Roman Truckenmüller

An ambient-temperature micro-cold-forming technique enables straightforward, rapid, and low-cost fabrication of polymer microcavities for 3D organoid culture and toxicity testing.

In this proof‐of‐concept study, we develop a novel 3D cell patterning and culture platform. The “Floor‐Ceiling‐Chip” (FC‐Chip) simply consists of two opposing 2D substrates in the form of ion track‐etched membranes, creating a pseudo‐3D microenvironment for the cells between them. This allows the stimulation of both the dorsal and ventral sides of cells, thereby also eliminating the artificial polarization, for example, of stromal cells, observed in standard culture dishes and inserts. By providing the membranes with micropatterned cell‐adhesive islands of varying geometries and sizes, the FC‐Chip enables control over cell shape and alignment in a 3D environment. Analysis of fluorescence microscopic images reveals distinct cellular and nuclear morphology, along with perinuclear actin organization, in the on‐chip cultures compared to cultures on traditional 2D substrates. Cells in the FC‐Chip exhibit fewer focal adhesions, lower expression of lamin A/C, and less nuclear localization of the yes‐associated protein 1. The chip demonstrates compatibility with standard biochemical assays and supports long‐term cultures up to 10 days, expanding its potential applications. Overall, the early version of the FC‐Chip presented here confirms the feasibility of a straightforward, accessible, and versatile future culture platform for the manipulation and modeling of cell morphology and organization in 3D.

Co‐assembly of cells and microsized, extracellular matrix (ECM)‐mimicking biomaterials, for example, in the form of microparticles, is a promising strategy for generating 3D microtissues. Additionally, microparticles, especially the porous ones, are known for their role as microcarriers in delivery systems, owing to their high specific surface area. Therefore, this work proposes the use of multifunctional, bioactive compound‐loaded porous microparticles, or microshuttles, that can simultaneously fulfill the roles of ECM‐mimicking scaffolding components and delivery vehicles in self‐assembled microtissues. This work presents a one‐step emulsification method, followed by a chemical etching step, for generating a library of porous poly(lactic ‐co‐ glycolic acid) (PLGA) microparticles with tunable pore sizes. The microparticles undergo cell‐guided assembly when co‐seeded with human mesenchymal stromal cells (HMSCs) in microwells, forming hybrid cell‐biomaterial microtissues. Additionally, the microparticles can be versatile microcarriers of various bone repair‐related factors, including bone morphogenetic protein 2 (BMP‐2), nanohydroxyapatite (nHA), and human umbilical vein endothelial cells (HUVECs). The results indicate enhanced expression of osteogenic genes and proteins in hybrid microtissues containing BMP‐2‐ and nHA‐loaded PLGA microparticles, and improved endothelial network formation in hybrid microtissues containing HUVEC‐loaded PLGA microparticles, as compared to HMSC‐only microtissues. These findings highlight the potential of the porous PLGA microshuttles in engineering potentially osteogenic, self‐assembled microtissues.

Cellular spheroids are considered a popular option for modeling healthy and diseased tissues in vitro and as injectable therapies. The formation and culture of spheroids can make use of different three-dimensional (3D) culture platforms, but the spheroids’ analysis often has to rely on endpoint assays. In this study, we propose a microfluidic bioreactor to culture and nondestructively monitor human mesenchymal stem cell (hMSC) spheroids over time using non-Faradaic electr­(ochem)­ical impedance spectroscopy (EIS). For this, an array of porous microwells thermoformed from ion track-etched thin films and a pair of sensing electrodes from transparent indium tin oxide are integrated into the flow and culture chamber of the bioreactor. To measure the spheroid’s electrical properties, the electrodes are connected to a frequency response analyzer (FRA), with a multiplexer in between to enable the operation of more than one bioreactor at the FRA at the same time. We find differences between the complex resistance/impedance and/or capacitance data of a reference condition without cells, a two-dimensional (2D) hMSC culture, hMSC spheroids, and hybrid spheroids aggregated from hMSCs and titanium or hydroxyapatite microparticles. We also found differences between different culture durations. These results suggest that our device can sense the presence and spatial arrangement of cells and micro­(sized) biomaterials as a function of time.

Enthesitis, the inflammation of the enthesis, which is the point of attachment of tendons and ligaments to bones, is a common musculoskeletal disease. The inflammation often originates from the fibrocartilage region of the enthesis as a consequence of mechanical overuse or ‐load and consequently tissue damage. During enthesitis, waves of inflammatory cytokines propagate in(to) the fibrocartilage, resulting in detrimental, heterotopic bone formation. Understanding of human enthesitis and its treatment options is limited, also because of lacking in vitro model systems that can closely mimic the pathophysiology of the enthesis and can be used to develop therapies. In this study, an enthes(it)is‐on‐chip model is developed. On opposite sides of a porous culture membrane separating the chip's two microfluidic compartments, human mesenchymal stromal cells are selectively differentiated into tenocytes and fibrochondrocytes. By introducing an inflammatory cytokine cocktail into the fibrochondrocyte compartment, key aspects of acute and chronic enthesitis, measured as increased expression of inflammatory markers, can be recapitulated. Upon inducing chronic inflammatory conditions, hydroxyapatite deposition, enhanced osteogenic marker expression and reduced secretion of tissue‐related extracellular matrix components are observed. Adding the anti‐inflammatory drug celecoxib to the fibrochondrocyte compartment mitigates the inflammatory state, demonstrating the potential of the enthesitis‐on‐chip model for drug testing.