Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks.

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-ß signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

Rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays.

Antibiotic resistance is a major global health concern that requires action across all sectors of society. In particular, to allow conservative and effective use of antibiotics clinical settings require better diagnostic tools that provide rapid determination of antimicrobial susceptibility. We present a method for rapid and scalable antimicrobial susceptibility testing using stationary nanoliter droplet arrays that is capable of delivering results in approximately half the time of conventional methods, allowing its results to be used the same working day. In addition, we present an algorithm for automated data analysis and a multiplexing system promoting practicality and translatability for clinical settings. We test the efficacy of our approach on numerous clinical isolates and demonstrate a 2-d reduction in diagnostic time when testing bacteria isolated directly from urine samples.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis.

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.

Dielectrophoretic characterization of cells in a stationary nanoliter droplet array with generated chemical gradients.

A novel design of reusable microfluidic platform that generates a stationary nanoliter droplet array (SNDA) for cell incubation and analysis, equipped with a complementary array of individually addressable electrodes for each microwell is studied. Various solute concentration gradients were generated between the wells where dielectrophoresis (DEP) was used to characterize the effect of the gradients on the cell's response. The feasibility of generating concentration gradients and observation of DEP responses was demonstrated using a gradient of salts in combination with microparticles and viable cells. L1210 Lymphoma cells were used as the model cells in these experiments. Lymphoma cells' cross-over frequency (COF) decreased with increasing stress conditions. Specifically, a linear decrease in the cell COF was measured as a function of solution tonicity and blebbistatin dose. Lymphoma cells were incubated under a gradient of the chemotherapeutic agent doxorubicin (DOX), which led to saturation in the cell-COF response at 30 nM DOX, demonstrating the potential of the platform in screening of label-free drugs.

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges.

Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high-quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties.