Biology subjects

Lee, A. P.

Publications and source records attributed to Lee, A. P..

7 recordsLinked to original sources

bioRxiv2024

A Microphysiological HHT-on-a-Chip Platform Recapitulates Patient Vascular Lesions

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations focally develop in multiple organs. These can be small (telangiectasias) or large (arteriovenous malformations, AVMs) and may rupture leading to frequent, uncontrolled bleeding. There are few treatment options and no cure for HHT. Most HHT patients are heterozygous for loss-of-function mutations for Endoglin (ENG) or Alk1 (ACVRL1), however, why loss of these genes manifests as vascular malformations remains poorly understood. To complement ongoing work in animal models, we have developed a microphysiological system model of HHT. Based on our existing vessel-on-a-chip (VMO) platform, our fully human cell-based HHT-VMO recapitulates HHT patient vascular lesions. Using inducible ACVRL1 (Alk1)-knockdown, we control timing and extent of endogenous Alk1 expression in primary human endothelial cells (EC) in the HHT-VMO. HHT-VMO vascular lesions develop over several days, and are dependent upon timing of Alk1 knockdown. Interestingly, in chimera experiments AVM-like lesions can be comprised of both Alk1-intact and Alk1-deficient EC, suggesting possible cell non-autonomous effects. Single cell RNA sequencing data are consistent with microvessel pruning/regression as contributing to AVM formation, while loss of PDGFB expression implicates mural cell recruitment. Finally, lesion formation is blocked by the VEGFR inhibitor pazopanib, mirroring the positive effects of this drug in patients. In summary, we have developed a novel HHT-on-a-chip model that faithfully reproduces HHT patient lesions and that is sensitive to a treatment effective in patients. The VMO-HHT can be used to better understand HHT disease biology and identify potential new HHT drugs. SignificanceThis manuscript describes development of an organ-on-a-chip model of Hereditary Hemorrhagic Telangiectasia (HHT), a rare genetic disease involving development of vascular malformations. Our VMO-HHT model produces vascular malformations similar to those seen in human HHT patients, including small (telangiectasias) and large (arteriovenous malformations) lesions. We show that VMO-HHT lesions are sensitive to a drug, pazopanib, that appears to be effective in HHT human patients. We further use the VMO-HHT platform to demonstrate that there is a critical window during vessel formation in which the HHT gene, Alk1, is required to prevent vascular malformation. Lastly, we show that lesions in the VMO-HHT model are comprised of both Alk1-deficient and Alk1-intact endothelial cells.

cell biology↗

bioRxiv2023

Three-dimensional Isotropic Imaging of Live Suspension Cells Enabled by Droplet Microvortices

Three-dimensional (3D) imaging of non-adherent cells in suspension media is challenging due to their propensity to drift when not fixed to a substrate, as required by optical sectioning technologies. Resolution differences in the lateral versus depth directions typically present in those systems further complicates single-cell morphometry of cellular features indicative of effector functions, such as cytosol and organelle volumetric distribution, and cell membrane topography. Here, we present a method for 3D fluorescent isotropic imaging of live, non-adherent single cells encapsulated in picoliter droplets using Optical Projection Tomography (OPT) enabled by droplet microvortices. Our microfluidic platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intra-droplet microvortices, which in turn are modulated to rotate single-cells on their axis to enable OPT-based imaging. This strategy allows observation of cells encapsulated inside non-toxic isotonic buffer droplets and facilitates scalable OPT acquisition by the simultaneous spinning of hundreds of cells. Specifically, we demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells--small cells prone to movement in their suspended phenotype. In addition, morphometry of primary T cells under different immunological activation states allowed us to identify six distinct nuclear content distributions, which differ from the conventional 2D images depicting spheroid and bean-like nuclear shapes commonly associated with lymphocytes. This Arrayed-Droplet Optical Projection Tomography (ADOPT) technology is capable of isotropic, single live-cell 3D imaging and has the potential to perform large-scale morphometry of immune cell effector function states, while providing compatibility with microfluidic droplet operations.

biophysics↗

bioRxiv2023

Titering of Chimeric Antigen Receptors on CAR T Cells enabled by a Microfluidic-based Dosage-Controlled Intracellular mRNA Delivery Platform

