fabrication of square pyramidal microwells for tumor spheroid fabrication

Fabbricazione seguita da incapsulamento 3D di sferoidi tumorali in idrogel 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.

Autore in primo piano: Modello in vitro di idrogel per lo studio del microambiente del glioblastoma

Fabbricazione e incapsulamento di sferoidi tumorali in idrogel di polietilenglicole per lo studio delle interazioni sferoide-matrice

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.

Fabbricazione di micropozzetti piramidali quadrati per la fabbricazione di sferoidi tumorali

Per iniziare, versare due grammi o circa un millilitro di soluzione di precursore PDMS negativo su un pozzetto di uno stampo master parametal quadrato a sei pozzetti. Dopo aver coperto lo stampo principale con PDMS, posizionare la piastra in un essiccatore sottovuoto per degassare la soluzione. Quindi, metti la piastra in un forno a 60 gradi Celsius per 24 ore per polimerizzare il PDMS.Una volta che il PDMS si è indurito ma è ancora caldo, utilizzare una spatola per rimuovere con cautela lo stampo PDMS dallo stampo principale. Quindi, con un punzone per biopsia, taglialo in una lastra di 35 millimetri di diametro. Metti lo stampo PDMS tagliato correttamente in una capsula di Petri e coprilo con il coperchio per continuare a polimerizzarlo a temperatura ambiente per altre 24 ore.Per preparare lo stampo PDMS positivo, posizionare una lastra di 35 millimetri dello stampo PDMS negativo in una capsula di Petri da 35 millimetri con i micropozzetti testurizzati rivolti verso l'alto. Versare 2,5 grammi o circa 1,2 millilitri di soluzione positiva di precursore PDMS sullo stampo negativo nella capsula di Petri. Degassare la soluzione precursore per 30 minuti prima di metterla in forno a 60 gradi Celsius per tre o quattro ore.Dopo l'indurimento, rimuovere gli stampi dalla capsula di Petri. Staccare immediatamente lo stampo positivo dallo stampo negativo. Utilizzare un punzone per biopsia da 10 millimetri per tagliare gli stampi positivi in lastre.Quindi, usa le pinzette per immergere delicatamente il lato piatto di ogni stampo positivo da 10 millimetri nella soluzione precursore della colla PDMS pre-preparata. Posizionare con cura uno stampo per pozzetto in un pozzetto di una piastra da 48 pozzetti. Premere delicatamente ogni stampo per incollarlo al fondo del pozzo.Incubare la piastra assemblata a 60 gradi Celsius per 4-24 ore per polimerizzare la colla.

fabrication-square-pyramidal-microwells-for-tumor-spheroid

Research

JoVE Journal

Bioengineering

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

66 Views.  Saint Louis University. To begin pour two grams or about one milliliter of negative PDMS precursor solution onto one well of a six well square parametal master mold. After covering the master mold with PDMS, place the plate in a vacuum desiccate to degas the solution. Next, place the plate in an oven at 60 degrees Celsius for 24 hours to cure the PDMS.Once PDMS cures but is still warm, use a spatula to carefully remove the PDMS mold from the master mold. Then with a biopsy punch, cut it into a 35 millimeter d...