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

Fabricação seguida de encapsulamento 3D de esferoides tumorais em hidrogéis 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 em destaque: Modelo de hidrogel in vitro para estudo de microambiente de glioblastoma

Fabricação e encapsulamento de esferoides tumorais em hidrogéis de polietilenoglicol para estudo de interações esferóide-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.

Fixação por imunofluorescência, coloração e clareamento de esferoides encapsulados para microscopia confocal

Para começar, aspirar o meio dos poços onde as células encapsuladas em hidrogel são cultivadas na placa de 24 poços. Enxágue os hidrogéis pipetando 500 microlitros de PBS diretamente em cada poço contendo os hidrogéis e, em seguida, aspirar suavemente o PBS. Usando uma pipeta de 1.000 microlitros, adicione 500 microlitros de solução fixadora por poço para fixar os esferoides na placa de 24 poços.Deixe o fixador de molho por 30 minutos à temperatura ambiente, antes de pipetar a solução fixadora e descartá-la em um recipiente de resíduos designado. Em seguida, lave os hidrogéis com PBS três vezes, conforme demonstrado anteriormente. Se não for usado imediatamente, armazene os hidrogéis em 500 microlitros de PBS por poço a quatro graus Celsius por até uma semana.Para corar as células encapsuladas com hidrogel, primeiro, prepare anticorpos primários adequadamente diluídos para nestina e SOX2. Após aspirar o PBS dos poços, adicione 50 microlitros dos anticorpos diluídos a cada poço. Incubar as células com os anticorpos por 24 horas para corá-las completamente.Em seguida, usando uma pipeta de 1.000 microlitros, remova a solução corante e descarte os resíduos adequadamente. Depois de enxaguar os hidrogéis três vezes, armazene-os em PBS a quatro graus Celsius por até duas semanas antes da aquisição de imagens ou imagem imediatamente. Para limpar o esferoide corado para melhorar a transparência da imagem, primeiro aspirar o PBS de cada poço.Em seguida, tratar sequencialmente os esferoides com 500 microlitros de 20%40% e 80%formamida por poço por 90 minutos cada. Finalmente, incubar os hidrogéis em formamida a 100% por 24 horas antes da obtenção de imagens. Os esferoides foram fotografados em diferentes profundidades de z-stack, permitindo a avaliação da viabilidade celular em cada local dentro da z-stack.Uma projeção máxima utilizando a pilha esferoide representou o ponto mais alto de intensidade de luz dentro de cada local, simplificado em uma imagem. Imagens representativas de esferoides corados revelaram que o fator de transcrição de auto-renovação, SOX2, foi co-localizado com DAPI no núcleo. Ao contrário, o marcador de células-tronco nestina estava presente em todas as células.Não houve diferença entre o esferoide livre flutuante sem gel e os esferoides encapsulados, possivelmente devido à inércia do gel de PEG utilizado. As imagens representativas de seções ópticas a cerca de 90 mícrons em esferoides limpos e não limpos revelaram que, quando a limpeza era realizada, o núcleo esferoide podia ser fotografado cerca de 30 mícrons mais profundo em comparação com um esferoide não limpo.

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 ...

Bioengenharia

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

Fabricação de Micropoços Piramidais Quadrados para Fabricação de Esferoides Tumorais

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

Formação, Colheita e Encapsulamento de Esferoides Tumorais Multicelulares em Hidrogéis