In vitro studies of cancer cell behavior are often limited in translational capability due to the absence of in vivo characteristics, such as the tumor microenvironment (TME), that largely govern this behavior. The TME includes a dense, highly organized network of proteins that forms the extracellular matrix (ECM). In many cancers, the ECM of the TME is distinct from that of healthy tissue in that it is highly aligned and crosslinked, forming a stiff matrix that serves as a structural scaffold for cancer cells. Notably, the TME ECM and its unique topography at the micro‐ and nanoscale have demonstrated profound influence on cell polarity, migration, invasion, and proliferation capabilities. Thus, proper recapitulation of the TME ECM is crucial for in vitro assessment of cancer cell behavior that accurately models in vivo processes. Nanofabricated biomaterials have emerged as versatile tools for the recapitulation of the nanoscale topography of the TME ECM. In this review, we discuss the various geometries of nanofabricated biomaterial scaffolds employed for 2D in vitro assays and their benefits and limitations. We additionally introduce our collagen‐coated nanogrooved array and its utility in modeling the ECM of understudied pediatric malignancies, including neuroblastoma.
Achieving selective tumor targeting remains a major challenge in heterogeneous tumors like glioblastoma (GBM), the most aggressive brain cancer. To address this, cell‐derived magnetic nanovectors, hybrid lipid nanoparticles integrating iron oxide nanoparticles (IONPs) cloaked with plasma membranes from patient‐derived GBM cells, were introduced. This nanoarchitecture exploits homotypic recognition to combine magnetic hyperthermia with magnetic resonance imaging (MRI) contrast within a customizable theranostic platform. However, preserving membrane protein conformation during nanoparticle fabrication remains a challenge that affects targeting specificity. In this work, a label‐free spectroscopic platform combining fluorescence emission, Fourier transform infrared spectroscopy, and Raman spectroscopy was developed to probe protein conformation at the nanoparticle interface. With this approach, an optimized ultrasonication protocol was shown to ensure protein integrity, enabling homotypic interactions and preventing hard protein corona formation, a determinant of nanoparticle fate in biological environments. Consistent with these findings, preserved membrane proteins enhance nanoparticle accumulation in patient‐derived GBM spheroids and support MRI contrast performance. Additionally, oxidative stress in cells upon IONP internalization induces a compensatory increase in glycolytic metabolism, underscoring the importance of tumor specificity to reduce off‐target effects. This work provides a framework for probing protein conformation on nanoparticles, aiding the refinement of biomimetic nanotheranostics for precision GBM treatment.
In vitro models play a vital role in elucidating cellular behavior and disease mechanisms within reproducible, ethical, and physiologically relevant microenvironments. While 2D models are easier to use and cost‐efficient, they often alter physiological cell morphology and function. In contrast, 3D models more accurately recapitulate in vivo‐like architecture, cell–cell interactions, and tissue‐specific behavior. However, scaffold‐based 3D culture systems are commonly fabricated on planar substrates, which can lead to cell attachment and proliferation on the surrounding 2D surface, depending on factors such as substrate chemistry, surface functionalization, scaffold geometry, and culture conditions. Therefore, minimizing influence from 2D‐grown cells on devices including 3D‐engineered cell microenvironments is crucial in assays such as gene expression analysis, to avoid undesired 2D‐cell contributions in biological readouts. In this work, we report the combination of ad hoc biochemical cell‐repellent Lipidure coatings with two‐photon polymerized 3D‐engineered glioblastoma (GBM) microenvironments, able to substantially exclude the formation of a 2D GBM cell monolayer in the context of proton radiobiology studies. The proposed platform enables more reliable biological characterization of GBM cells in 3D, precise detection of DNA damage markers following proton radiotherapy, and sufficient RNA yield for gene expression profiling.
Electrical pacing is a key strategy for enhancing the functional maturity of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) by preserving the contractile phenotype of explanted cardiomyocytes or promoting the development of adult‐like features in immature ones. Electrical stimulation platforms typically employ opaque electrodes positioned at the periphery of the culture dish, limiting the area that can be stimulated and necessitating high currents to activate all the cells. Here, we systematically evaluated how electrode material and spatial configuration influence the response of hPSC‐CMs. We compared transparent indium–tin‐oxide and gold electrodes arranged in three different configurations to vary electrode–cell distance, active surface area, and field orientation. Using electrochemical impedance spectroscopy and finite element modeling, we characterized the electrical properties of each setup. We assessed the two‐dimensional (2D) hPSC‐CM monolayer contractility under identical stimulation protocols across all configurations. Among all tested setups, the vertical configuration was the most efficient in eliciting robust contractile responses of mature hPSC‐CMs, and gold electrodes showed higher efficiency. However, electrical pacing did not enhance maturation markers in hPSC‐CMs when applied as a training method. These findings provide design principles for optimizing electrical stimulation systems in cardiac tissue engineering, with implications for disease modeling and cell‐based regenerative therapies.
Melioidosis, caused by Burkholderia pseudomallei , is a life‐threatening disease and a potential biological warfare agent. Currently, no licensed vaccine is available for prevention. To address this unmet need, we bioengineered an endotoxin‐free Escherichia coli strain to assemble biopolymer inclusions coated with B. pseudomallei vaccine candidate antigens such as LolC, Omp85, OmpW, TssM, BopA, BimA, Hcp 1, Hcp 2, Hcp 3, and Hcp 6. These biopolymer particle (BP)‐based vaccines were formulated with the safe poly(I:C) adjuvant for intramuscular and intranasal administration in BALB/c mice. The vaccines were safe and induced specific and strong antibody responses. Additionally, the vaccines mediated secretion of Th1 and Th2 cytokines. No significant weight changes were observed in vaccinated groups compared with controls. In animal trials, immunized mice were challenged with a lethal dose of B. pseudomallei strain K96243 via the intraperitoneal route. Mice receiving BimA‐BP‐BopA and COM1 (a combination of BimA‐BP‐BopA, LolC‐BP‐Hcp1, and ME‐BM‐BP‐83 vaccine candidates) survived the challenge, with reduced bacterial burden in their livers and lungs when compared with empty BPs.
