Droplet Interfacial Tensions and Phase Transitions Measured in Microfluidic Channels.

Measurements of droplet phase and interfacial tension (IFT) are important in the fields of atmospheric aerosols and emulsion science. Bulk macroscale property measurements with similar constituents cannot capture the effect of microscopic length scales and highly curved surfaces on the transport characteristics and heterogeneous chemistry typical in these applications. Instead, microscale droplet measurements ensure properties are measured at the relevant length scale. With recent advances in microfluidics, customized multiphase fluid flows can be created in channels for the manipulation and observation of microscale droplets in an enclosed setting without the need for large and expensive control systems. In this review, we discuss the applications of different physical principles at the microscale and corresponding microfluidic approaches for the measurement of droplet phase state, viscosity, and IFT.

Engineering T cell response to cancer antigens by choice of focal therapeutic conditions.

Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (-80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the 'quantity' (i.e., amount) and 'quality' (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.

The Role of Protein Loss and Denaturation in Determining Outcomes of Heating, Cryotherapy, and Irreversible Electroporation on Cardiomyocytes.

Atrial fibrillation (AF) currently affects millions of people in the U.S. alone. Focal therapy is an increasingly attractive treatment for AF that avoids the debilitating effects of drugs for disease control. Perhaps the most widely used focal therapy for AF is heat-based radiofrequency (heating), although cryotherapy (cryo) is rapidly replacing it due to a reduction in side effects and positive clinical outcomes. A third focal therapy, irreversible electroporation (IRE), is also being considered in some settings. This study was designed to help guide treatment thresholds and compare mechanism of action across heating, cryo, and IRE. Testing was undertaken on HL-1 cells, a well-established cardiomyocyte cell line, to assess injury thresholds for each treatment method. Cell viability, as assessed by Hoechst and propidium iodide (PI) staining, was found to be minimal after exposure to temperatures ≤-40 °C (cryo), ≥60 °C (heating), and when field strengths ≥1500 V/cm (IRE) were used. Viability was then correlated to protein denaturation fraction (PDF) as assessed by Fourier transform infrared (FTIR) spectroscopy, and protein loss fraction (PLF) as assessed by bicinchoninic acid (BCA) assay after the three treatments. These protein changes were assessed both in the supernatant and the pellet of cell suspensions post-treatment. We found that dramatic viability loss (≥50%) correlated strongly with ≥12% protein change (PLF, PDF or a combination of the two) in every focal treatment. These studies help in defining both cellular thresholds and protein-based mechanisms of action that can be used to improve focal therapy application for AF.

A Microfluidic Device for Automated High Throughput Detection of Ice Nucleation of Snomax®.

Measurement of ice nucleation (IN) temperature of liquid solutions at sub-ambient temperatures has applications in atmospheric, water quality, food storage, protein crystallography and pharmaceutical sciences. Here we present details on the construction of a temperature-controlled microfluidic platform with multiple individually addressable temperature zones and on-chip temperature sensors for high-throughput IN studies in droplets. We developed, for the first time, automated droplet freezing detection methods in a microfluidic device, using a deep neural network (DNN) and a polarized optical method based on intensity thresholding to classify droplets without manual counting. This platform has potential applications in continuous monitoring of liquid samples consisting of aerosols to quantify their IN behavior, or in checking for contaminants in pure water. A case study of the two detection methods was performed using Snomax® (Snomax International, Englewood, CO, USA), an ideal ice nucleating particle (INP). Effects of aging and heat treatment of Snomax® were studied with Fourier transform infrared (FTIR) spectroscopy and a microfluidic platform to correlate secondary structure change of the IN protein in Snomax® to IN temperature. It was found that aging at room temperature had a mild impact on the ice nucleation ability but heat treatment at 95 °C had a more pronounced effect by reducing the ice nucleation onset temperature by more than 7 °C and flattening the overall frozen fraction curve. Results also demonstrated that our setup can generate droplets at a rate of about 1500/min and requires minimal human intervention for DNN classification.

Ultrarapid Inductive Rewarming of Vitrified Biomaterials with Thin Metal Forms.

Arteries with 1-mm thick walls can be successfully vitrified by loading cryoprotective agents (CPAs) such as VS55 (8.4 M) or less concentrated DP6 (6 M) and cooling at or beyond their critical cooling rates of 2.5 and 40 °C/min, respectively. Successful warming from this vitrified state, however, can be challenging. For example, convective warming by simple warm-bath immersion achieves 70 °C/min, which is faster than VS55's critical warming rate of 55 °C/min, but remains far below that of DP6 (185 °C/min). Here we present a new method that can dramatically increase the warming rates within either a solution or tissue by inductively warming commercially available metal components placed within solutions or in proximity to tissues with non-invasive radiofrequency fields (360 kHz, 20 kA/m). Directly measured warming rates within solutions exceeded 1000 °C/min with specific absorption rates (W/g) of 100, 450 and 1000 for copper foam, aluminum foil, and nitinol mesh, respectively. As proof of principle, a carotid artery diffusively loaded with VS55 and DP6 CPA was successfully warmed with high viability using aluminum foil, while standard convection failed for the DP6 loaded tissue. Modeling suggests this approach can improve warming in tissues up to 4-mm thick where diffusive loading of CPA may be incomplete. Finally, this technology is not dependent on the size of the system and should therefore scale up where convection cannot.

Biomaterial scaffolds for non-invasive focal hyperthermia as a potential tool to ablate metastatic cancer cells.

Currently, there are very few therapeutic options for treatment of metastatic disease, as it often remains undetected until the burden of disease is too high. Microporous poly(ε-caprolactone) biomaterials have been shown to attract metastasizing breast cancer cells in vivo early in tumor progression. In order to enhance the therapeutic potential of these scaffolds, they were modified such that infiltrating cells could be eliminated with non-invasive focal hyperthermia. Metal disks were incorporated into poly(ε-caprolactone) scaffolds to generate heat through electromagnetic induction by an oscillating magnetic field within a radiofrequency coil. Heat generation was modulated by varying the size of the metal disk, the strength of the magnetic field (at a fixed frequency), or the type of metal. When implanted subcutaneously in mice, the modified scaffolds were biocompatible and became properly integrated with the host tissue. Optimal parameters for in vivo heating were identified through a combination of computational modeling and ex vivo characterization to both predict and verify heat transfer dynamics and cell death kinetics during inductive heating. In vivo inductive heating of implanted, tissue-laden composite scaffolds led to tissue necrosis as seen by histological analysis. The ability to thermally ablate captured cells non-invasively using biomaterial scaffolds has the potential to extend the application of focal thermal therapies to disseminated cancers.