Chimeric antigen receptor (CAR) T-cell therapy shows unprecedented efficacy for cancer treatment, particularly in treating patients with various blood cancers, most notably B-cell acute lymphoblastic leukemia (B-ALL). In recent years, CAR T-cell therapies are being investigated for treating other hematologic malignancies and solid tumors. Despite the remarkable success of CAR T-cell therapy, it has unexpected side effects that are potentially life threatening. Here, we demonstrate the delivery of approximately the same amount of CAR gene coding mRNA into each T cell propose an acoustic-electric microfluidic platform to manipulate cell membranes and achieve dosage control via uniform mixing, which delivers approximately the same amount of CAR genes into each T cell. We also show that CAR expression density can be titered on the surface of primary T cells under various input power conditions using the microfluidic platform.

bioengineering↗

bioRxiv2023

3D Confinement-enabled Priming of Synaptic Activation Promotes Primary T Cell Expansion

The success of autologous cell therapy, which depends highly on T lymphocyte expansion efficiency, is often hindered by suboptimal interactions between T-cell receptors and peptide-MHC molecules. Here, we demonstrate 3D confinement-enabled priming of T cell-MHC immune synapse junctions based on cytoskeletal forces within minutes, which is 200-fold faster than conventional 24 h bulk shaking method. Using T cell-Dynabead binding skeletons in the starting culture, two- to six-fold greater T cell expansion was achieved over the conventional T cell expansion approach without inducing excessive cell exhaustion. Under 3D force-confinement, T-cell division (G1, S, and G2 phases) was increased to be twice as fast. Creating 3D T cell-Dynabead skeletons as the "booster" material enables highly efficient T cell expansion, without requiring complex surface modification of antigen-presenting cells. This method can be modularly adapted to existing T cell expansion processes for a wide range of applications including adoptive cell therapies. Teaser3D confinement-enabled priming of synaptic activation enables radically faster autologous cell production.

immunology↗

bioRxiv2021

High-throughput and dosage-controlled intracellular delivery of large cargos by an acoustic-electric micro-vortices platform

Intracellular delivery of cargos for cell engineering plays a pivotal role in transforming medicine and biomedical discoveries. Recent advances in microfluidics and nanotechnology have opened up new avenues for efficient, safe, and controllable intracellular delivery, as they improve precision down to the single-cell level. Based on this capability, several promising micro- and nanotechnology approaches outperform viral and conventional non-viral techniques in offering dosage-controlled delivery and/or intracellular delivery of large cargos. However, to achieve this level of precision and effectiveness, they are either low in throughput, limited to specific cell types (e.g., adherent vs. suspension cells), or complicated to operate with. To address these challenges, here we introduce a versatile and simple-to-use intracellular delivery microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP). Hundreds of acoustic microstreaming vortices form the production line of the AESOP platform, wherein hundreds of thousands of cells are trapped, permeabilized, and mixed with exogenous cargos. Using AESOP, we show intracellular delivery of a wide range of molecules (from <1 kDa to 2 MDa) with high efficiency, cell viability, and dosage-controlled capability into both suspension and adherent cells and demonstrate throughput at 1 million cells/min per single chip. In addition, we demonstrate AESOP for two gene editing applications that require delivery of large plasmids: i) eGFP plasmid (6.1 kbp) transfection, and ii) CRISPR-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and sgRNA. Compared to alternative platforms, AESOP not only offers dosage-controlled intracellular delivery of large plasmids (>6kbp) with viabilities over 80% and comparable delivery efficiencies, but also is an order of magnitude higher in throughput, compatible with both adherent and suspension cell lines, and simple to operate.

bioengineering↗

bioRxiv2020

An In Vitro Vascularized Micro-Tumor Model of Human Colorectal Cancer Recapitulates In Vivo Drug Responses

Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated the VMT platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment response in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional (2D) monolayer culture and 3-dimensional (3D) spheroids.

cancer biology↗

bioRxiv2019

Multi-omics profiling of CHO parental hosts reveals cell line-specific variations in bioprocessing traits

Chinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human-like post-translational modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines have been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO-K1, CHO-DXB11 and CHO-DG44) in suspension cultures and further report their growth-related characteristics, and N- and O-glycosylation patterns of host cell proteins (HCPs). The comparative multi-omics analysis indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N- and O-glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced.

systems biology↗