engineering microvasculature-on-chip models as research-enabling systems for hemostasis and thrombosis

Transcription

1 engineering microvasculature-on-chip models as research-enabling systems for hemostasis and thrombosis Wilbur A. Lam, MD, PhD Associate Professor Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University Department of Pediatrics Aflac Cancer and Blood Disorders Center Division of Pediatric Hematology/Oncology Children s Healthcare of Atlanta/Emory University School of Medicine

2 overview of laboratory approach microfluidics microfabrication cell mechanics of hematology in health and disease microvasculature-on-achip protein nano/microcontact printing development of research-enabling nano/microsystems translation to our patients point-of-care anemia diagnostic smartphone-based diagnostics diagnostic and medical device development

3 endothelial-ization of microfluidics as microvasculature-on-a-chip systems microvascular dimensions (lumen size: 15 to 30 µm diameter) cultured endothelial cells line entire 3D inner surface of microchannels provides integration of: endothelial cell adhesion cell deformability cell aggregation hemolytic byproducts cytokines thrombosis controlled flow conditions DAY 21 3

4 in vitro endothelialized microfluidics to study hematologic cell-cell interactions macroscale image of PDMS microfluidic device phase contrast of endothelialized microdevice 100 µm Tsai et al, J Clin Invest, 2012 Myers et al, J of Vis Exp, 2012

5 3D images of the microvasculature-on-a-chip 20 µm red = endothelial cell membrane blue = endothelial cell nuclei Tsai et al, J Clin Invest, 2012

6 computer fluid dynamic modeling confirms regular, laminar flow patterns wall shear stress and microchannel velocity profiles are consistent with in vivo microvascular conditions Tsai et al, J Clin Invest, 2012

7 biologic characterization: nitric oxide production and VE-cadherin expression NO production VE-cadherin at cell junctions VE-cadherin-FITC Tsai et al, J Clin Invest, 2012

8 sickle cell patient blood causes in vitro vascular occlusion in endothelialized microfluidics 20 µm whole blood from sickle cell (Hgb SS) patient whole blood from sickle cell (Hgb SS) patient on hydroxyurea Tsai et al, J Clin Invest, 2012

9 quantification of microfluidic data from sickle cell patient samples Tsai et al, J Clin Invest, 2012

10 application of the microvasculature-on-a-chip to hemolytic uremic syndrome (HUS) enterohemorrhagic E. Coli (O157:H7) release of Shigatoxins bloody diarrhea pathologic secretion of large chains of von willebrand factor (vwf) microvascular thrombosis kidney failure

11 microvasculature-on-a-chip as an in vitro HUS model R6G leukocytes & plt VWF - FITC Tsai et al, J Clin Invest, 2012 brightfield addition of shigatoxin-2 (STX2) to HUVECs and 1 dyne/cm 2

12 microvascular thrombosis and aggregation may be shear dependent in HUS R6G leukocytes & plt VWF - FITC Tsai et al, J Clin Invest, 2012 brightfield addition of shigatoxin-2 (STX2) to HUVECs and 10 dyne/cm 2

13 using endothelialized microfluidics for drug discovery: studying effects of eptifibatide (Integrilin) on HUS shear stress (dyne/cm 2 ) shear stress eptifibatide inhibits integrin α IIb β 3 (glycoprotein IIb/IIIa) and platelet aggregation Tsai et al, J Clin Invest, 2012

14 can we integrate whole blood, endothelial cells, and flow into a single microfluidic hemostasis model? Sakurai et al, Nature Communications, 2018

15 incorporating pneumatic valves with endothelialized microfluidics enable in vitro mechanical bleeding model 35μm Yumiko Sakurai, MS Elaissa Hardy, PhD Sakurai et al, Nature Communications, 2018

16 incorporating pneumatic valves with endothelialized microfluidics enable in vitro mechanical bleeding model minimal anticoagulation w/ corn trypsin inhibitor elapsed time: 15 min 35μm vascularized channel collagencoated bleeding channel shear stress: 5 dyne/cm 2 50 µm platelets red fibrin/ogen green endothelial cells - blue

17 endothelialized bleeding model enables spatiotemporal resolution of multiple aspects of clot formation endothelial cells platelets vwf co-localized with endothelial cells vwf accumulation at injury vwf co-localized with platelets microfluidic system allows visualization of vwf-mediated processes from different sources (endothelial cells, platelets, plasma) Sakurai et al, Nature Communications, 2018

