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

Tillverkning följt av 3D-inkapsling av tumörsfäroider 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.

Författare i fokus: In vitro hydrogelmodell för mikromiljöstudie av glioblastom

Tillverkning och inkapsling av tumörsfäroider i polyetylenglykolhydrogeler för studier av sfäroid-matrisinteraktioner

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.

Immunofluorescensfixering, färgning och rensning av inkapslade sfäroider för konfokalmikroskopi

Till att börja med aspirerar du mediet från brunnarna där de hydrogelinkapslade cellerna odlas i 24-hålsplattan. Skölj hydrogelerna genom att pipettera 500 mikroliter PBS direkt på varje brunn som innehåller hydrogelerna och aspirera sedan försiktigt PBS. Använd en 1 000 mikroliters pipett och tillsätt 500 mikroliter fixativ lösning per brunn för att fixera sfäroiderna i plattan med 24 hål.Låt fixeringsmedlet blötlägga gelerna i 30 minuter i rumstemperatur innan du pipetterar ut fixeringslösningen och slänger den i en avsedd avfallsbehållare. Skölj sedan hydrogelerna med PBS tre gånger som visats tidigare. Om hydrogelerna inte används omedelbart, förvara dem i 500 mikroliter PBS per brunn vid fyra grader Celsius i upp till en vecka.För att färga i de hydrogelinkapslade cellerna förbereder man först lämpligt utspädda primära antikroppar för nestin och SOX2. Efter att ha aspirerat PBS från brunnarna, tillsätt 50 mikroliter av de utspädda antikropparna till varje brunn. Inkubera cellerna med antikropparna i 24 timmar för att färga dem helt.Använd sedan en 1 000 mikroliters pipett, ta bort färglösningen och kassera avfallet på lämpligt sätt. Efter att ha sköljt hydrogelerna tre gånger, förvara dem i PBS vid fyra grader Celsius i upp till två veckor före avbildning, eller avbilda omedelbart. För att rensa den färgade sfäroiden för att förbättra bildtransparensen, aspirera först PBS från varje brunn.Behandla sedan sfäroiderna sekventiellt med 500 mikroliter 20 % 40 % och 80 % formamid per brunn i 90 minuter vardera. Inkubera slutligen hydrogelerna i 100 % formamid i 24 timmar före avbildning. Sfäroiderna avbildades på olika djup i z-stacken, vilket gjorde det möjligt att bedöma cellviabiliteten på varje plats i z-stacken.En maximal projektion med hjälp av sfäroidstapeln representerade den högsta punkten för ljusintensitet inom varje plats, förenklat till en bild. Representativa bilder av färgade sfäroider avslöjade att den självförnyande transkriptionsfaktorn, SOX2, var samlokaliserad med DAPI i kärnan. Tvärtom fanns stamcellsmarkören nestin i hela cellerna.Det fanns ingen skillnad mellan den fritt flytande sfäroiden utan gel och de inkapslade sfäroiderna, möjligen på grund av trögheten hos den använda PEG-gelen. De representativa bilderna av optiska snitt på cirka 90 mikrometer in i rensade och oclearade sfäroider avslöjade att när rensning utfördes kunde sfäroidkärnan avbildas cirka 30 mikrometer djupare jämfört med en orensad sfäroid.

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

Tillverkning av fyrkantiga pyramidala mikrobrunnar för tillverkning av tumörsfäroider

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Multicellulär tumörsfäroidbildning, skörd och inkapsling i hydrogeler