Research ArticleHEALTH AND MEDICINE

Biophysical and biomolecular interactions of malaria-infected erythrocytes in engineered human capillaries

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Science Advances  17 Jan 2020:
Vol. 6, no. 3, eaay7243
DOI: 10.1126/sciadv.aay7243
  • Fig. 1 Photoablation-guided capillary growth in lithography-based microvessel devices.

    (A) Diagram of device assembly and capillary fabrication. Main channels were generated by soft lithography in acrylic jigs followed by capillary generation by photoablation and endothelial ingrowth. (B) Array (2 × 2) of 20-μm-diameter vessels demonstrates stable vessel lumens. Green: F-actin; blue: nuclei. (C) Endothelial ingrowth over 18 hours demonstrates complete vessel formation. (D and E) Cryosectioned capillaries were stained with (D) hematoxylin and eosin as well as (E) type IV collagen, demonstrating lumen formation by single endothelial cells and robust basement membrane deposition. (F) Constriction vessel design allows for the generation of sub–10-μm-diameter capillaries shown in both projected and cross-sectional views. Red: VE-cadherin; blue: nuclei. (G to I) Ultrastructural analysis of capillary vessel regions by transmission electron microscopy (TEM) shows vessels at varying diameters (from 40 to 10 μm) in cross-sectional (G and I) and longitudinal (H) views and varying wall thicknesses and junctions at cell-cell contact in zoomed views (I). Cross-sectional views of vessel regions near the connection. Col: collagen substrate; *: lumen; C: cells. (J) Stitched confocal image demonstrates complete vessel network consisting of 33 capillaries. Green: von Willebrand factor; blue: nuclei; red: VE-cadherin; purple: F-actin.

  • Fig. 2 Motion and deformation of RBCs while traversing capillary constriction zones.

    (A) Superimposed time lapse image series of RBC motion through an endothelialized vessel. Time lapse interval: 0.1 s. Scale bar, 25 μm. (B) Schematic showing RBC perfusion through constriction-shaped vessel. wc = 10 μm; wm = 40 μm. (C) Representative examples of parachute-, slipper-, and discocyte-like RBC deformations in capillary constrictions. Scale bar, 10 μm. (D) Representative traces of RBC major axis length and orientation for (i) stretching and (ii) tumbling RBCs, where x = 0, y = 0 is the center of the capillary; gray boxes: capillary extents. (E) Normalized extent of deformation, defined as the ratio of major axis length at a given position to the average major axis length in the precapillary region for (i) normal RBCs and (ii) knobless malaria-IRBCs (n = 3; each biological replicate is represented by one symbol, ■, ♦, ×, or ▲).

  • Fig. 3 Population dynamics analysis of RBC flow and accumulation within constriction capillaries.

    (A) Bright-field image of RBCs flowing through a constriction vessel. Dotted lines demonstrate capillary outline trace. (B) Flow velocity model of a single capillary based on fabrication dimensions. (C) Left: Schematic illustrations of the different types of perfused RBCs and IRBCs. Middle: Representative image of binding kinetics within the capillary constriction zones. IRBCs are shown in red. Right: Spatial distribution of accumulation after 20 min of perfusion represented by a combined heat map of average fluorescence from multiple experiments. Normal RBC (n = 24), IT4VAR19 (n = 26), trypsinized IT4VAR19 (n = 19), and 2G2 (n = 19). (D and E) Quantification of average intensity as a measure of x position (D) and comparison among precapillary, capillary, and postcapillary regions (E). n = 24, 26, 17, and 19 ACV units quantified from N = 4 independent experiments for each condition of perfusion: normal RBC, IT4VAR19, trypsinized IT4VAR19, and 2G2, respectively. Analysis of variance (ANOVA) F = 11.61, P < 0.00001 for precapillary region, ANOVA F = 47.83, P < 0.00001 for capillary region, and ANOVA F = 6.41, P < 0.001 for postcapillary region.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/3/eaay7243/DC1

    Supplementary Materials and Methods

    Fig. S1. Photoablation-guided fabrication of acellular capillary-sized microchannels.

    Fig. S2. Cellular limitations of capillary fabrication.

    Fig. S3. Endothelialization strategies for capillary network.

    Fig. S4. Ultrastructural analysis of vessels at capillary regions across the ACV units imaged via TEM.

    Fig. S5. Edge detection workflow for studying traversal of single RBCs through a capillary-sized constriction.

