September 14-15, 2023
La Jolla, CA, USA
September 14-15, 2023
La Jolla, CA, USA
Microfluidic T Cell Engineering for Immunotherapies – Abe Lee, Ph.D. (University of California Irvine)
Adoptive cell therapy (ACT) is a type of immunotherapy that involves the processing of blood from a donor to isolate immune cells (e.g. T cells) for genetic manipulation followed by reinfusion of the cells into patients. Specifically for CAR T cell therapy, genetic coding material (e.g. DNA, mRNA) is inserted into the T cells to express chimeric antigen receptors to target biomarkers of cancer cells and trigger an activated immune response towards the tumor of interest. This process that starts from blood drawn from one person and ends with specialized engineered cells delivered to the same patient includes multiple tedious and costly steps, and can require a long time that the patient may not have. Microfluidics techniques are being developed that can address all steps of this cell manufacturing process, including cell harvesting, cell isolation, cell activation and expansion, and cell transfection. In this talk I will introduce two microfluidic platforms in my lab, one is the lateral cavity acoustic transducer (LCAT) and the other is droplet microfluidics. LCAT was used for processing blood samples, isolating T cells, transfecting T cells, and finally expanding T cells to scale up for treatment. Based on LCAT, we developed the acoustic electric shear orbiting poration (AESOP) device to uniformly deliver genetic cargo dosage into a large population of cells simultaneously. Based on droplet microfluidics we constructed a single cell artificial antigen presenting cells (aAPCs) for T cell activation. By trapping single cells in microfluidic compartments, we are able to study the cell morphology and cell-cell communications to further understand immune cell activation and immune cell synapses.
Tissue Chip with Gravity-Driven Flow for Drug Efficacy and Toxicity Studies – Hossein Tavana, Ph.D.
(University of Akron)
Organs-on-chips or tissue chips enable culturing fluidically connected three-dimensional cell cultures representing different organs or tissues. These devices are promising to evaluate systemic drug responses to cancer therapies and associated toxicities in preclinical studies. However, tissue chips are often complex in design, assembly, and fabrication, and are incompatible with robotic liquid handling for high throughput drug testing. To address this problem, we developed a novel tissue chip in the format of a standard 96-well plate using 3D printing. The plate contains 16 identical units, each with three main tissue compartments that represent liver, tumor, and bone marrow, and two inlet and outlet wells as media reservoirs. Fluid flow and exchange of media among compartments occurs due to gravity and by rocking of the plate at predefined frequencies. We selected liver and bone marrow because they are common sites of off-target toxicity from cancer drugs and because liver is a major site of drug metabolism. We validated that standard imaging and biochemical assays can conveniently be performed on different tissue compartments. Our results with a molecular inhibitor, trametinib, and a conventional chemotherapeutic, 5-fluorouracil, showed the sensitivity of the tissue chip to simultaneously determine efficacy of the drugs against cancer compartment and toxicity to liver and bone marrow compartments. This novel tissue chip offers a simple, yet enabling technology to evaluate both the efficacy and toxicity of cancer therapeutics in early discovery stages.
Single-Cell Interactive Cytometry using Made-to-Order Droplet Ensembles – Russell Cole, Ph.D.
(Scribe Biosciences)
Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for the precise study of single-cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multi-cell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a new paradigm in cell interaction profiling, Made-to-Order-Droplet-Ensembles (MODEs), that allows for the construction of precision cell ratio assays and characterization of their interactions for tens to hundreds of thousands of combinations. MODE technology leverages high throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the efficacy of MOD by building different formats of assays with a variety of cell types (NKs, CAR-T) and measuring single cell functional readouts such as degranulation and granzyme B activity, cytokine secretion, cytotoxicity, and antibody detection, enabling single-cell screening at scale.
Multiparametric Analysis of Small Extracellular Vesicles Purified by a Rapid and Label-free Lab on a
Chip Device – Leyla Esfandiari, Ph.D. (University of Cincinnati)
Abstract: Introduction: Conventional purification methods of small extracellular vesicles (sEVs) suffer from significant shortcomings including low purity, low capture efficiency, long processing times, large sample volume requirement, need for specialized equipment and trained personnel, and high costs. Our group has previously developed a label-free insulator-based dielectrophoretic (iDEP) device for rapid and selective entrapment of sEVs based on their unique dielectric properties and size1-3. Here we report a comprehensive three-fold characterization of sEVs isolated using the iDEP device from human biofluids including serum, plasma, and urine by utilizing conventional flow cytometry (cFCM), advanced imaging flow cytometry (iFCM), and next generation miRNA sequencing indicating high yield and purity.
Results: cFCM indicated 55% exosomes from serum, 31% from plasma, and 30% from urine to be CD63+. Similar analysis showed 22% exosomes from serum, 41% from plasma, and 34% from urine to be CD81+. iFCM revealed high CD63+ expressions with 3.26 x 107 EVs/mL for serum, 5.08 x 106 EVs/mL for plasma, and 1.3 x 107 EVs/mL for urine. CD81+ expressions also revealed high yield with 1.32 x 108 EVs/mL, 2.05 x 106 EVs/mL, and 4.02 x 106 EVs/mL for serum, plasma, and urine, respectively. Percentage of sEVs positive for each marker were comparable to expressions for sEVs purchased from ATCC Inc. Following miRNA sequencing, hsa-miR-6236, hsa-miR-148a, and hsa-let7b were found to be most highly enriched across samples. PCA, with 54% coverage, indicated urine sEVs segregated to +PC1, while serum and plasma sEVs intermixed in a -PC1 cluster. Plasma samples were furthermore found to cluster on -PC2, while most coming from serum remained on +PC2.
Discussion: Analysis of sEVs isolated using the iDEP device was found comparable to those isolated using conventional techniques, including differential ultracentrifugation (DU) and size-exclusion chromatography (SEC). cFCM analysis has previously indicated 38.1% exosomes positive for both CD63 and CD81 when using DU for isolation from Broncho Alveolar Lavage fluid samples4. iFCM analyses have reported comparable CD63+ expressions (~1 x 107 EVs/mL for DU and 3 X 107 EVs/mL for SEC) and CD81+ (1.5 x 106 EVs/mL for DU) from plasma and culture media, respectively5,6. Additionally, miRNAs known to be highly expressed in cancers were observed to be enriched across sample types7. This affirms our isolation methodology as a viable alternative to those currently established.
Conclusion: The utility of a label-free iDEP device in isolating sEVs from serum, plasma, and urine was demonstrated by performing NTA and multiparametric characterization using cFCM, iFCM, and miRNA sequencing. The comprehensive characterization verified that sEVs were successfully isolated from biofluids with minimal impact to vesicles’ integrity. The iDEP device hence has potential to be further evolved as a liquid biopsy platform for rapid isolation of sEVs based on their size and dielectric properties in clinical settings.
1. Shi et al., Sci Rep, 2018
2. Shi et al., Lab Chip, 2019
3. Shi et al., Plos One, 2019
4. Hough et al., Methods, 2020
5. Mastoridis et al., Front Immunol, 2018
6. Gorgens et al., J Extracell Vesicles, 2019
7. Bertoli et al., Theranostics, 2015
From Prototype to Product: Pitfalls and Possibilities – Nancy Schoenbrunner, Ph.D. (AmplifiDx)
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.