Biology subjects

Poepping, T. L.

Publications and source records attributed to Poepping, T. L..

4 recordsLinked to original sources

bioRxiv2026

Development of a rocking bioreactor strategy to augment pro-angiogenic factor secretion by human adipose-derived stromal cells

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-{gamma} and TNF-. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

bioengineering↗

bioRxiv2024

Intussusceptive angiogenesis-on-a-chip: Evidence for transluminal vascular bridging by endothelial delamination

Intussusceptive angiogenesis is an increasingly recognized vessel duplication process that generates and reshapes microvascular beds. However, the mechanism by which a vessel splits into two is poorly understood. Particularly vexing is formation of the hallmark transluminal endothelial cell bridge. How an endothelial cell comes to cross a flowing lumen rather than line it is enigmatic. To elucidate this, we used a microvessel-on-a-chip strategy, creating a micro-conduit coherently lined with flow-sensitive endothelial cells but in which transluminal bridges also formed. Bridge morphologies ranged from filamentous strand to multicellular columns with a central core. These bridge architectures were found to recapitulate those in microvessels in embryos, tumours, diseased organs, and the dermis of patients with limb-threatening ischemia. Time-lapse, multi-plane, 3D microscopy of the micro-physiologic conduit revealed that bridges arose from endothelial cells oriented orthogonal to flow that partially released from the wall while retaining attachments at the ends. This delamination process was blocked by hyperactivation of Rho and augmented by interventions that weaken cell-substrate interactions, including inhibiting non-muscle myosin II and blocking 5{beta}1 integrin but, interestingly, not v{beta}3 integrin. Thus, endothelial cells can leave their monolayer and transect a flowing lumen through controlled delamination. This previously unrecognized lumen entry program could explain the launch of intussusceptive angiogenesis and opens a framework for intervening. Significance StatementRapid generation of small blood vessels is vital for embryonic development and many diseases. An efficient means of creating a new microvessel is for an existing vessel to split into two, a process recognized for over 35 years. However, the cellular events underlying vessel splitting remain largely a mystery. The challenge is how to look inside a microvessel and capture transient events. We address this challenge using a microvessel-on-a-chip strategy. We discovered that select endothelial cells lining the wall can partially lift to transect the lumen. This resolves the paradox of an adhesion-dependent cell reaching across a pressurized lumen. It also reframes microvascular therapy considerations, including for diabetics with leg ulcers, a condition we show has hallmarks of microvessel splitting.

physiology↗

bioRxiv2024

A simple, cost-effective microfluidic device using a 3D cross-flow T-junction for producing decellularized extracellular matrix-derived microcarriers

Cell therapies using human mesenchymal stromal cells (MSCs) are promising for a wide variety of clinical applications. However, broad-scale clinical translation is limited by conventional culture methods for MSC expansion within 2D tissue-culture flasks. MSC expansion on ECM-derived microcarriers within stirred bioreactor systems offers a promising approach to support MSC growth. Previously, our team established methods for fabricating ECM-derived microcarriers from a variety of decellularized tissue sources using electrospraying techniques. However, these microcarriers are relatively large and have a broad size distribution, which may limit their utility. Smaller and more uniform microcarriers may be favorable for MSC growth within bioreactors and have greater potential to serve as a minimally invasive injectable cell delivery platform. To address these limitations, the current project focused on the development of a new microfluidic-based approach enabling both uniform and small microcarrier production. Using a novel, modified 3D T-junction design, we successfully generated microcarriers using human decellularized adipose tissue (DAT) as the ECM source. Our new cost-effective device produced microbeads that were small and monodisperse, at a range of flow rate combinations and with high production rates. Photo-crosslinking using rose bengal allowed for the generation of microcarriers that were stable following rehydration, with a mean diameter of 196 {+/-} 47 {micro}m. Following methods optimization and microcarrier characterization, in vitro studies confirmed that the new microcarriers supported human adipose-derived stromal cell (hASC) attachment and growth, as well as ECM production, across 14 days within spinner flask bioreactors. Overall, this study demonstrates the feasibility of using our novel, cost-effective, and reusable microfluidics device to generate cell-supportive microcarriers comprised exclusively of ECM in a size range that could be injected via a small gauge needle and were stable in long-term culture.

bioengineering↗

bioRxiv2018

Membrane Lipid-KIR2.x Channel Interactions Enable Hemodynamic Sensing in Cerebral Arteries

Inward rectifying (KIR) K+ channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. We explored whether vascular KIR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. A KIR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries and molecular analyses confirmed KIR2.1/KIR2.2 mRNA and protein expression. Electrophysiology next revealed that endothelial KIR was sensitive to phosphatidylinositol 4,5- bisphosphate (PIP2), with depletion impairing flow-induced activation of the channel. In contrast, smooth muscle KIR was sensitive to membrane cholesterol, with sequestration blocking pressures ability to inhibit this channel. Membrane lipids helped confer KIR mechanosensitivity to intact arteries; virtual models then reconceptualised KIR as a dynamic regulator of basal tone development. We conclude that specific membrane lipid-KIR interactions enable unique channel populations to sense hemodynamic stimuli and set brain perfusion.

neuroscience↗