Poster Presentations from the 2023 Microscale Innovation in Life Science Symposium

The next generation of single cell analysis will involve the measurement of functional properties of living cells, including growth, death, protein secretions, and the interactions between multiple cell types. Similar to transcriptomic measurements, there is a need for measuring high dimensional functional properties of single cells so that the relevant subpopulations can be identified and retrieved for follow-on applications, including cell line development and immune marker discovery. However, existing platforms to date have either hand insufficient scale, low dimensionality and/or are too high cost to be practically implemented for routine biology experiments. In this talk, I will summarize our recent advances in using high dimensional time lapse imaging to measure the functional heterogeneity of living cells at the scale of 100,000 single clones per experiment. I will provide several examples of the applications of our platform in the ability to find rare drug-resistant cells, existing at frequencies of less than 1 in 10,000 in the parental population, as well as in measuring the potency of immune cells that are both effective at secreting cytokines and also in killing cancer cells in a target dependent manner. Ultimately, this platform can be used in the development of better drugs that more effectively suppress resistance, as well as better cell-based therapies that have greater clinical effectiveness.

Presentation from the Next Generation Miniaturization Technologies session

The investigation of radiation’s effects on the GI tract, the identification of dosimetry biomarkers, and the testing of new radiation countermeasure drugs are all limited in humans due to the restricted access to human samples or the use of animal models that are not representative of human physiology. Thus, there is a need for improved in vitro models to elucidate the effects of x-ray radiation on the human GI system that mimic the in vivo physiological environment and interaction between human GI epithelium and gut microbiome. Here, we developed a Gut-on-Chip system (HuMiX) to reproduce multiple in vivo parameters classically associated with human GI following acute irradiation. The HuMiX device is a co-culture system consisting of three chambers separated by two porous polycarbonate membranes, delimiting the microbial, intestinal, and vascular compartments. Previous design iterations of HuMiX have demonstrated that the system can imitate the in vivo immunologic, metabolic, and transcriptional responses to commensal gut bacteria [Shah et al, 2016]. In the newly designed system, ~16,000 Caco-2 cells/mm2 were seeded on a collagen-treated membrane in the middle chamber and incubated under normal cell culture incubator conditions. The middle and bottom chambers were then flowed with Caco-2 cell medium at 67 ul/min for 6 days, when 8 x 107/mL of mixed bacterial flora, isolated from human stool samples from 6 different donors, were injected into the top chamber. Post 12 hours co-culture, the devices were sham- or 8 Gy-irradiated at 1 Gy/min using 320 keV x-ray beam with 2 mm Al filter. Twenty-four hours after irradiation, the supernatant was collected for inflammatory cytokine detection, the bacterial cells were collected from the top chamber for microbiome profiling and Caco2 cells were stained. Preliminary microbiome profiling identified the presence of three phyla: Bacteroides, FAFV (Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia), and Proteobacteria. DNA concentration, species richness, and species diversity obtained from the devices decreased as compared to those profiles isolated from direct fecal bacteria isolation and/or cultured in bacterial culture broths, but still maintained highly diverse profiles. Current analyses are on-going to identify if radiation modified the Firmicutes/Bacteriodetes ratio as shown by in vivo studies. The Caco2 phenotype revealed the formation of villi, the presence of cell-cell tight junctions and the production of mucus, demonstrating the establishment of physiological intestinal hallmarks. Irradiation showed ability to disrupt cell junctions and to permeabilize the intestinal barrier as suggested by dextran assay. Citrulline, an amino acid whose plasma level decrease following intestinal radiation-induced damage, was also quantified. This study is an ongoing project and the injection of immune cells will be performed in the future to assess if the HuMiX can recapitulate the expression change of well-known radiation dosimetry biomarkers in blood. Overall, our data suggest that the HuMiX offers a promising tool to study radiation effect on human gut and test radiation countermeasure approaches.

AmplifiDx is developing a high-performance, low-cost microfluidic cartridge-based point-of-care testing system. The speaker will describe how the company has distinguished itself in an extremely crowded space, and how it continues to defy the odds even in this challenging economy for start-ups. This involves differentiated technology, but more importantly, creative partnerships and an understanding of the market.

When it comes to diagnostic devices, what is better? An implantable device? A wearable device? Or a point-of-care device, that takes a sample from you and measures in-vitro? They all have pros and cons, but the underlying answer is innovations of technology available for that particular diagnostic. In this talk, I will present my work starting companies from in-vitro to implantable -- from breast pumps , to a device that measures fetal-maternal hemorrhaging, to point-of-care virus detection, and finally on my newest endeavor, engineering a painless, minimally invasive, small form-factor continuous wearable glucose monitor for my daughter who was recently diagnosed with Type I diabetes. We are developing this platform to sense multiple analytes in a real-time, continuous manner, thus changing the paradigm for on-body biosensing and continuous biomonitoring for medicine.

Encoded library technologies typically involve affinity selection to identify hits, which identifies binding but not necessarily function. Transfer of encoded libraries to solid-phase enables activity-based hit generation using high-throughput screening assays. This has been accomplished for DNA-encoded libraries, but not other encoded library formats. This lecture will discuss the first steps toward enabling activity-based mRNA display library screening. DNA-functionalized magnetic microspheres are used to template via bulk emulsification the formation of uniform hydrogel particles. The DNA templates are transcribed and the product mRNA is captured locally in the gel via hybridization. The captured mRNA is then translated and puromycin capture results in polyvalent, monoclonal display of the translated peptide (100 nM peptide in gel by HiBiT quantitation). The beads are analyzed by FACS and enrichments of > 50-fold were observed in a model screen. The ultra-miniaturized gel particle library format scalably transfers output from selection to focused libraries for functional screening.

Entropic Biosciences, Inc. has designed a proprietary scaffold-free technology to grow high-throughput, automated, self-standing 3D cellular aggregates. We have used this platform technology to generate spheroids from various cancer cell types. This enables precision medicine and testing potential chemotherapeutics against 3D models derived from individual patients’ cancer and other verticles, including cellular agriculture and tissue engineering applications.

Microfluidic technologies were always at the forefront of innovations in liquid biopsies. There were several cornerstone microfluidic technologies that enabled sensitive and yet specific identification of blood-based biomarkers. The confluence of technology advancement and rapid developments in molecular characterization techniques pushed the boundaries of liquid biopsies thus enhancing the clinical utility of blood biopsies. Several such key enabling technologies will be presented. How isolation of circulating tumor cells and tumor derived vesicles using novel microfluidic technologies can enable precision diagnostics will be demonstrated with the example clinical case studies.

“Context of Use”. A little-known phrase but one that has profound implications. This phrase underscores the importance of considering the practical and real-world aspects of how something will be used or applied. This understanding helps guide decision-making, assessment, and optimization to ensure that products and processes are safe, effective, and fit for their intended purpose. Understanding the context of use has profound implications across the drug discovery continuum that focusses on developing the right in vitro model to answer a specific biological question while maintaining the desired balance between physiological relevance and throughput. The InSphero 3D Insight (TM) platform delivers robust, reproducible, scalable and actionable data across different disease pathologies to provide solutions for specific contexts of use.