18 quantifying hemostasis in an endothelialized bleeding model of von Willebrand Disease AVW3 - inhibits A1 domain of vwf Sakurai et al, Nature Communications, 2018

19 α IIb β 3 integrin inhibition attenuates platelet contraction and density of in vitro hemostatic plug control eptifibatide (Intregrilin) Sakurai et al, Nature Communications, 2018

20 microengineered bleeding model demonstrates that endothelial cells initiate phosphatidylserine (PS) exposure endothelial cells Annexin V merge before mechanical injury after mechanical injury Sakurai et al, Nature Communications, 2018

21 hemophilia A patients exhibit prolonged in vitro bleeding time and impaired fibrin formation hemophilia A whole blood: hemostasis not achieved hemophilia A hema n=4 subjects time: 1200 s healthy control whole blood: hemostasis achieved healthy controls healthy n=3 subjects time: 1200 s fibrin/ogen endothelial cells Sakurai et al, Nature Communications, 2018 platelets (CD41) in vitro bleeding time (sec)

22 hydrogel-based substrates improve the physiologic conditions of endothelialized microfluidics 25 μm a collagen-based microfluidic enables: gelatin/agarose quantifying endothelial permeability under 10 physiologic μm flow (not conditions PDMS-based) gel long term culture (>1 month) self-healing endothelial barrier function resolution of phenomenon (sickle cell vaso-occlusion, thrombosis)

23 microvascular endothelial cells self-deposit extracellular matrix in hydrogel-based microfluidic human pulmonary microvascular endothelial cells Yongzhi Qiu, PhD Qiu et al, Nature Biomedical Engineering, (accepted)

24 hydrogel-based microvasculature-on-a-chip serves as a novel perfusable endothelial permeability assay acellular endothelialized

25 perfusing irreversibly sickled RBCs into gel-based microvasculature-on-a-chip induces channel occlusion ~ 10 % irreversibly sickled RBCs (ISCs) in vitro vaso-occlusion Qiu et al, Nature Biomedical Engineering, (accepted)

26 sickle RBC occlusion induces in situ endothelial permeability in gel-based microvasculature-on-a-chip blue: nuclei yellow: sickle red cells green: BSA-488 Qiu et al, Nature Biomedical Engineering, (accepted)

27 perfusing ISCs increases endothelial permeability but system self-heals after RBC de-adhesion perfuse washed RBCs for 4 hrs perfuse medium for overnight 1 st permeability assay 2 nd permeability assay 3 rd permeability assay 1 st leakage caused by occlusion 2 nd 3 rd system selfheals over time after RBCs de-adhere

28 hydrogel-based microvasculature-on-a-chip enables monitoring of thrombosis resolution Day 14 of culture 10 ng/ml TNF-α overnight, 30 min perfusion of whole blood, perfusion of 50% PPP and 50% culture medium

29 hydrogel-based microvasculature-on-a-chip enables monitoring of thrombosis resolution Day 2 Day 3 Day 4 Day 5 Day 6 50 μm 50 μm

30 hydrogel-based microvasculature-on-a-chip enables monitoring of thrombosis resolution Day 2 40 μm Day 3 Day 4 Day 5 Day 7 Day 10

31 endothelial permeability after thrombosis in hydrogelbased microvasculature-on-a-chip Day 10

32 University of Wisconsin Michael Graham, PhD acknowledgements collaborators Emory/Georgia Tech Tom Barker, PhD Todd Sulchek, PhD Clint Joiner, MD, PhD BIDMC/ Harvard Medical School Rob Flaumenhaft, MD, PhD Blood Center of Wisconsin Shawn Jobe, MD, PhD Boston University Michael Smith, PhD Marcus Carden, Jordan Ciciliano, Meredith Fay, Caroline Hansen, Elaissa Hardy, Rob Mannino, David Myers, Yumiko Sakurai, Yongzhi Qiu, Margo Rollins, Reggie Tran, Erika Tyburski, Kendall Williams funding Rice University Joel Moake, MD Cincinnati Children s Russell Ware, MD, PhD University of North Carolina Alisa Wolberg, PhD University of Pennsylvania John Weisel, PhD