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

PEG 하이드로겔에서 종양 스페로이드의 3D 캡슐화 후

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.

저자 스포트라이트: 교모세포종 미세환경 연구를 위한 체외 하이드로겔 모델

스페로이드-매트릭스 상호 작용 연구를 위한 폴리에틸렌 글리콜 하이드로겔의 종양 스페로이드 제조 및 캡슐화

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(공초점 현미경을 위한 캡슐화된 스페로이드의 면역형광 고정, 염색 및 투명화)

시작하려면 하이드로겔 캡슐화된 세포가 24웰 플레이트에서 배양되는 웰에서 배지를 흡입합니다. 하이드로겔이 들어 있는 각 웰에 500마이크로리터의 PBS를 직접 피펫팅하여 하이드로겔을 헹구고 PBS를 부드럽게 흡입합니다. 1, 000 마이크로리터 피펫을 사용하여, 웰당 500 마이크로리터의 고정 용액을 추가하여 24웰 플레이트에 스페로이드를 고정합니다.정착액을 실온에서 30분 동안 담가두었다가 정착액을 피펫팅하여 지정된 폐기물 용기에 버리십시오. 다음으로, 이전에 설명한 대로 PBS로 하이드로겔을 세 번 헹굽니다. 즉시 사용하지 않을 경우 하이드로겔을 섭씨 4도에서 웰당 500마이크로리터의 PBS에 최대 1주일 동안 보관하십시오.하이드로겔로 캡슐화된 세포를 염색하려면 먼저 nestin 및 SOX2에 대해 적절하게 희석된 1차 항체를 준비합니다. 웰에서 PBS를 흡입한 후 각 웰에 희석된 항체 50마이크로리터를 추가합니다. 항체로 세포를 24시간 동안 배양하여 완전히 염색합니다.그런 다음 1, 000 마이크로 리터 피펫을 사용하여 염색 용액을 제거하고 폐기물을 적절하게 폐기하십시오. 하이드로겔을 세 번 헹군 후 이미징 또는 즉시 이미징 전에 섭씨 4도의 PBS에 최대 2주 동안 보관하십시오. 이미징 투명도를 개선하기 위해 염색된 스페로이드를 제거하려면 먼저 각 웰에서 PBS를 흡입합니다.그런 다음 스페로이드를 웰당 500마이크로리터의 20%40% 및 80%포름아미드로 각각 90분 동안 순차적으로 처리합니다. 마지막으로 이미징 전 24시간 동안 100% 포름아미드에 하이드로겔을 배양합니다. 스페로이드는 다양한 z-stack 깊이에서 이미지화되어 z-stack 내의 각 위치에서 세포 생존율을 평가할 수 있었습니다.스페로이드 스택을 활용한 최대 투영은 각 위치 내에서 가장 높은 빛의 강도를 나타내며 하나의 이미지로 단순화되었습니다. 염색된 스페로이드의 대표 이미지는 자가 재생 전사 인자인 SOX2가 핵에서 DAPI와 함께 국소화되어 있음을 보여주었습니다. 반대로, 줄기세포 마커 네스틴은 세포 전체에 존재했습니다.겔이 없는 자유 부유 스페로이드와 캡슐화된 스페로이드 사이에는 차이가 없었는데, 이는 아마도 사용된 PEG 겔의 불활성 때문일 수 있습니다. 약 90미크론에서 투명화된 스페로이드와 투명화되지 않은 스페로이드로 나뉜 광학 절편의 대표 이미지는 투명화가 수행될 때 투명화되지 않은 스페로이드에 비해 스페로이드 코어를 약 30미크론 더 깊게 이미징할 수 있음을 보여주었습니다.

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 ...

연구

생체공학

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

종양 스페로이드 제작을 위한 정사각형 피라미드형 마이크로웰 제작

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

하이드로겔의 다세포 종양 스페로이드 형성, 수확 및 캡슐화