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

Fabricación seguida de encapsulación 3D de esferoides tumorales en hidrogeles 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.

Autor destacado: Modelo de hidrogel in vitro para estudio de microambiente de glioblastoma

Fabricación y encapsulación de esferoides tumorales en hidrogeles de polietilenglicol para el estudio de las interacciones esferoide-matriz

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.

Fijación, tinción y eliminación por inmunofluorescencia de esferoides encapsulados para microscopía confocal

Para comenzar, aspire los medios de los pocillos donde se cultivan las células encapsuladas en hidrogel en la placa de 24 pocillos. Enjuague los hidrogeles pipeteando 500 microlitros de PBS directamente en cada pocillo que contenga los hidrogeles y luego aspire suavemente el PBS. Con una pipeta de 1.000 microlitros, añada 500 microlitros de solución fijadora por pocillo para fijar los esferoides en la placa de 24 pocillos.Deje que el fijador remoje los geles durante 30 minutos a temperatura ambiente, antes de pipetear la solución fijadora y desecharla en un contenedor de residuos designado. A continuación, enjuague los hidrogeles con PBS tres veces como se demostró anteriormente. Si no se usan inmediatamente, almacene los hidrogeles en 500 microlitros de PBS por pocillo a cuatro grados centígrados hasta por una semana.Para teñir las células encapsuladas en hidrogel, primero prepare anticuerpos primarios adecuadamente diluidos para la nestina y SOX2. Después de aspirar el PBS de los pocillos, agregue 50 microlitros de los anticuerpos diluidos a cada pocillo. Incubar las células con los anticuerpos durante 24 horas para teñirlas por completo.A continuación, con una pipeta de 1.000 microlitros, retire la solución de tinción y deseche los residuos de forma adecuada. Después de enjuagar los hidrogeles tres veces, guárdelos en PBS a cuatro grados centígrados hasta dos semanas antes de la obtención de imágenes, o tome imágenes inmediatamente. Para eliminar el esferoide teñido y mejorar la transparencia de las imágenes, primero aspire el PBS de cada pocillo.A continuación, trate secuencialmente los esferoides con 500 microlitros de formamida al 20%, 40% y 80% por pocillo durante 90 minutos cada uno. Finalmente, incubar los hidrogeles en formamida al 100% durante 24 horas antes de la toma de imágenes. Los esferoides se obtuvieron imágenes a diferentes profundidades de la pila z, lo que permitió la evaluación de la viabilidad de la celda en cada ubicación dentro de la pila z.Una proyección máxima utilizando la pila de esferoides representaba el punto más alto de intensidad de luz dentro de cada ubicación, simplificado en una sola imagen. Las imágenes representativas de los esferoides teñidos revelaron que el factor de transcripción de autorrenovación, SOX2, estaba colocalizado con DAPI en el núcleo. Por el contrario, el marcador de células madre nestina estaba presente en todas las células.No hubo diferencia entre el esferoide de flotación libre sin gel y los esferoides encapsulados, posiblemente debido a la inercia del gel PEG utilizado. Las imágenes representativas de secciones ópticas a unas 90 micras en esferoides aclarados y no aclarados revelaron que cuando se realizaba el aclarado, el núcleo del esferoide podía obtener imágenes a unas 30 micras más profundas en comparación con un esferoide no aclarado.

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 ...

Investigación

Bioingeniería

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

Fabricación de micropocillos piramidales cuadrados para la fabricación de esferoides tumorales

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Formación, recolección y encapsulación de esferoides tumorales multicelulares en hidrogeles