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

Fabbricazione seguita da incapsulamento 3D di sferoidi tumorali in idrogel PEG

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.

Autore in primo piano: Modello in vitro di idrogel per lo studio del microambiente del glioblastoma

Fabbricazione e incapsulamento di sferoidi tumorali in idrogel di polietilenglicole per lo studio delle interazioni sferoide-matrice

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.

Fissazione dell'immunofluorescenza, colorazione e pulizia di sferoidi incapsulati per microscopia confocale

Per iniziare, aspirare il terreno dai pozzetti in cui vengono coltivate le cellule incapsulate in idrogel nella piastra a 24 pozzetti. Sciacquare gli idrogel pipettando 500 microlitri di PBS direttamente su ciascun pozzetto contenente gli idrogel, quindi aspirare delicatamente il PBS. Utilizzando una pipetta da 1.000 microlitri, aggiungere 500 microlitri di soluzione fissativa per pozzetto per fissare gli sferoidi nella piastra a 24 pozzetti.Lasciare in ammollo i gel per 30 minuti a temperatura ambiente, prima di pipettare la soluzione fissativa e gettarla in un apposito contenitore per rifiuti. Successivamente, sciacquare gli idrogel con PBS tre volte come dimostrato in precedenza. Se non utilizzati immediatamente, conservare gli idrogel in 500 microlitri di PBS per pozzetto a quattro gradi Celsius per un massimo di una settimana.Per colorare le cellule incapsulate in idrogel, preparare prima gli anticorpi primari opportunamente diluiti per la nestina e SOX2. Dopo aver aspirato il PBS dai pozzetti, aggiungere 50 microlitri di anticorpi diluiti a ciascun pozzetto. Incubare le cellule con gli anticorpi per 24 ore per colorarle completamente.Quindi, utilizzando una pipetta da 1.000 microlitri, rimuovere la soluzione colorante e gettare i rifiuti in modo appropriato. Dopo aver risciacquato gli idrogel tre volte, conservarli in PBS a quattro gradi Celsius per un massimo di due settimane prima dell'imaging o creare immediatamente un'immagine. Per eliminare lo sferoide colorato e migliorare la trasparenza dell'imaging, aspirare prima il PBS da ciascun pozzetto.Quindi trattare in sequenza gli sferoidi con 500 microlitri di 20%40% e 80%formamide per pozzetto per 90 minuti ciascuno. Infine, incubare gli idrogel in 100% formammide per 24 ore prima dell'imaging. Gli sferoidi sono stati sottoposti a imaging a diverse profondità dello z-stack, consentendo la valutazione della vitalità cellulare in ogni posizione all'interno dello z-stack.Una proiezione massima che utilizza la pila di sferoidi rappresentava il punto più alto di intensità luminosa all'interno di ogni posizione, semplificato in un'unica immagine. Immagini rappresentative di sferoidi colorati hanno rivelato che il fattore di trascrizione di auto-rinnovamento, SOX2, era co-localizzato con DAPI nel nucleo. Al contrario, il marcatore delle cellule staminali nestina era presente in tutte le cellule.Non c'era differenza tra lo sferoide fluttuante senza gel e gli sferoidi incapsulati, probabilmente a causa dell'inerzia del gel PEG utilizzato. Le immagini rappresentative delle sezioni ottiche a circa 90 micron in sferoidi chiari e non chiariti hanno rivelato che quando è stato eseguito il clearing, il nucleo sferoidale poteva essere ripreso circa 30 micron più in profondità rispetto a uno sferoide non chiarito.

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 ...

Ricerca

Bioingegneria

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

Fabbricazione di micropozzetti piramidali quadrati per la fabbricazione di sferoidi tumorali

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Formazione, raccolta e incapsulamento di sferoidi tumorali multicellulari in idrogel