Fluorescence-Based Quantification of Hepatic Ldl Uptake and Donor Hepatocyte Engraftment Using ATTO 700–Labeled LDL

Author

• Victor Agustin Cortes Flores, Clemson UniversityFollow

Date of Award

5-2026

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Bioengineering

Committee Chair/Advisor

Dr. Renee Cottle

Committee Member

Dr. Alexis Stamatikos

Committee Member

Dr. Naren Vyavahare

Abstract

Familial hypercholesterolemia (FH) is an inherited disorder characterized by elevated low-density lipoprotein cholesterol (LDL-C) that increases the risk of premature atherosclerotic cardiovascular disease. In severe FH, including low-density lipoprotein receptor (LDLR), hepatic LDL clearance is profoundly impaired and many lipid lowering treatment strategies have limited effectiveness. Current advances in ex vivo CRISPR–Cas9 hepatocyte gene editing in vivo and ex vivo approaches for treatment of FH are under development.

However, a central barrier to translation is the ability to quantitatively measure the percentage of healthy gen edited hepatocytes in the liver while simultaneously assessing functional hepatic LDL handling. This thesis establishes a fluorescence-based hepatic LDL uptake and imaging platform using near-infrared ATTO 700–labeled human LDL (ATTO 700–LDL) to enable quantitative, longitudinal assessment of LDL uptake dynamics and to support engraftment studies following hepatocyte transplantation of gene modified healthy hepatocytes. In our studies, human LDL was fluorescently labeled via NHS-ester chemistry under defined buffering conditions, with multiple dye concentrations evaluated to optimize fluorescence while maintaining stability.

Excess unbound dye was removed using 100 kDa centrifugal filtration and standardized handling procedures were implemented to minimize aggregation and preserve functional LDL uptake behavior. Probe function was validated in vitro using Hepa 1-6 hepatocytes. Incubation with ATTO 700–LDL produced robust cellular fluorescence quantified by flow cytometry, while heparin-treated controls demonstrated reduced signal, supporting receptor-mediated internalization and confirming that labeling preserved biologically relevant uptake. The assay was then translated in vivo using C57BL/6J and Ldlr−/− mice following intravenous administration of ATTO 700–LDL (5 mg/kg) conjugates and whole-body and explanted organ fluorescence imaging on an IVIS Spectrum system across early kinetic and extended post-injection time points using an optimized acquisition workflow.

To enable robust quantification of engraftment and cell fate, this platform is designed to integrate with hepatocyte transplantation experiments in which LDLR+/+ hepatocytes isolated from mT/mG donor mice are transplanted into Ldlr−/− recipients. This approach enables visualization and quantitative measurement of the localization, persistence, and fate of transplanted, gene-edited hepatocytes in vivo, providing a disease-relevant context for assessing how donor-cell engraftment influences hepatic LDL uptake.

Ex vivo organ imaging further supported tissue-level quantification, with fluorescence localized predominantly to liver and gallbladder, consistent with hepatobiliary processing and enabling standardized region-of-interest analyses across animals. Collectively, these studies establish ATTO 700–LDL conjugates as a stable, functional biomolecule and define a standardized workflow for in vitro validation, in vivo kinetic imaging, and ex vivo biodistribution to quantify LDL uptake and processing. This platform provides a quantitative foundation for tracking gene-edited hepatocyte engraftment and evaluating functional hepatic LDL uptake in FH mouse models, thereby supporting the development of gene-editing therapies for familial hypercholesterolemia.

Recommended Citation

Cortes Flores, Victor Agustin, "Fluorescence-Based Quantification of Hepatic Ldl Uptake and Donor Hepatocyte Engraftment Using ATTO 700–Labeled LDL" (2026). All Theses. 4749.
https://open.clemson.edu/all_theses/4749