyramid.......

Discussion

Hydrogel-based multicellular tumor spheroid models are increasingly being developed to advance cancer therapeutic discoveries^11,13,29. They are beneficial because they emulate key parameters of the tumor microenvironment in a controlled manner and, despite their complexity, are simpler and cheaper to use than in vivo models, and many are compatible with high-throughput screening technologies. The hydrogel biomaterials can be tun.......

Materials

Name	Company	Catalog Number	Comments
70% Ethanol	Fisher Scientific	LC22210-4
15 mL Conicals	FALCON	352097
24-Well Plate Ultra Low Attachment plates	Fisher Scientific	07-200-602
35 mm Petri Dish	Amazon	706011
4-arm poly(ethylene glycol)-acrylate (4-arm PEG-Ac; 10 kDa)	Laysan Bio	ACRL-PEG-ACRL-10K-5g
50 mL Conicals	Fisher Scinetific	3181345107
6-well AggreWell 400	StemCell Technologies, Vancouver, Canada	34421	Square pyramidal microwells
anti-adherence rinsing solution	StemCell Technologies, Vancouver, Canada	Cat #: 07010
Aspartic Acid-Arginine-Cysteine-Glycine-Valine-Proline-Methionine-Serine-Methionine-Arginine-Glycine-Cysteine-Arginine- Aspartic Acid (DRCG-VPMSMR-GCRD) peptide	Genic Bio, Shanghai, China	n/a	Custom synthesis
Chemical Fume Hood	KEWAUNEE	99151
Corning Matrigel Basement Membrane Matrix, LDEV Free	Corning	356234	Basement membrane matrix
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)	Thermo Scientific	62247
Detergent - Triton-X	Sigma Aldrich	T8787	Nonionic surfactant
Dimethyl sulfoxide (DMSO)	Fisher Scientific	BP231-100
Disposable Pipettes (1 mL, 2 mL, 5 mL, 10 mL, 25 mL, 50 mL)	Fisher Scinetific	1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F
Fetal Bovine Serum	HyClone	SH30073-03
Formaldehyde 37% Solution	Sigma Aldrich	F1635
Glass Plates	Slumpys	GBS4100SFSL
Glass Transfer Pipettes	Fisher Scinetific	5 3/4": 1367820A, 9":136786B
Glycine-Arginine-Cysteine-Aspartic Acid-Arginine-Glycine-Aspartic Acid-Serine (GRCD-RGDS) peptide	Genic Bio, Shanghai, China	n/a	Custom synthesis
Hemacytometer	Bright-Line	383684
Hydrophobic solution - Repel Silane	GE Healthcare Bio-Sciences	17-1332-01
Incubator	NUAIRE	NU-8500
Inverted Microscope (Axiovert 25)	Zeiss	663526
Invitrogen DiOC16(3) (3,3'-Dihexadecyloxacarbocyanine Perchlorate)	Fisher Scientific	D1125
Leica Confocal SP8	Leica Microsystems Inc.
Light and Flourescent Microscope (Axiovert 200M)	Zeiss	3820005619
Micro centrifuge tubes	Fisher Scientific	2 mL: 02681258
Microscope Software	Zeiss	AxioVision Rel. 4.8.2
Nestin Alexa Fluor 594	Santa Cruz Biotechnology	sc-23927
Parafilm	PARAFILM	PM992
PBS (1x), pH 7.4	HyClone	SH30256.01
Penicillin Streptomycin	MP Biomedicals	1670046
Pipette Aid	Drummond Scientific Co.	P-76864
Pipette Tips (1–200 µL, 101–1000 µL)	Fisher Scinetific	2707509
Plastic Standard Disposable Transfer Pipettes	Fisher Scientific	13-711-9D
Plastic Weigh Boats (100 mL)	Amazon	mdo-azoc-1030
poly(ethylene glycol)-dithiol (PEG-diSH; 3.4 kDa)	Laysan Bio	SH-PEG-SH-3400-5g
Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]	Elsworth Adhesives	3097358-1004	Polydimethylsiloxane
Powder Free Examination Gloves	Quest	92897
Propidium iodide, 1 mg/mL aqueous soln.	Fisher Scientific	AAJ66584AB
RPMI-1640 Medium (1x)	HyClone	SH30027-02
Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm	Grace Bio-Labs	JTR-S-0.5
SOX2 Alexa Fluor 488	Santa Cruz Biotechnology	sc-365823
Tissue Culture Hood	NUAIRE	NU-425-600
Triethanolamine, ≥99.0% (GC)	Sigma Aldrich	90279
Trypsin 0.25% (1x)	Sigma Aldrich	SH30042.01
U-87 MG human glioblastoma cells	American Type Culture Collection	HTB-14