Biology subjects

Botchwey, E.

Publications and source records attributed to Botchwey, E..

7 recordsLinked to original sources

bioRxiv2026

Volumetric Scattering Microscopy

Optical scattering in biological tissues fundamentally limits fluorescence imaging by disrupting spatial and angular information, thereby restricting volumetric visualization. Although hardware-intensive and computational approaches have advanced scattering microscopy, practical three-dimensional imaging through tissue remains constrained by instrumental complexity and axial ambiguity. Here, we present volumetric scattering microscopy (VSM), a scan-free, optical-computational framework for three-dimensional fluorescence imaging in scattering biological media. VSM captures angularly resolved speckle-encoded fluorescence using an aperture-segmented Fourier light-field configuration and reconstructs volumetric structure through adaptive feature-based descattering and joint sub-pupil alignment. This hybrid strategy preserves angular information embedded in scattered light without wavefront measurement or mechanical scanning, while maintaining the simplicity of a standard epi-fluorescence architecture. We demonstrate high-fidelity volumetric reconstruction across phantoms, engineered cellular systems, ex vivo tissues with volumetric muscle loss, and intact Xenopus embryos, achieving preserved spatial resolution, enhanced optical sectioning, and quantitative accuracy under strong scattering conditions. VSM supports large-field, robust volumetric imaging in both layered and fully embedded scattering environments. By transforming scattered light into a structured encoding resource, VSM establishes a scalable pathway toward routine three-dimensional fluorescence imaging in complex biological systems.

bioengineering↗

bioRxiv2026

PAVR: High-Resolution Cellular Imaging via a Physics-Aware Volumetric Reconstruction Framework

The rapid convergence of advanced microscopy and deep learning is transforming cell biology by enabling imaging systems in which optical encoding and computational inference are jointly optimized for volumetric information capture and interpretation. However, broadly accessible three-dimensional imaging at high spatiotemporal resolution remains constrained by volumetric reconstruction throughput, susceptibility to artifacts, and the burden of collecting modality-matched training data. Here, we introduce PAVR, a physics-aware light-field imaging platform that integrates single-shot volumetric acquisition with fast, end-to-end volumetric reconstruction. PAVR is trained entirely using in silico system responses, avoiding reliance on external high-resolution ground-truth modalities and enabling sample-independent reconstruction across diverse biological contexts. Using fixed and live mammalian cells, we demonstrate multicolor volumetric imaging of subcellular organelles, three-dimensional tracking of autofluorescent particles, and high-speed visualization of organelle remodeling and interactions. We further extend PAVR to quantify coupled morphological and functional dynamics in beating human induced pluripotent stem cell-derived cardiomyocytes under pharmacological perturbation. Together, PAVR establishes a scalable hardware-software platform for high-throughput volumetric imaging and quantitative analysis of dynamic cellular systems in both basic and translational settings.

bioengineering↗

bioRxiv2025

Sphingomyelinase Licensing of Mesenchymal Stromal Cells Alters Lipid and Protein Metabolites for Immunomodulation

Mesenchymal stromal cells (MSCs) are widely studied for their immunomodulatory and tissue reparative capabilities, but clinical translation has been hampered by inconsistent efficacy and limited standardization in manufacturing. While cytokine-based priming methods, such as interferon-gamma (IFN-{gamma}) stimulation, have shown promise in enhancing MSC potency, alternative approaches targeting distinct biological metabolism integral to secretome and membrane architecture have not been explored in MSCs. In this study, we investigate sphingomyelinase (SMase), an enzyme that generates ceramide from sphingomyelin, as a novel lipid-based priming strategy to modulate MSC function. Here, human MSCs were treated with SMase and high-content imaging and morphological profiling revealed that SMase-treated cells adopted a phenotype overlapping with IFN-{gamma}-licensed MSCs, including increased cell compactness and solidity. Lipidomic analysis showed broad alterations in sphingolipid species, and dynamic flux estimation (DFE) modeling predicted distinct metabolic shifts in SMase-treated cells compared to untreated controls. These changes were sustained up to 35 hours post-stimulation, indicating stable metabolic reprogramming. SMase priming also altered the MSC secretome, enriching for factors implicated in immune regulation. Functionally, SMase-primed MSCs retained the ability to suppress T-cell activation and promote anti-inflammatory macrophage phenotypes. Collectively, these findings demonstrate that SMase stimulation induces a durable, immunomodulatory-like state in MSCs through coordinated changes in lipid metabolism and secretory activity. This lipid-centric priming approach represents a promising alternative to cytokine-based licensing strategies and may support therapeutic MSC products.

bioengineering↗

bioRxiv2025

Resolving vs. Non-resolving Sphingolipid Dynamics During Macrophage Activation: A Time-resolved Metabolic Analysis

