immunofluorescence fixation, staining, and clearing of encapsulated spheroids for confocal microscopy

Fabrikasjon etterfulgt av 3D-innkapsling av tumorsfæroider i PEG-hydrogeler

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 encapsulated3,4. Such hydrogel-based spheroid models enable the elucidation of cell-cell and cell-matrix interactions on various cell behaviors, such as viability, proliferation, stemness, or therapy responsiveness3.
Here, we describe a protocol for the encapsulation of glioblastoma spheroids in polyethylene glycol (PEG) hydrogels. There are multiple literature reports of glioblastoma cell spheroid encapsulation in hydrogels. For example, spheroids were formed by encapsulating U87 cells in PEG hydrogels decorated with an RGDS adhesive ligand and crosslinked with an enzymatically cleavable peptide to determine the effect of hydrogel stiffness on cell behavior5. U87 cells have also been formed in other PEG-based or hyaluronic acid-based hydrogels to expand the cancer stem cell population6 or to explore matrix-mediated mechanisms of chemotherapy resistance7,8,9. Glioblastoma spheroids have also been encapsulated in gelatin hydrogels to study the crosstalk between microglia and cancer cells and its effect on cell invasion10. Overall, such studies have demonstrated the utility of hydrogel-based in vitro models in understanding glioblastoma pathology and devising treatments.
Further, there are different methods for tumor spheroid fabrication and hydrogel encapsulation11. For example, dispersed cells could be seeded in hydrogels and allowed to form spheroids over time5,12. One drawback of such a method is the polydispersity of the formed spheroids, which could lead to differential cell responses. To produce uniform spheroids, cells could be encapsulated in microgels and cultured for extended periods until they invade and remodel the gel13, or cells could be deposited in templated gels with spherical &#39;holes&#39; and allowed to aggregate14. The drawback of these methods is their relative complexity, the need for a droplet generator or other means to form microgels or the &#39;holes&#39; in the gel, and the time it takes for spheroids to grow and mature. Alternatively, spheroids could be pre-formed in microwells9,15,16 or in hanging-drop plates17,18 and then encapsulated in a hydrogel, similar to the technique described here. These methods are simpler and can be done in a higher throughput fashion. Interestingly, it has been shown that the method of spheroid formation can affect spheroid cell behaviors, such as gene expression, cell proliferation, or drug responsiveness19,20.
Here, we focus on glioblastoma since it is a solid tumor whose native environment is the soft, nanoporous brain matrix21, which can be mimicked by a soft, nanoporous hydrogel. Glioblastoma is also the deadliest brain cancer for which there is no available cure22. However, the protocol described here can be used for the encapsulation of spheroids representing any solid tumor. We chose to use PEG hydrogels that are formed through a Michael-type addition reaction23. PEG is a synthetic, non-degradable, and biocompatible hydrogel that is inert and serves as scaffolding and physical cell support but does not support cell attachment23. Cell adhesiveness can be added separately via tethering of whole proteins or adhesive ligands24, and degradability can be added via chemical modifications of the PEG polymer chain or hydrolytically or enzymatically degradable crosslinkers25,26. This allows for biochemical properties to be tuned independently of mechanical or physical hydrogel properties, which could be advantageous in studying cell-matrix interactions. The Michael-type gelation chemistry is selective and happens at physiological conditions; hence, it allows for spheroid encapsulation by simply mixing the spheroids with the hydrogel precursor solution.
Overall, the methodology presented here has several notable characteristics. First, fabricating tumor spheroids in a multiwell assembly is efficient, quick, and the cost of the required materials is low. Second, the spheroids are produced in large batches in a variety of sizes with low polydispersity. Finally, only commercially available materials are required. The utility of the methodology is illustrated by exploring the effect of substrate properties on spheroid cell viability, circularity, and cell stemness.

