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

在 PEG 水凝胶中制备肿瘤球状体后进行 3D 封装

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.

作者聚焦：胶质母细胞瘤微环境研究的体外水凝胶模型

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

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.

用于共聚焦显微镜的包封球体的免疫荧光固定、染色和清除

首先，从在24孔板中培养水凝胶封装细胞的孔中吸出培养基。通过将 500 μL PBS 直接移液到每个含有水凝胶的孔上冲洗水凝胶，然后轻轻吸出 PBS。使用 1， 000 微升移液管，每孔加入 500 微升固定溶液，以将球状体固定在 24 孔板中。让固定剂在室温下浸泡凝胶 30 分钟，然后移出固定剂溶液并将其丢弃在指定的废物容器中。接下来，如前所述，用PBS冲洗水凝胶三次。如果不立即使用，则将水凝胶储存在每孔 500 微升 PBS 中，温度为 4 摄氏度，最多一周。要在水凝胶封装的细胞中染色，首先，制备适当稀释的巢蛋白和 SOX2 一抗。从孔中吸出PBS后，向每个孔中加入50微升稀释的抗体。将细胞与抗体一起孵育 24 小时以完全染色。然后使用1， 000微升移液管，除去染色溶液并适当丢弃废物。冲洗水凝胶三次后，在成像前将其储存在 4 摄氏度的 PBS 中长达两周，或立即成像。为了清除染色的球状体以提高成像透明度，首先从每个孔中抽吸PBS。然后依次用每孔 500 微升 20%40% 和 80% 甲酰胺处理球状体，每次 90 分钟。最后，在成像前将水凝胶在 100% 甲酰胺中孵育 24 小时。在不同的 z 堆栈深度下对球体进行成像，从而可以在 z 堆栈内的每个位置进行细胞活力评估。利用球体堆栈的最大投影表示每个位置内的最高光强度点，简化为一个图像。染色球体的代表性图像显示，自更新转录因子 SOX2 与细胞核中的 DAPI 共定位。相反，干细胞标记物巢蛋白存在于整个细胞中。没有凝胶的自由漂浮球体和封装的球体之间没有区别，这可能是由于所用PEG凝胶的惰性。在清除和未清除的球体中约90微米处的光学切片的代表性图像显示，当进行清除时，与未清除的球体相比，球体核心的成像深度约为30微米。