Mitigating reactive oxygen species production and increasing gel porosity improves lymphocyte motility and fibroblast spreading in photocrosslinked gelatin-thiol hydrogels.

On-chip 3D culture systems that incorporate immune cells such as lymphocytes and stromal cells are needed to model immune organs in engineered systems such as organs-on-chip. Photocrosslinking is a useful tool for creating such immune-competent hydrogel cultures with spatial cell organization. However, loss of viability and motility in photocrosslinked gels can limit its utility, especially when working with fragile primary cells. We hypothesized that optimizing photoexposure-induced ROS production, hydrogel porosity or a combination of both factors was necessary to sustain cell viability and motility during culture in photocrosslinked gelatin-thiol (GelSH) hydrogels. Jurkat T cells, primary human CD4+ T cells and human lymphatic fibroblasts were selected as representative lymphoid immune cells to test this hypothesis. Direct exposure of these cells to 385 nm light and LAP photoinitiator dramatically increased ROS levels. Pretreatment with an antioxidant, ascorbic acid (AA), protected the cells from light + LAP-induced ROS and was non-toxic at optimized doses. Furthermore, scanning electron microscopy showed that native GelSH hydrogels had limited porosity, and that adding collagen to GelSH precursor before crosslinking markedly increased gel porosity. Next, we tested the impact of AA pretreatment and increasing gel porosity, alone or in combination, on cell viability and function in 3D GelSH hydrogel cultures. Increasing gel porosity, rather than AA pretreatment, was more critical for rescuing viability of Jurkat T cells and spreading of human lymphatic fibroblasts in GelSH-based gels, but both factors improved the motility of primary human CD4+ T cells. Increased porosity enabled formation of spatially organized co-cultures of primary human CD4+ T cells and human lymphatic fibroblasts in photo-crosslinked gels in a multi-lane microfluidic chip, towards modeling the lymphoid organ microenvironment. Some optimization is still needed to improve homogeneity between regions on the chip. These findings will enable researchers utilizing photocrosslinking methods to develop immunocompetent 3D culture models that support viability and function of sensitive lymphoid cells.

A Scalable, Modular Degasser for Passive In-Line Removal of Bubbles from Biomicrofluidic Devices.

Bubbles are a common cause of microfluidic malfunction, as they can perturb the fluid flow within the micro-sized features of a device. Since gas bubbles form easily within warm cell culture reagents, degassing is often necessary for biomicrofluidic systems. However, fabrication of a microscale degasser that can be used modularly with pre-existing chips may be cumbersome or challenging, especially for labs not equipped for traditional microfabrication, and current commercial options can be expensive. Here, we address the need for an affordable, accessible bubble trap that can be used in-line for continuous perfusion of organs-on-chip and other microfluidic cultures. We converted a previously described, manually fabricated PDMS degasser to allow scaled up, reproducible manufacturing by commercial machining or fused deposition modeling (FDM) 3D printing. After optimization, the machined and 3D printed degassers were found to be stable for >2 weeks under constant perfusion, without leaks. With a ~140 µL chamber volume, trapping capacity was extrapolated to allow for ~5-20 weeks of degassing depending on the rate of bubble formation. The degassers were biocompatible for use with cell culture, and they successfully prevented bubbles from reaching a downstream microfluidic device. Both degasser materials showed little to no leaching. The machined degasser did not absorb reagents, while the FDM printed degasser absorbed a small amount, and both maintained fluidic integrity from 1 µL/min to >1 mL/min of pressure-driven flow. Thus, these degassers can be fabricated in bulk and allow for long-term, efficient bubble removal in a simple microfluidic perfusion set-up.

Towards spatially-organized organs-on-chip: Photopatterning cell-laden thiol-ene and methacryloyl hydrogels in a microfluidic device.

Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but the combination of patterned hydrogels with organs-on-chip to generate organized 3D cultures under microfluidic perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning protocol that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing, and tested the impact of crosslinking chemistry on stability and spatial resolution. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities (≥ 107/mL) did not impede thiol-norbornene gelation, but decreased the storage moduli of methacryloyl hydrogels. Hydrogel composition and light dose were selected to match the storage moduli of soft tissues. To generate the desired pattern on-chip, the cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask. The on-chip 3D cultures were self-standing and the designs were interchangeable by simply swapping out the photomask. Thiol-ene hydrogels yielded highly accurate feature sizes from 100 - 900 µm in diameter, whereas methacryloyl hydrogels yielded slightly enlarged features. Furthermore, only thiol-ene hydrogels were mechanically stable under perfusion overnight. Repeated patterning readily generated multi-region cultures, either separately or adjacent, including non-linear boundaries that are challenging to obtain on-chip. As a proof-of-principle, primary human T cells were patterned on-chip with high regional specificity. Viability remained high (> 85%) after 12-hr culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D co-cultures to mimic organ-like structures that were previously difficult to obtain.

Engineering in vitro immune-competent tissue models for testing and evaluation of therapeutics.

Advances in 3D cell culture, microscale fluidic control, and cellular analysis have enabled the development of more physiologically-relevant engineered models of human organs with precise control of the cellular microenvironment. Engineered models have been used successfully to answer fundamental biological questions and to screen therapeutics, but these often neglect key elements of the immune system. There are immune elements in every tissue that contribute to healthy and diseased states. Including immune function will be essential for effective preclinical testing of therapeutics for inflammatory and immune-modulated diseases. In this review, we first discuss the key components to consider in designing engineered immune-competent models in terms of physical, chemical, and biological cues. Next, we review recent applications of models of immunity for screening therapeutics for cancer, preclinical evaluation of engineered T cells, modeling autoimmunity, and screening vaccine efficacy. Future work is needed to further recapitulate immune responses in engineered models for the most informative therapeutic screening and evaluation.

Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR.

3D cell culture in protein-based hydrogels often begins with chemical functionalization of proteins with cross-linking agents such as methacryloyl or norbornene. An important and variable characteristic of these materials is the degree of functionalization (DoF), which controls the reactivity of the protein for cross-linking and therefore impacts the mechanical properties and stability of the hydrogel. Although 1H NMR has emerged as the most accurate technique for quantifying absolute DoF of chemically modified proteins, colorimetric techniques still dominate in actual use and may be more useful for quantifying fractional or percent DoF. In this work, we sought to develop an optimized colorimetric assay for DoF of common gelatin-based biomaterials and validate it versus NMR; along the way, we developed a set of best practices for both methods and considerations for their most appropriate use. First, the amine-reactive ninhydrin assay was optimized in terms of solvent properties, temperature, ninhydrin concentration, and range of gelatin standards. The optimized assay produced a linear response to protein concentration in a convenient, 96-well plate format and yielded a fractional DoF similar to NMR in most cases. In comparing with NMR, we identified that DoF can be expressed as fractional or absolute, and that fractional DoF can be inaccurate if the amino acid content of the parent protein is not properly accounted for. In summary, the fractional DoF of methacryloyl- and norbornene-functionalized gelatins was quantified by an optimized colorimetric ninhydrin assay and orthogonally by 1H NMR. These methods will be valuable for quality control analysis of protein-based hydrogels and 3D cell culture biomaterials. Graphical abstract.