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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 encapsulated3,

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.......

Representative Results

Spheroid-based drug screening platforms to study chemotherapeutic effects are increasingly sought after due to the emphasis on modulating the tumor microenvironment upon spheroid encapsulation in biomaterials replicating native tissue. Here we developed a method for multicellular tumor spheroid preparation and subsequent encapsulation and imaging in a 3D hydrogel. The spheroids are prepared in microwell molds (Figure 3A,B), which result in spheroids with spherical shapes and.......

Discussion

Hydrogel-based multicellular tumor spheroid models are increasingly being developed to advance cancer therapeutic discoveries11,13,29. 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.......

Acknowledgements

This work was funded by start-up funds provided to Dr. Silviya P Zustiak by Saint Louis University as well as by a seed grant from the Henry and Amelia Nasrallah Center for Neuroscience at Saint Louis University awarded to Dr. Silviya P Zustiak.

....

Materials

NameCompanyCatalog NumberComments
70% EthanolFisher Scientific LC22210-4
15 mL ConicalsFALCON352097
24-Well Plate Ultra Low Attachment platesFisher Scientific07-200-602
35 mm Petri DishAmazon706011
4-arm poly(ethylene glycol)-acrylate (4-arm PEG-Ac; 10 kDa)Laysan BioACRL-PEG-ACRL-10K-5g
50 mL ConicalsFisher Scinetific3181345107
6-well AggreWell 400 StemCell Technologies, Vancouver, Canada34421Square pyramidal microwells 
anti-adherence rinsing solutionStemCell Technologies, Vancouver, CanadaCat #: 07010
Aspartic Acid-Arginine-Cysteine-Glycine-Valine-Proline-Methionine-Serine-Methionine-Arginine-Glycine-Cysteine-Arginine- Aspartic Acid (DRCG-VPMSMR-GCRD) peptideGenic Bio, Shanghai, Chinan/aCustom synthesis
Chemical Fume HoodKEWAUNEE99151
Corning Matrigel Basement Membrane Matrix, LDEV Free Corning356234Basement membrane matrix
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)Thermo Scientific62247
Detergent - Triton-XSigma AldrichT8787Nonionic surfactant
Dimethyl sulfoxide (DMSO)Fisher Scientific BP231-100
Disposable Pipettes (1 mL, 2 mL, 5 mL, 10 mL, 25 mL, 50 mL)Fisher Scinetific1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F
Fetal Bovine SerumHyCloneSH30073-03
Formaldehyde 37% SolutionSigma AldrichF1635
Glass PlatesSlumpysGBS4100SFSL
Glass Transfer PipettesFisher Scinetific5 3/4": 1367820A, 9":136786B
Glycine-Arginine-Cysteine-Aspartic Acid-Arginine-Glycine-Aspartic Acid-Serine (GRCD-RGDS) peptideGenic Bio, Shanghai, Chinan/aCustom synthesis
HemacytometerBright-Line383684
Hydrophobic solution - Repel Silane GE Healthcare Bio-Sciences17-1332-01
IncubatorNUAIRENU-8500
Inverted Microscope (Axiovert 25)Zeiss663526
Invitrogen DiOC16(3) (3,3'-Dihexadecyloxacarbocyanine Perchlorate)Fisher Scientific D1125
Leica Confocal SP8Leica Microsystems Inc.
Light and Flourescent Microscope (Axiovert 200M)Zeiss3820005619
Micro centrifuge tubesFisher Scientific2 mL: 02681258
Microscope SoftwareZeissAxioVision Rel. 4.8.2
Nestin Alexa Fluor 594 Santa Cruz Biotechnologysc-23927
ParafilmPARAFILM PM992
PBS (1x), pH 7.4HyCloneSH30256.01
Penicillin StreptomycinMP Biomedicals1670046
Pipette AidDrummond Scientific Co.P-76864
Pipette Tips (1–200 µL, 101–1000 µL)Fisher Scinetific2707509
Plastic Standard Disposable Transfer PipettesFisher Scientific13-711-9D
Plastic Weigh Boats (100 mL)Amazon mdo-azoc-1030
poly(ethylene glycol)-dithiol (PEG-diSH; 3.4 kDa)Laysan BioSH-PEG-SH-3400-5g
Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]Elsworth Adhesives3097358-1004Polydimethylsiloxane
Powder Free Examination GlovesQuest92897
Propidium iodide, 1 mg/mL aqueous soln. Fisher Scientific AAJ66584AB
RPMI-1640 Medium (1x)HyCloneSH30027-02
Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cmGrace Bio-LabsJTR-S-0.5
SOX2 Alexa Fluor 488 Santa Cruz Biotechnologysc-365823
Tissue Culture HoodNUAIRENU-425-600
Triethanolamine, ≥99.0% (GC) Sigma Aldrich90279
Trypsin 0.25% (1x) Sigma AldrichSH30042.01
U-87 MG human glioblastoma cellsAmerican Type Culture Collection HTB-14

References

  1. Hirschhaeuser, F., et al. Multicellular tumor spheroids: an underestimated tool is catching up again. Journal of Biotechnology. 148 (1), 3-15 (2010).
  2. Costa, E. C., de Melo-Diogo, D., Moreira, A. F., Carvalho, M. P., Correia, I. J.

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Tumor SpheroidPolyethylene Glycol HydrogelsTumor Extracellular MicroenvironmentGlioblastoma3D EncapsulationIn Vitro ModelMicrowell MoldsSpheroid FabricationHydrogel PropertiesConfocal Microscopy

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