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

Herstellung mit anschließender 3D-Verkapselung von Tumorsphäroiden in PEG-Hydrogelen

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.

Autor im Rampenlicht: In-vitro-Hydrogel-Modell für Glioblastom-Mikroumgebungsstudie

Herstellung und Verkapselung von Tumorsphäroiden in Polyethylenglykol-Hydrogelen zur Untersuchung von Sphäroid-Matrix-Wechselwirkungen

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.

Immunfluoreszenzfixierung, -färbung und -reinigung von verkapselten Sphäroiden für die konfokale Mikroskopie

Saugen Sie zunächst das Medium aus den Wells an, in denen die Hydrogel-verkapselten Zellen in der 24-Well-Platte kultiviert werden. Spülen Sie die Hydrogele, indem Sie 500 Mikroliter PBS direkt in jede Vertiefung mit den Hydrogelen pipettieren und dann das PBS vorsichtig absaugen. Fügen Sie mit einer 1.000-Mikroliter-Pipette 500 Mikroliter Fixierlösung pro Vertiefung hinzu, um die Sphäroide in der 24-Well-Platte zu fixieren.Lassen Sie das Fixiermittel die Gele 30 Minuten lang bei Raumtemperatur einweichen, bevor Sie die Fixierlösung herauspipettieren und in einem dafür vorgesehenen Abfallbehälter entsorgen. Spülen Sie anschließend die Hydrogele dreimal mit PBS, wie zuvor gezeigt. Wenn Sie die Hydrogele nicht sofort verwenden, lagern Sie sie in 500 Mikrolitern PBS pro Vertiefung bei vier Grad Celsius bis zu einer Woche.Um in den Hydrogel-verkapselten Zellen zu färben, bereiten Sie zunächst entsprechend verdünnte Primärantikörper für Nestin und SOX2 vor. Nachdem Sie das PBS aus den Vertiefungen abgesaugt haben, geben Sie 50 Mikroliter der verdünnten Antikörper in jede Vertiefung. Inkubieren Sie die Zellen 24 Stunden lang mit den Antikörpern, um sie vollständig zu färben.Entfernen Sie dann mit einer 1.000-Mikroliter-Pipette die Färbelösung und entsorgen Sie den Abfall entsprechend. Nachdem Sie die Hydrogele dreimal gespült haben, lagern Sie sie vor der Bildgebung bis zu zwei Wochen lang bei vier Grad Celsius in PBS oder bilden Sie sie sofort ab. Um das gefärbte Sphäroid zur Verbesserung der Bildtransparenz zu entfernen, saugen Sie zuerst das PBS aus jeder Vertiefung ab.Behandeln Sie dann die Sphäroide nacheinander mit 500 Mikrolitern 20%40% und 80% Formamid pro Vertiefung für jeweils 90 Minuten. Schließlich inkubieren Sie die Hydrogele vor der Bildgebung 24 Stunden lang in 100%igem Formamid. Die Sphäroide wurden in unterschiedlichen Z-Stapel-Tiefen abgebildet, was die Bewertung der Zellviabilität an jeder Stelle innerhalb des Z-Stapels ermöglichte.Eine maximale Projektion unter Verwendung des Sphäroidstapels stellte den höchsten Punkt der Lichtintensität an jedem Ort dar, vereinfacht in einem Bild. Repräsentative Bilder von gefärbten Sphäroiden zeigten, dass der Transkriptionsfaktor SOX2 zur Selbsterneuerung mit DAPI im Zellkern kolokalisiert war. Im Gegenteil, Stammzellmarker Nestin war überall in den Zellen vorhanden.Es gab keinen Unterschied zwischen dem frei schwebenden Sphäroid ohne Gel und den verkapselten Sphäroiden, möglicherweise aufgrund der Inertheit des verwendeten PEG-Gels. Die repräsentativen Bilder von optischen Schnitten bei etwa 90 Mikrometern in geklärten und ungeklärten Sphäroiden zeigten, dass der Sphäroidkern beim Clearing etwa 30 Mikrometer tiefer abgebildet werden konnte als ein ungeklärtes Sphäroid.

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 ...

Forschung

Biotechnik

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

Herstellung von quadratischen pyramidalen Mikrowells für die Herstellung von Tumorsphäroiden

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Bildung, Ernte und Verkapselung von multizellulären Tumorsphäroiden in Hydrogelen