Biology subjects

Nichols, L.

Publications and source records attributed to Nichols, L..

5 recordsLinked to original sources

bioRxiv2025

Continuous FACS sorting of double emulsion picoreactors with a 3D printed vertical mixer.

High-throughput screening and directed evolution using microfluidic picoreactors have produced high-activity enzymes. In this approach, a substrate is co-encapsulated with a candidate enzyme and individual picoreactors are sorted based on an activity reporter. While many approaches use water-in-oil droplets (single emulsions) for fluorescence-activated droplet sorting (FADS) on custom-fabricated microfluidic devices that require integrated optics and electronics, recent approaches have lowered the engineering barriers to adoption by using simple microfluidic droplet generators to produce water-in-oil-in-water droplets (double emulsion picoreactors, DEs) that can be sorted with commercial FACS (fluorescence-activated cell sorting). Despite the simplified engineering requirements, high variability in loading rates and low yield during loading are barriers to efficient DE FACS sorting. Here, we optimized surfactants to enhance DE stability and demonstrated that a 3D-printed corkscrew on the sample line acts as a vertical mixer to enable more continuous loading. With these optimized loading conditions, we analyzed 1.17 million DEs in four 10-minute sorting rounds with a mean frequency of 480 Hz (390 Hz including sample exchanges); in a mock sort of 10% fluorescent DEs, we achieved 89.2{+/-}1.1% accuracy and 78{+/-}0.9% recovery with our optimized loading protocol. Overall, improved ease-of-use and throughput for FACS sortable DEs should expand the accessibility of directed evolution in controlled in vitro environments.

bioengineering↗

bioRxiv2024

Utilization of a Human Liver Tissue Chip for Drug-Metabolizing Enzyme Induction Studies of Perpetrator and Victim Drugs

Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. Increasing number of therapeutic drugs on the market underscores the necessity to accurately assess the new drug combinations during pre-clinical evaluation for DDIs. In vitro primary human hepatocyte (PHH)-only models are commonly used only for perpetrator DDI studies due to their rapid loss of metabolic function. But co-culturing non-human cells with human PHHs can stabilize metabolic activity and be utilized for both perpetrator and victim studies, though this raises concerns about human specificity for accurate clinical assessment. In this study, we evaluated Liver Tissue Chip (LTC) with PHH-only liver microphysiological system (MPS) for DDI induction studies. Liver MPS from three individual donors maintained their functional and metabolic activity for up to 4 weeks demonstrating suitability for long-term pharmacokinetics (PK) studies. The responses to rifampicin induction of three PHH donors were assessed using CYP activity and mRNA changes. Additionally, victim PK studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following rifampicin-mediated induction which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values respectively compared to the untreated liver MPS. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on CYP activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study demonstrates the utility of the LTC for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs. SIGNIFICANCE STATEMENTThis study demonstrates the utility of the Liver Tissue Chip (LTC) with primary human hepatocyte (PHH)-only liver microphysiological system (MPS) for drug-drug interaction (DDI) induction studies. This unique in vitro system with continuous recirculation maintains long-term functionality and metabolic activity for up to 4 weeks, enabling the study of perpetrator and victim drug pharmacokinetics, quantification of drug-induced CYP mRNA and activity levels, investigation of patient variability, and ultimately clinical predictions.

bioengineering↗

bioRxiv2023

A Novel Milli-fluidic Liver Tissue Chip with continuous Recirculation for Predictive pharmacokinetics Applications

A crucial step in lead selection during drug development is accurate estimation and optimization of hepatic clearance using in vitro methods. However, current methods are limited by factors such as lack of physiological relevance, short culture/incubation times that are not consistent with drug exposure patterns in patients, use of drug absorbing materials, and evaporation during long-term incubation. To address these technological needs, we developed a novel milli-fluidic human liver tissue chip (LTC) that was designed with continuous media recirculation and optimized for hepatic cultures using human primary hepatocytes. Here, we characterized the LTC using a series of physiologically relevant metrics and test compounds to demonstrate that we could accurately predict the PK of both low and high clearance compounds. The non-biological characterization indicated that the cyclic olefin copolymer (COC)-based LTC exhibited negligible evaporation and minimal non-specific binding of drugs of varying ionic states and lipophilicity. Biologically, the LTC exhibited functional and polarized hepatic culture with sustained metabolic CYP activity for at least 15 days. This long-term culture was then used for drug clearance studies for low and high-clearance compounds for at least 12 days and clearance was estimated for a range of compounds with high in vitro in vivo correlation (IVIVC). We also demonstrated that LTC can be induced by rifampicin, and the culture age had insignificant effect on depletion kinetic and predicted clearance value. Thus, we used advances in bioengineering to develop a novel purpose-built platform with high reproducibility and minimal variability to address unmet needs for PK applications.

pharmacology and toxicology↗

bioRxiv2019

Optimized double emulsion flow cytometry with high-throughput single droplet isolation

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 m nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

bioengineering↗

bioRxiv2018

Ecological analyses of mycobacteria in showerhead biofilms and their relevance to human health

Bacteria within the genus Mycobacterium can be abundant in showerheads, and the inhalation of aerosolized mycobacteria while showering has been implicated as a mode of transmission in nontuberculous mycobacterial (NTM) lung infections. Despite their importance, the diversity, distributions, and environmental predictors of showerhead-associated mycobacteria remain largely unresolved. To address these knowledge gaps, we worked with citizen scientists to collect showerhead biofilm samples and associated water chemistry data from 656 households located across the U.S. and Europe. Our cultivation-independent analyses revealed that the genus Mycobacterium was consistently the most abundant genus of bacteria detected in residential showerheads, yet mycobacterial diversity and abundances were highly variable. Mycobacteria were far more abundant, on average, in showerheads receiving municipal versus well water, and in U.S. households as compared to European households, patterns that are likely driven by differences in the use of chlorine disinfectants. Moreover, we found that water source, water chemistry, and household location also influenced the prevalence of specific mycobacterial lineages detected in showerheads. We identified geographic regions within the U.S. where showerheads have particularly high abundances of potentially pathogenic lineages of mycobacteria and these hot spots generally overlapped with those regions where NTM lung disease is most prevalent. Together these results emphasize the public health relevance of mycobacteria in showerhead biofilms. They further demonstrate that mycobacterial distributions in showerhead biofilms are often predictable from household location and water chemistry, knowledge that advances our understanding of NTM transmission dynamics and the development of strategies to reduce exposures to these emerging pathogens.

microbiology↗