tumor-spheroid-fabrication-encapsulation-polyethylene-glycol

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 ...

Saint Louis University

Volumetric muscle loss (VML) is traumatic or surgical loss of skeletal muscle with resultant functional impairment. Skeletal muscle's innate capacity for regeneration is lost with VML due to a critical loss of stem cells, extracellular matrix, and neuromuscular junctions. Consequences of VML include permanent disability or delayed amputations of the affected limb. Currently, a successful clinical therapy has not been identified. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and their three-dimensional aggregation can further enhance therapeutic efficacy. In this study, MSC aggregation into spheroids was optimized in vitro based on cellular viability, spheroid size, and trophic factor secretion. The regenerative potential of the optimized MSC spheroid therapy was then investigated in a murine model of VML injury. Experimental groups included an untreated VML injury control, intramuscular injection of MSC spheroids, and MSC spheroids encapsulated in a fibrin-laminin hydrogel. Compared to the untreated VML group, the spheroid encapsulating hydrogel group enhanced myogenic marker (i.e., MyoD and myogenin) protein expression, improved muscle mass, increased presence of centrally nucleated myofibers as well as small fibers (<500 μm ), modulated pro- and anti-inflammatory macrophage marker expression (i.e., iNOS and Arginase), and increased the presence of CD146 pericytes and CD31 endothelial cells in the VML injured muscles. Future studies will evaluate the extent of functional recovery with the spheroid encapsulating hydrogel therapy.

Co-delivery of fibrin-laminin hydrogel with mesenchymal stem cell spheroids supports skeletal muscle regeneration following trauma.

Glioblastoma (GBM) is the deadliest brain tumor for which there is no cure. Bioengineered GBM models, such as hydrogel-encapsulated spheroids, that capture both cell-cell and cell-matrix interactions could facilitate testing of much needed therapies. Elucidation of specific microenvironment properties on spheroid responsiveness to therapeutics would enhance the usefulness of GBM models as predictive drug screening platforms. Here, GBM spheroids consisting of U87 or patient-derived GBM cells were encapsulated in soft (∼1 kPa), stiff (∼7 kPa), and dual-stiffness polyethylene glycol-based hydrogels, with GBM spheroids seeded at the stiffness interface. Spheroids were cultured for 7 days and examined for viability, size, invasion, laminin expression, hypoxia, proliferation, and response to the chemotherapeutic temozolomide (TMZ). We noted excellent cell viability in all hydrogels, and higher infiltration in soft compared to stiff hydrogels for U87 spheroids. In dual gels spheroids mostly infiltrated away from the stiffness interface with minimal crossing over it and some individual cell migration along the interface. U87 spheroids were equally responsive to TMZ in the soft and stiff hydrogels, but cell viability in the spheroid periphery was higher than the core for stiff hydrogels whereas the opposite was true for soft hydrogels. HIF1A expression was higher in the core of spheroids in the stiff hydrogels, while there was no difference in cell proliferation between spheroids in the stiff vs soft hydrogels. Patient-derived GBM spheroids did not show stiffness-dependent drug responses. U87 cells showed similar laminin expression in soft and stiff hydrogels with higher expression in the spheroid periphery compared to the core. Our results indicate that microenvironment stiffness needs to be considered in bioengineered GBM models including those designed for use in drug screening applications. STATEMENT OF SIGNIFICANCE: Recent work on tumor models engineered for use in drug screening has highlighted the potential of hydrogel-encapsulated spheroids as a simple, yet effective platform that show drug responses similar to native tumors. It has also been shown that substrate stiffness, in vivo and in vitro, affects cancer cell responses to drugs. This is particularly important for glioblastoma (GBM), the deadliest brain cancer, as GBM cells invade by following the stiffer brain structures such as white matter tracks and the perivascular niche. Invading cells have also been associated with higher resistance to chemotherapy. Here we developed GBM spheroid models using soft, stiff and dual-stiffness hydrogels to explore the connection between substrate stiffness, spheroid invasion and drug responsiveness in a controlled environment.

Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels.

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose-response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

Article	Year
Co-delivery of fibrin-laminin hydrogel with mesenchymal stem cell spheroids supports skeletal muscle regeneration following trauma. Journal of tissue engineering and regenerative medicine\| PubMed ID: 34551191
Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels. Acta biomaterialia\| PubMed ID: 35659918	2023
Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture. Scientific reports\| PubMed ID: 36273031	2022

Joseph Bruns

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