Forfatter i søkelyset: In vitro hydrogelmodell for glioblastom-mikromiljøstudie

Tumor sfæroid fabrikasjon og innkapsling i polyetylenglykolhydrogeler for å studere sfæroid-matriksinteraksjoner

Our lab focuses on designing in vitro hydrogel models for glioblastoma steroid encapsulation to mimic the tumor extracellular microenvironment. The effective substrate, mechanical and biochemical properties on steroid cell viability, circularity, stemness, and spreading are investigated with such models. Current experimental challenges in tumor module development revolve around a desire for complexity to mimic all aspects of the tumor microenvironment for physiological relevance, and also a desire for simplicity to make the model accessible, robust, low cost, and easy to use, and compatible with multiplex screening approaches.We present a protocol that enables fast, robust, and low-cost fabrication of tumor steroids and encapsulation in hydrogels made to mimic the tumor microenvironment. The model does not require any specialized equipment. It will be particularly useful for exploring steroid matrix interactions in building in vitro tissue physiology or pathology models.Understanding the interactions between glioblastoma cancer cells and their microenvironment can encourage development of new therapies to better treat this cancer. Furthermore, development of an in vitro model for glioblastoma cancer can enhance development of this model for other cancer diseases.

Immunfluorescensfiksering, farging og rydding av innkapslede sfæroider for konfokalmikroskopi

For å begynne, aspirer mediet fra brønnene der hydrogel-innkapslede celler dyrkes i 24-brønnplaten. Skyll hydrogelene ved å pipetere 500 mikroliter PBS direkte på hver brønn som inneholder hydrogelene, og aspirer deretter PBS forsiktig. Bruk en 1.000 mikroliter pipette, tilsett 500 mikroliter fikseringsløsning per brønn for å fikse sfæroidene i 24-brønnplaten.La fikseringsmiddelet bløtlegge gelene i 30 minutter ved romtemperatur, før du pipetterer ut fikseringsløsningen og kaster den i en egnet avfallsbeholder. Skyll deretter hydrogelene med PBS tre ganger som vist tidligere. Hvis de ikke brukes umiddelbart, må hydrogelene oppbevares i 500 mikroliter PBS per brønn ved fire grader Celsius i opptil en uke.For å farge i hydrogel-innkapslede celler, må du først forberede passende fortynnede primære antistoffer for nestin og SOX2. Etter å ha aspirert PBS fra brønnene, tilsett 50 mikroliter av de fortynnede antistoffene til hver brønn. Inkuber cellene med antistoffene i 24 timer for å flekke dem helt.Bruk deretter en 1.000 mikroliter pipette, fjern fargeløsningen og kast avfallet på riktig måte. Etter å ha skyllet hydrogelene tre ganger, oppbevar dem i PBS ved fire grader Celsius i opptil to uker før avbildning, eller bilde umiddelbart. For å fjerne den fargede sfæroiden for å forbedre bildegjennomsiktigheten, må du først aspirere PBS fra hver brønn.Deretter behandles sfæroidene sekvensielt med 500 mikroliter 20% 40% og 80% formamid per brønn i 90 minutter hver. Til slutt inkuber hydrogelene i 100% formamid i 24 timer før avbildning. Sfæroidene ble avbildet på varierende z-stack dybder, noe som muliggjorde cellens levedyktighetsvurdering på hvert sted i z-stakken.En maksimal projeksjon ved hjelp av sfæroidstakken representerte det høyeste punktet med lysintensitet på hvert sted, forenklet til ett bilde. Representative bilder av fargede sfæroider avslørte at den selvfornyelsestranskripsjonsfaktoren, SOX2, var samlokalisert med DAPI i kjernen. Tvert imot var stamcellemarkøren nestin tilstede i hele cellene.Det var ingen forskjell mellom den frittflytende sfæroiden uten gel og de innkapslede sfæroidene, muligens på grunn av inertiteten til PEG-gelen som ble brukt. De representative bildene av optiske seksjoner ved ca. 90 mikron i ryddede og uklare sfæroider viste at når clearing ble utført, kunne sfæroidkjernen avbildes omtrent 30 mikron dypere sammenlignet med en uryddet sfæroid.

Research

JoVE Journal

Bioengineering

Immunofluorescence Fixation, Staining, and Clearing of Encapsulated Spheroids for Confocal Microscopy

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

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

Fabrikasjon av kvadratpyramidale mikrobrønner for tumor sfæroid fabrikasjon

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Multicellulær tumor sfæroid dannelse, høsting og innkapsling i hydrogeler