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

PEGハイドロゲルにおける腫瘍スフェロイドの作製とそれに続く3Dカプセル化

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.

著者スポットライト:膠芽腫微小環境研究のためのin vitroハイドロゲルモデル

スフェロイド-マトリックス相互作用研究のためのポリエチレングリコールハイドロゲルにおける腫瘍スフェロイドの作製とカプセル化

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.

共焦点顕微鏡用のカプセル化スフェロイドの免疫蛍光固定、染色、および透明化(英語)

まず、ハイドロゲルでカプセル化した細胞を24ウェルプレートで培養するウェルから培地を吸引します。500マイクロリットルのPBSをヒドロゲルを含む各ウェルに直接ピペッティングしてハイドロゲルをすすぎ、PBSを静かに吸引します。1, 000 マイクロリットルのピペットを使用して、ウェルあたり 500 マイクロリットルの固定液を添加し、スフェロイドを 24 ウェルプレートに固定します。固定液を室温で30分間ゲルに浸してから、固定液をピペッティングして指定の廃液容器に廃棄します。次に、前述のように、ハイドロゲルをPBSで3回すすぎます。すぐに使用しない場合は、ハイドロゲルをウェルあたり500マイクロリットルのPBSに入れ、摂氏4度で最大1週間保管してください。ハイドロゲルでカプセル化した細胞で染色するには、まず、ネスチンおよびSOX2用の適切に希釈した一次抗体を調製します。ウェルからPBSを吸引した後、希釈した抗体を50マイクロリットル各ウェルに加えます。細胞を抗体と24時間インキュベートして、完全に染色します。次に、1, 000マイクロリットルのピペットを使用して、染色液を除去し、廃棄物を適切に廃棄します。ハイドロゲルを3回すすぎた後、イメージングまたはイメージングの前に、摂氏4度のPBSで最大2週間保存するか、すぐにイメージングします。染色されたスフェロイドを透明化してイメージングの透明性を向上させるには、まず各ウェルからPBSを吸引します。次に、スフェロイドをウェルあたり500マイクロリットルの20%40%および80%ホルムアミドでそれぞれ90分間順次処理します。最後に、イメージング前にハイドロゲルを100%ホルムアミド中で24時間インキュベートします。スフェロイドは、さまざまなzスタック深度でイメージングされ、zスタック内の各位置での細胞生存率の評価が可能になりました。回転楕円体スタックを利用した最大投影法は、各位置内の光強度の最高点を表し、1 つの画像に単純化されました。染色したスフェロイドの代表的な画像から、自己複製転写因子であるSOX2が核内でDAPIと共局在していることが明らかになりました。それどころか、幹細胞マーカーネスチンは細胞全体に存在していました。ゲルを含まない自由浮遊スフェロイドとカプセル化されたスフェロイドの間に違いはありませんでしたが、これはおそらく使用したPEGゲルの不活性によるものと考えられます。透明な回転楕円体と透明でない回転楕円体の約90ミクロンの光学切片の代表的な画像から、透明化を行うと、透明化されていない回転楕円体と比較して、回転楕円体コアを約30ミクロン深く画像化できることが明らかになりました。

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 ...

生物工学

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

腫瘍スフェロイド作製のための角錐型マイクロウェルの作製

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

多細胞腫瘍スフェロイドの形成、採取、およびハイドロゲルへのカプセル化