Biology subjects

Pratt, S. L.

Publications and source records attributed to Pratt, S. L..

3 recordsLinked to original sources

bioRxiv2025

Single-cell bacterial culturing and antibiotic susceptibility testing using permeable hydrogel-shelled microcapsules

Microbial communities, such as biofilms, consist of bacteria that exhibit cell-to-cell heterogeneity in their physiological properties, including enzyme activity and gene expression. This single-cell heterogeneity influences the communitys overall metabolic activity and contributes to stress tolerance, such as antimicrobial resistance. To study the impact of single-cell heterogeneity on population-level behaviors, methods have been developed to isolate and characterize bacteria at the single-cell level. One such method includes water-in-oil drop-based microfluidics. However, a limitation of this approach is that the droplet contents cannot be exchanged during experiments, making it difficult to investigate how cells respond to changing environmental conditions. To address this limitation, we developed a drop-based microfluidic technique called Bioflex, which creates permeable hydrogel-shell microcapsules that act as growth chambers for individual bacterial cells. The microcapsules are formed by crosslinking a shell made from a PEG-based hydrogel (4-arm PEG-maleimide) around a dextran core. After cross-linking, the dextran core diffuses out of the capsules and is replaced with buffer or growth medium. By adjusting fluid flow rates during the process, we can control the size and shell thickness of the microcapsules, allowing the production of different capsule architectures. The Bioflex capsules are biocompatible, supporting the encapsulation and growth of Pseudomonas aeruginosa by allowing nutrient transport across the capsule shell. Moreover, the capsules remained permeable to antimicrobial treatments introduced during P. aeruginosa incubation. For example, P. aeruginosa cells in Bioflex capsules responded to externally delivered ciprofloxacin treatments, with their responses varying depending on the timing of the antibiotic introduction. In summary, Bioflex capsules provide a novel, high-throughput platform for isolating single-cells and testing how single-cell derived communities react to time-dependent stresses, such as antibiotic treatments. Graphical AbstractBacterial cultivation using permeable microscale Bioflex capsules. (A) P. aeruginosa PAO1 expressing eGFP cultured in a Bioflex capsule starting from a single cell. (B) Bioflex capsules are permeable to nutrient and waste transport. (C) The capsules allow for exchange of media to study the responses of single cells or small populations of cells within the drops using time-lapse imaging. O_FIG O_LINKSMALLFIG WIDTH=101 HEIGHT=200 SRC="FIGDIR/small/647129v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@69d8bforg.highwire.dtl.DTLVardef@11507b6org.highwire.dtl.DTLVardef@1ad6649org.highwire.dtl.DTLVardef@1167ae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Neratinib Synergizes with Trastuzumab Antibody Drug Conjugate or with Vinorelbine to Treat HER2 Mutated Breast Cancer Patient Derived Xenografts and Organoids.

HER2 (ERBB2) is a major therapeutic drug target in breast cancer and The Cancer Genome Atlas (TCGA) Breast Cancer project and other studies have identified HER2 activating mutations in breast cancers without HER2 gene amplification. HER2 activating mutations occur in 2-5% of metastatic breast cancer patients (MBC), and clinical trials have shown that the irreversible pan-HER tyrosine kinase inhibitor, neratinib, produces a 31-40% clinical benefit rate for HER2 mutated MBC patients. We developed breast cancer patient-derived xenografts (PDX) from ER+, HER2 mutated MBC patients and used them to test neratinib-based drug combinations. Using organoid culture of these PDX breast cancer cells, we performed rapid, high-throughput ex vivo screening assays to test novel drug combinations. These organoid culture experiments identified drug synergy with the neratinib plus ado-trastuzumab emtansine (T-DM1) and neratinib plus vinorelbine combinations and we validated these results with in vivo PDX experiments. Statement of SignificancePDXs are a ready source of human cancer organoids, and with thousands of PDXs already available worldwide, PDX derived organoids (PDxOs) can dramatically accelerate cancer drug testing. This strategy of PDxO drug testing is particularly useful for rare cancer subtypes or mutations to identify the most promising treatment strategies for clinical trials testing.

cancer biology↗

bioRxiv2023

Single-cell Herpes Simplex Virus type-1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics

Single-cell analyses of viral infections often reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of HSV-1 infection of neurons at the single-cell level. We used micron-scale Matrigel beads, termed microgels, to culture individual murine Superior Cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are subsequently enclosed in individual media-in-oil droplets with a dual fluorescent-reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. The innovative techniques presented here for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

bioengineering↗