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

Fabricage gevolgd door 3D-inkapseling van tumorsferoïden in PEG-hydrogels

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.

Auteur in de schijnwerpers: in vitro hydrogelmodel voor onderzoek naar glioblastoom micro-omgeving

Fabricage en inkapseling van tumorsferoïden in polyethyleenglycolhydrogels voor het bestuderen van sferoïde-matrixinteracties

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.

Fixatie, kleuring en verwijdering van immunofluorescentie van ingekapselde sferoïden voor confocale microscopie

Zuig om te beginnen de media op uit de putjes waar de met hydrogel ingekapselde cellen worden gekweekt in de plaat met 24 putjes. Spoel de hydrogels door 500 microliter PBS rechtstreeks op elk putje met de hydrogels te pipetteren en zuig de PBS vervolgens voorzichtig op. Voeg met een pipet van 1.000 microliter 500 microliter fixeeroplossing per putje toe om de sferoïden in de plaat met 24 putjes te fixeren.Laat het fixeermiddel de gels 30 minuten op kamertemperatuur weken, voordat u de fixeeroplossing eruit pipettert en weggooit in een daarvoor bestemde afvalcontainer. Spoel vervolgens de hydrogels drie keer met PBS, zoals eerder is aangetoond. Als ze niet onmiddellijk worden gebruikt, bewaar de hydrogels dan maximaal een week in 500 microliter PBS per putje bij vier graden Celsius.Om de met hydrogel ingekapselde cellen te kleuren, bereidt u eerst op de juiste manier verdunde primaire antilichamen voor nestine en SOX2 voor. Voeg na het opzuigen van de PBS uit de putjes 50 microliter van de verdunde antilichamen toe aan elk putje. Incubeer de cellen met de antilichamen gedurende 24 uur om ze volledig te kleuren.Verwijder vervolgens met een pipet van 1.000 microliter de kleuroplossing en gooi het afval op de juiste manier weg. Nadat u de hydrogels drie keer hebt gespoeld, bewaart u ze maximaal twee weken voorafgaand aan de beeldvorming in PBS bij vier graden Celsius, of maakt u onmiddellijk beeld. Om de gekleurde sferoïde te verwijderen voor het verbeteren van de beeldtransparantie, zuigt u eerst de PBS uit elk putje op.Behandel de sferoïden vervolgens achtereenvolgens met 500 microliter van 20%40% en 80%formamide per putje gedurende elk 90 minuten. Incubeer ten slotte de hydrogels in 100% formamide gedurende 24 uur voorafgaand aan de beeldvorming. De sferoïden werden afgebeeld op verschillende z-stackdieptes, waardoor de levensvatbaarheid van de cel op elke locatie binnen de z-stack kon worden beoordeeld.Een maximale projectie met behulp van de sferoïde stapel vertegenwoordigde het hoogste punt van lichtintensiteit binnen elke locatie, vereenvoudigd tot één beeld. Representatieve beelden van gekleurde sferoïden onthulden dat de zelfvernieuwende transcriptiefactor, SOX2, co-gelokaliseerd was met DAPI in de kern. Integendeel, stamcelmarker nestine was in de cellen aanwezig.Er was geen verschil tussen de vrij zwevende sferoïde zonder gel en de ingekapselde sferoïden, mogelijk vanwege de inertie van de gebruikte PEG-gel. De representatieve beelden van optische secties op ongeveer 90 micron in geklaarde en niet-geklaarde sferoïden onthulden dat wanneer het opruimen werd uitgevoerd, de sferoïde kern ongeveer 30 micron dieper kon worden afgebeeld in vergelijking met een niet-geklaarde sferoïde.

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

Fabricage van vierkante piramidale microwells voor de fabricage van tumorsferoïden

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Meercellige tumorsferoïde vorming, oogst en inkapseling in hydrogels