Figure 8.

Adapted two-step seeding method for MVN formation in hybrid matrices. a) Schematics of adapted two-step seeding method for two different types of ECMs (ECM-A and ECM-B). To reduce the usage of the scarce ECMs (such as patient-derived or tissue specific ECM), common ECMs (such as bovine fibrin), can be used for outer layer EC seeding. After the uncured ECs/ECM-A liquids were carefully aspirated, ECs with FBs in scarce ECM-B were seeded to the central gel region to form MVNs in hybrid ECMs. b) Representative images of wide perfusable MVNs formed in fibrin at 3 mg mL⁻¹ (ECM-A) or 5 mg mL⁻¹ (ECM-B) using one-step seeding method (ImHUVECs expressing BFP, 8 × 10⁶ mL⁻¹; lung FBs, 1 × 10⁶ mL⁻¹). Green, ImHUVECs. Magenta, dextran (70 kDa). c) Confocal images of perfusable MVNs formed in hybrid fibrin (outer layer 3 mg mL⁻¹, central 5 mg mL⁻¹) using two-step seeding strategy (outer layer ImHUVECs expressing BFP, 10 × 10⁶ mL⁻¹; central ImHUVECs expressing BFP, 5 × 10⁶ mL⁻¹; and lung FBs 1.5 × 10⁶ mL⁻¹). Opening percentage is 94.33 ± 8.295%; central perfusable MVN percentage is 97.89 ± 3.63% (n = 3 devices, 5 ROIs each). Green, ImHUVECs. Magenta, dextran (70 kDa). White arrows point to the perfusable narrow microvessels. d) Experimental design of seeding MVNs in bovine or human fibrin. To discriminate between the two types of ECMs, Alexa Fluor 488 labeled human fibrin was added to non-labeled human fibrin. Left, one-step seeding method in bovine fibrin gel; middle, one-step seeding in human fibrin gel (containing 10% Alexa Fluor 488 labeled human fibrin); right, adapted two-step seeding for hybrid fibrin (outer layer, bovine fibrin; central, human fibrin mixed with 10% of Alexa Fluor 488 labeled human fibrin). e) Representative images of wide perfusable MVNs formed in fibrin derived from bovine (ECM-A) or from human (ECM-B) using one-step seeding method (ImHUVECs expressing BFP, 8 × 10⁶ mL⁻¹; lung FBs, 1 × 10⁶ mL⁻¹). f) Representative images of perfusable MVNs formed in hybrid ECMs (outer layer bovine fibrin, central human fibrin) using two-step seeding method (outer layer ImHUVECs 10 × 10⁶ mL⁻¹, central ImHUVECs 5 × 10⁶ mL⁻¹, and lung FBs 1.5 × 10⁶ mL⁻¹). Opening percentage is 92.07 ± 10.88%; central perfusable MVN percentage is 98.25 ± 3.99% (n = 3 devices, 5 ROIs each). Cyan, ImHUVECs expressing BFP. Green, human fibrin. Magenta, dextran (70 kDa). White arrows point to the perfusable narrow microvessels.