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

Fabrikasyon ve ardından tümör sferoidlerinin PEG hidrojellerinde 3D kapsüllenmesi

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.

Yazar Spotlight: Glioblastoma Mikroçevre Çalışması için İn Vitro Hidrojel Modeli

Sferoid-Matriks Etkileşimlerini İncelemek için Polietilen Glikol Hidrojellerde Tümör Sferoid Üretimi ve Kapsüllenmesi

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.

Konfokal mikroskopi için immünofloresan fiksasyonu, boyanması ve kapsüllenmiş sferoidlerin temizlenmesi

Başlamak için, hidrojel kapsüllü hücrelerin 24 oyuklu plakada kültürlendiği oyuklardan ortamı aspire edin. 500 mikrolitre PBS'yi doğrudan hidrojelleri içeren her bir oyuğa pipetleyerek hidrojelleri durulayın ve ardından PBS'yi nazikçe aspire edin. 1.000 mikrolitrelik bir pipet kullanarak, 24 oyuklu plakadaki sferoidleri sabitlemek için oyuk başına 500 mikrolitre fiksatif çözelti ekleyin.Fiksatif solüsyonu pipetlemeden ve belirlenmiş bir atık kabına atmadan önce fiksatifin jelleri oda sıcaklığında 30 dakika ıslatmasına izin verin. Daha sonra, hidrojelleri daha önce gösterildiği gibi PBS ile üç kez durulayın. Hemen kullanılmazsa, hidrojelleri bir haftaya kadar dört santigrat derecede oyuk başına 500 mikrolitre PBS'de saklayın.Hidrojel kapsüllü hücrelerde boyama yapmak için önce nestin ve SOX2 için uygun şekilde seyreltilmiş primer antikorlar hazırlayın. PBS'yi kuyucuklardan aspire ettikten sonra, her bir oyuğa 50 mikrolitre seyreltilmiş antikor ekleyin. Hücreleri tamamen boyamak için 24 saat boyunca antikorlarla inkübe edin.Ardından 1.000 mikrolitrelik bir pipet kullanarak boyama solüsyonunu çıkarın ve atıkları uygun şekilde atın. Hidrojelleri üç kez duruladıktan sonra, görüntülemeden veya hemen görüntülemeden önce iki haftaya kadar dört santigrat derecede PBS'de saklayın. Görüntüleme şeffaflığını artırmak için lekeli sferoidi temizlemek için, önce PBS'yi her bir oyuktan aspire edin.Daha sonra sferoidlere her biri 90 dakika boyunca oyuk başına 500 mikrolitre% 20% 40 ve% 80 formamid ile sırayla muamele edin. Son olarak, görüntülemeden önce hidrojelleri 24 saat boyunca %100 formamid içinde inkübe edin. Sferoidler, değişen z-yığını derinliklerinde görüntülendi ve z-yığını içindeki her yerde hücre canlılığı değerlendirmesine izin verildi.Küresel yığını kullanan maksimum projeksiyon, her konumdaki en yüksek ışık yoğunluğu noktasını temsil etti ve tek bir görüntüde basitleştirildi. Boyanmış sferoidlerin temsili görüntüleri, kendini yenileyen transkripsiyon faktörü SOX2'nin çekirdekte DAPI ile birlikte lokalize olduğunu ortaya koydu. Aksine, kök hücre işaretleyici nestin hücrelerin tamamında mevcuttu.Jelsiz serbest yüzen sferoid ile kapsüllenmiş sferoidler arasında, muhtemelen kullanılan PEG jelinin eylemsizliğinden dolayı hiçbir fark yoktu. Optik kesitlerin yaklaşık 90 mikronluk temizlenmiş ve temizlenmemiş sferoidlere temsili görüntüleri, temizleme yapıldığında, sferoid çekirdeğin, temizlenmemiş bir sferoide kıyasla yaklaşık 30 mikron daha derin görüntülenebildiğini ortaya koydu.