    Fig. S6. Transcriptional profiling of var gene expression in the two parasite lines.

    Fig. S7. Ultrastructural analysis of capillary region after perfusion of RBCs imaged via TEM.

    Fig. S8. Trypsin cleaves the surface-expressed IT4VAR19 PfEMP1 variant.

    Movie S1. Endothelial ingrowth into 20-μm capillaries between two large vessels after photoablation.

    Movie S2. Representative video of normal RBCs perfused through endothelialized capillaries at physiological hematocrit.

    Movie S3. Representative video of normal RBCs perfused through acellular collagen capillary-shaped channels at physiological hematocrit accumulating a significant amount before entering the capillary region and jetting and leading to significantly lower hematocrit into the downstream.

    Movie S4. Representative video of normal RBCs stretching through capillaries (diameter, ~10 μm) when perfused at the single-cell scale.

    Movie S5. Representative video of normal RBCs tumbling through capillaries (diameter, ~10 μm) when perfused at the single-cell scale.

    Movie S6. Representative video of 2G2 IRBCs tumbling through capillaries (diameter, ~10 μm) when perfused at the single-cell scale.

    Movie S7. Representative video of normal RBCs perfused through the capillaries at physiological hematocrit with no cell accumulation in the lumen.

    Movie S8. Representative video of IT4VAR19 IRBCs perfused through the capillaries at physiological hematocrit accumulating a significant amount and blocking the capillary flow.

    Movie S9. Representative video of trypsinized IT4VAR19 perfused through the capillaries showing a significant decrease in cell accumulation and not blocking the capillary flow.

    Movie S10. Representative video of 2G2 IRBCs perfused through the capillaries at physiological hematocrit accumulating at postcapillary regions but not blocking the capillary flow.

    References (61, 62)

  • Supplementary Materials

    The PDFset includes:

    • Supplementary Materials and Methods
    • Fig. S1. Photoablation-guided fabrication of acellular capillary-sized microchannels.
    • Fig. S2. Cellular limitations of capillary fabrication.
    • Fig. S3. Endothelialization strategies for capillary network.
    • Fig. S4. Ultrastructural analysis of vessels at capillary regions across the ACV units imaged via TEM.
    • Fig. S5. Edge detection workflow for studying traversal of single RBCs through a capillary-sized constriction.
    • Fig. S6. Transcriptional profiling of var gene expression in the two parasite lines.
    • Fig. S7. Ultrastructural analysis of capillary region after perfusion of RBCs imaged via TEM.
    • Fig. S8. Trypsin cleaves the surface-expressed IT4VAR19 PfEMP1 variant.
    • Legends for movies S1 to S10
    • References (61, 62)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Endothelial ingrowth into 20-μm capillaries between two large vessels after photoablation.
    • Movie S2 (.mp4 format). Representative video of normal RBCs perfused through endothelialized capillaries at physiological hematocrit.
    • Movie S3 (.mp4 format). Representative video of normal RBCs perfused through acellular collagen capillary-shaped channels at physiological hematocrit accumulating a significant amount before entering the capillary region and jetting and leading to significantly lower hematocrit into the downstream.
    • Movie S4 (.mp4 format). Representative video of normal RBCs stretching through capillaries (diameter, ~10 μm) when perfused at the single-cell scale.
    • Movie S5 (.mp4 format). Representative video of normal RBCs tumbling through capillaries (diameter, ~10 μm) when perfused at the single-cell scale.
    • Movie S6 (.mp4 format). Representative video of 2G2 IRBCs tumbling through capillaries (diameter, ~10 μm) when perfused at the single-cell scale.
    • Movie S7 (.mp4 format). Representative video of normal RBCs perfused through the capillaries at physiological hematocrit with no cell accumulation in the lumen.
    • Movie S8 (.mp4 format). Representative video of IT4VAR19 IRBCs perfused through the capillaries at physiological hematocrit accumulating a significant amount and blocking the capillary flow.
    • Movie S9 (.mp4 format). Representative video of trypsinized IT4VAR19 perfused through the capillaries showing a significant decrease in cell accumulation and not blocking the capillary flow.
    • Movie S10 (.mp4 format). Representative video of 2G2 IRBCs perfused through the capillaries at physiological hematocrit accumulating at postcapillary regions but not blocking the capillary flow.

    Files in this Data Supplement:

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