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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering Undergraduate Fire Talks

Advancing cell therapies with in vitro barrier tissue models

Saturday, October 11, 2025

8:35 AM - 8:40 AM PDT

Location: Room 33B

Presenting Author(s)

Annika Deans, BS

Undergraduate Researcher
University of Rochester
Rochester, New York, United States

Co-Author(s)

Isabelle Linares, MS

PhD Candidate
University of Rochester
Penfield, New York, United States
LT

Lydia Taylor

Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
MM

Michael Malloy

Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester

Primary Investigator(s)

CM

Craig Morrell

Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester
TD

Tara Deans

Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
James McGrath, PhD

Department of Biomedical Engineering, University of Rochester, United States

Introduction: : There is an unmet need for medical technologies that can deliver therapeutic molecules or cellular treatments not possible with conventional pharmaceutical or surgical approaches. Platelets, derived from rare bone marrow megakaryocytes (MKs), are anucleate cells that circulate the bloodstream at 150K-450K platelets/µL. They are equipped with membrane proteins that bind to damaged vessels, receptors that trigger activation, and granules that release proteins upon activation. While primarily involved in hemostasis, platelets also interact closely with immune cells, tumor cells, bacteria, and vascular endothelial cells. These properties have inspired approaches that engineer platelets for use as tools for drug delivery.

We recently demonstrated that murine and human MKs can be genetically modified during differentiation to produce engineered platelets capable of delivering non-native proteins (NNP). These engineered platelets can naturally store, traffic, and release therapeutic payloads. However, for these proteins to reach diseased tissue, they must cross endothelial cells which line the interior surface of the blood vessels.

To study this, we developed an in vitro blood vessel model to quantify protein release and migration following platelet activation. This model compares protein migration under static and flow conditions using both wild-type and engineered platelets. It is built on the fluidic µSiM (microfluidic device enabled by silicon membrane), which uses an ultrathin nanoporous membrane in a microfluidic device featuring both a flow based ‘blood’ compartment and a static basal ‘tissue’ compartment (Figure 1). This model offers a controllable in vitro platform for testing engineered platelets designed for delivery of therapeutic cargo to diseased tissue.

Materials and

Methods: : We have developed a method to differentiate pluripotent stem cells into MKs. By plating mouse embryonic stem cells (mESCs) on a layer of OP9 cells for 5 days, the mESCs differentiate into hematopoietic cells (HSCs). Once the mESCs begin to differentiate, thrombopoietin (TPO) is added to the culture. TPO is an essential cytokine which stimulates the differentiation of HSCs to become MKs. From here, we have developed two packaging techniques to incorporate non-native proteins into engineered platelets. In one, we load NNPs into the cytoplasm of platelets for delivery to neighboring cells (Figure 2A). In the second strategy, we fuse the human chemokine RANTES to NNPs. RANTES is an intracellular sorting signal that results in NNP packaging into α-granules for release upon activation (Figure 2B). In both cases, the NNPs retain their functionality after being delivered.

To quantify the release of proteins from activated platelets, we first line the µSiM device with a layer of human umbilical vein endothelial cells (HUVECs). After 24 hrs, when the HUVECs reach confluency, platelets are activated with 0.25U/mL of thrombin and placed at a concentration of 50,000 platelets/uL on the top channel of the device. The device is incubated for 30 minutes to allow the transit of proteins through the endothelial layer. The medium on the bottom channel is then collected and an ELISA is performed to quantify protein release.

Results, Conclusions, and Discussions:: Following our recently developed method of differentiating mESCs to MKs, we successfully obtained MKs and transfected them with GFP-expressing transgenes resulting in 1) an even distribution of GFP in the MK cytoplasm (Figure 2A), and 2) localized GFP expression in the α-granules (Figure 2B). Confocal imaging shows the localization of GFP throughout the differentiated MKs and the native protein, Von Willebrand factor (VWF) (Figure 2C,D). This protein distribution is transferred to platelets to enable the two methods for protein delivery. These data show that we can successfully engineer platelets and load them with non-native proteins to be used for therapeutic applications.

To first demonstrate that native proteins can cross the endothelium, we loaded pre-activated WT murine platelets into our in vitro blood vessel model seeded with HUVECs (Figure 3A,B). Following incubation, the medium from the bottom channel of the device was collected and an ELISA was performed to quantify the amount of platelet factor 4 (PF4) that crossed the endothelium. PF4, like VWF (Figure 2C,D), is a native protein that is packaged and stored in the α-granules and released upon platelet activation. The ELISA results (Figure 3C) demonstrate that proteins do in fact cross the endothelium, supporting the initiative to engineer platelets to deliver non-native proteins as cell therapies.

Additional studies are being performed to study the release of native and non-native proteins from engineered platelets in our blood vessel model. These studies will incorporate devices in both static and flow conditions to simulate blood flow (Figure 1).

Acknowledgements and/or References (Optional): :