Sphingolipids are increasingly recognized as critical regulators of inflammation and cell fate decisions, with metabolites such as ceramide and sphingosine 1{square}phosphate exerting contrasting effects on cell survival and proliferation. In macrophages, this balance is especially important, given their central role in host defense, pathogenesis and wound healing. Here, we present a time{square}resolved model of sphingolipid metabolism in RAW 264.7 macrophages stimulated with KdO{square}-Lipid A. By integrating measured metabolite concentrations with dynamic flux estimation and established enzyme kinetics, we systematically map dynamic changes in the sphingolipid network during inflammation. Our results reveal a three{square}phase pattern of sphingolipid remodeling that correlates with distinct functional states of the cell. Moreover, metabolites can be classified into "resolving" or "non{square}resolving" lipids based on whether they return to basal levels or remain dysregulated through the later phases of the inflammatory response. This partitioning suggests that targeted modulation of specific metabolic nodes may influence the resolution of inflammation. Importantly, our computational approach can assist in the rational design of experimental studies by pinpointing putative drug targets with maximal impact on sphingolipid homeostasis. Such targeted interventions may prevent the pathological amplification of inflammatory signals without globally suppressing essential sphingolipid functions. These findings highlight the utility of an integrative systems{square}level analysis for elucidating sphingolipid dynamics in macrophages and underscore its potential to guide therapeutic strategies against conditions involving dysregulated inflammation.

immunology↗

bioRxiv2023

Harnessing Bilayer Biomaterial Delivery of FTY720 as an Immunotherapy to Accelerate Oral Wound Healing

Orofacial clefts are the most common craniofacial congenital anomaly. Following cleft palate repair, up to 60% of surgeries have wound healing complications leading to oronasal fistula (ONF), a persistent connection between the roof of the mouth and the nasal cavity. The current gold standard methods for ONF repair use human allograft tissues; however, these procedures have risks of graft infection and/or rejection, requiring surgical revisions. Immunoregenerative therapies present a novel alternative approach to harness the bodys immune response and enhance the wound healing environment. We utilized a repurposed FDA-approved immunomodulatory drug, FTY720, to reduce the egress of lymphocytes and induce immune cell fate switching toward pro-regenerative phenotypes. Here, we engineered a bilayer biomaterial system using Tegaderm, a liquid-impermeable wound dressing, to secure and control the delivery of FTY720- nanofiber scaffolds (FTY720-NF). We optimized release kinetics of the bilayer FTY720-NF to sustain drug release for up to 7d with safe, efficacious transdermal absorption and tissue biodistribution. Through comprehensive immunophenotyping, our results illustrate a pseudotime pro-regenerative state transition in recruited hybrid immune cells to the wound site. Additional histological assessments established a significant difference in full thickness ONF closure in mice on Day 7 following treatment with bilayer FTY720-NF, compared to controls. These findings demonstrate the utility of immunomodulatory strategies for oral wound healing, better positing the field to develop more efficacious treatment options for pediatric patients. One Sentence SummaryLocal delivery of bilayer FTY720-nanofiber scaffolds in an ONF mouse model promotes complete wound closure through modulation of pro-regenerative immune and stromal cells.

bioengineering↗

bioRxiv2022

UNDESRTANDING MESENCHYMAL STEM CELL IMMUNE POTENCY:A MORPHOLOMIC AND LIPIDOMIC PERSPECTIVE

Interest in human mesenchymal stem cells (MSCs) as an immune therapy has been on the rise for the past two decades with cutting edge research yielding promising results, but there are currently no MSC therapies approved by the food and drug administration (FDA). Failure of MSCs to translate as a therapy has been reported by the National Cell Manufacturing Consortium (NCMC) to be due to a lack of reliable potency metrics and sufficient understanding of the mechanism of action. Here we show that cell membrane components are a good candidate to interrogate the MSC immunomodulatory mechanism of action and provide a method to increase MSC potency through the sphingolipid pathway. We found that high and low indolamine-2,3-deoxygenase (IDO) potency cells have distinct morphological signatures that is also reflected in the sphingolipid activity, with low IDO potency cell lines having low sphingomyelinase activity and high IDO potency cell lines having high sphingomyelinase activity. Perturbation of the salvage pathway with the addition of exogenous neutral sphingomyelinase not only shifted morphological signatures to a high potency profile, but also significantly increased IDO activity within both high and low IDO potency donors. These results provide a proof of concept for the engineering of MSC immunomodulation and provides further evidence for the role sphingolipids in MSC immunomodulation that can enable further investigation.

cell biology↗

bioRxiv2021

Characterizing Human Mesenchymal Stromal Cells Immune Modulatory Potency Using Targeted Lipidomic Profiling of Sphingolipids

Cell therapies are expected to increase over the next decade due to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized bone-marrow (BM), adipose derived and umbilical cord tissue MSCs sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography - tandem mass spectrometry. We found ceramide levels differed based upon donors sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all 3 sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed IFN-{gamma} primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low IDO MSCs, indicating sphingomeylinase or ceramidase enzymatic activity may be involved in their immune potency.

bioengineering↗