A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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,
1. Solutions preparation
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.......
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.......
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.
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved