• Skip to content

Lam Lab

Wilbur A. Lam lab

  • People
  • Research
  • Publications
  • HealthReach
  • iCLOTS software
  • Contact

Research

Significant differences in single-platelet biophysics exist across species but attenuate during clot formation

Interspecies platelet differences
When observed collectively in 5 single-platelet assays (ie, including single-platelet contraction cytometry, adhesion on fibrinogen and collagen, and spreading area on fibrinogen and collagen), the murine platelet exhibited the most similarity to the human platelet, with a relative similarity of 95.4%.

Lead researcher: Oluwamayokun “Mo” Oshinowo

Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas human and murine platelets are similar. Multiple biophysical parameters integrate during clot formation, measured by bulk clot contraction, and attenuate biophysical differences.


A microengineered vascularized bleeding model that integrates the principal components of hemostasis

Microfluidic bleeding device to investigate clinical clotting mechanisms
Endothelial cells are cultured to confluence in the vascular channel while the pneumatic valve is in the closed position (left). When the valve layer is pulled down by negative pressure in valve actuator channel (pull) and mechanical fluidic pressure (push) is applied through outlet channel, the vascular channel is disrupted and endothelial injury is mechanically induced (middle). Blood is perfused while the valve is maintained in the open position and flows through the vascular channel (horizontal) as well as the newly created “bleeding” wound channel, which leads to the outlet channel (right).

Lead researchers: Yumiko Sakurai, MS, Elaissa Hardy, PhD, and Byungwook Ahn, PhD

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an “endothelialized” microfluidic system coupled with a microengineered pneumatic valve that induces a vascular “injury”. With perfusion of whole blood, hemostatic plug formation is visualized and “in vitro bleeding time” is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.


Microvasculature-on-a-chip for the long-term study of endothelial barrier dysfunction and microvascular obstruction in disease

Long-term microvasculature-on-a-chip
Representative 3D confocal microscope images of endothelialized microchannels occluded by sickle RBCs (pre-stained with R18; pointed by arrows) and the resultant, co-localized increased endothelial permeability and leakage of BSA-AF488 in situ.

Lead researcher: Yongzhi Qiu, PhD

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and hematologic disorders such as sickle cell disease (SCD) and malaria often lead to increased endothelial permeability, haemolysis, and microvascular obstruction. However, the associations among these pathological phenomena remain unknown. Here, we report a perfusable, endothelialized microvasculature-on-a-chip featuring an interpenetrating-polymer-network hydrogel that recapitulates the stiffness of blood-vessel intima, basement membrane self-deposition and self-healing endothelial barrier function for longer than 1 month. The microsystem enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found how extracellular heme, a hemolytic byproduct, induces delayed but reversible endothelial permeability in a dose-dependent manner, and demonstrate that endothelial interactions with SCD or malaria-infected erythrocytes cause reversible microchannel occlusion and increased in situ endothelial permeability. The microvasculature-on-a-chip enables mechanistic insight into the endothelial barrier dysfunction associated with SCD, malaria and other inflammatory and haematological diseases.


Interdigitated microelectronic bandage augments hemostasis and clot formation at low applied voltage in vitro and in vivo

Electric bandage device for clinical use
Electrical bandage prototype with gold interdigitated electrical leads.

Lead researcher: Elaissa Hardy, PhD

Hemorrhage or uncontrolled bleeding can arise either due to a medical condition or from a traumatic injury and are typically controlled with the application of a hemostatic agent. Hemostatic agents are currently derived from animal or human products, which carry risks of blood borne infections and immune dysregulation. Therefore, the need exists for novel biomedical therapies not derived from animal or human products to achieve hemostasis. Accordingly, we created an interdigitated microelectronic bandage that applies low voltage electrical stimulation to an injury site, resulting in faster clot formation without excessive heating, accelerated fibrin formation, and hemostasis overall. Our interdigitated microelectronic bandage found fibrin formed 1.5× faster in vitro. In vivo, total cessation of bleeding was 2.5× faster, resulting in 2× less blood loss. Electricity has been used in medical applications such as defibrillation, cauterization, and electrosurgery, but scant research has focused on hemostasis. Here we report a novel surface treatment using an interdigitated microelectronic device that creates rapid hemostasis in both in vitro and in vivo bleeding models with low applied voltages, representing a new and novel class of hemostatic agents that are electrically-based.


Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency

Microfluidic transduction
Microfluidics bring LV particles within closer proximity to target cells without requiring large quantities that would otherwise be wasted due to their short half-life.

Lead researcher: Reginald Tran, PhD

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1+) and human (CD34+) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.


Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts

Leukocyte margination in microfluidic devices after clinical drug treatment
Under homeostatic conditions, leukocytes marginate via two main mechanisms: sequestration within the microvascular capillary beds and movement toward the vessel wall in veins.

Lead researchers: Meredith Fay and David Myers, PhD

Abstract: Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.

Copyright © 2023 · Author Pro on Genesis Framework · WordPress · Log in