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

Fabrication Followed by 3D Encapsulation of Tumor Spheroids 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.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

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.

Immunofluorescence Fixation, Staining, and Clearing of Encapsulated Spheroids for Confocal Microscopy

To begin, aspirate the media from the wells where the hydrogel-encapsulated cells are cultured in the 24-well plate. Rinse the hydrogels by pipetting 500 microliters of PBS directly onto each well containing the hydrogels, and then gently aspirate the PBS. Using a 1, 000 microliter pipette, add 500 microliters of fixative solution per well to fix the spheroids in the 24-well plate.Allow the fixative to soak the gels for 30 minutes at room temperature, before pipetting out the fixative solution and discarding it in a designated waste container. Next, rinse the hydrogels with PBS three times as demonstrated previously. If not used immediately, store the hydrogels in 500 microliters of PBS per well at four degrees Celsius for up to one week.To stain in the hydrogel-encapsulated cells, first, prepare appropriately diluted primary antibodies for nestin and SOX2. After aspirating the PBS from the wells, add 50 microliters of the diluted antibodies to each well. Incubate the cells with the antibodies for 24 hours to stain them completely.Then using a 1, 000 microliter pipette, remove the staining solution and discard the waste appropriately. After rinsing the hydrogels three times, store them in PBS at four degrees Celsius for up to two weeks prior to imaging, or image immediately. To clear the stained spheroid for improving imaging transparency, first aspirate the PBS from each well.Then sequentially treat the spheroids with 500 microliters of 20%40%and 80%formamide per well for 90 minutes each. Finally incubate the hydrogels in 100%formamide for 24 hours prior to imaging. The spheroids were imaged at varying z-stack depths, allowing for the cell viability assessment at each location within the z-stack.A maximum projection utilizing the spheroid stack represented the highest point of light intensity within each location, simplified into one image. Representative images of stained spheroids revealed that the self-renewal transcription factor, SOX2, was co-localized with DAPI in the nucleus. On the contrary, stem cell marker nestin was present throughout the cells.There was no difference between the free floating spheroid without gel and the encapsulated spheroids, possibly due to the inertness of the PEG gel used. The representative images of optical sections at about 90 microns into cleared and uncleared spheroids revealed that when clearing was performed, the spheroid core could be imaged about 30 microns deeper compared to an uncleared spheroid.

immunofluorescence-fixation-staining-clearing-encapsulated-spheroids

Research

JoVE Journal

Bioengineering

92 vistas.  Saint Louis University. 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 tem...