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3D Printing and Vascularized Organ Construction
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M N O P

Figure 10. 3D bioprinting of adipose-derived stem cell (ASC)-laden gelatin/alginate/fibrin hydrogel for organ manufacturing at Prof. Wang’s
laboratory in Tsinghua University: (A) A pioneering double-nozzle 3D bioprinter made in this laboratory; (B) schematic description of the cell-laden
gelatin/alginate/fibrin hydrogel and pancreatic islets being printed into a grid construct using the 3D bioprinter; (C) a large scale-up 3D-printed grid
construct containing ASC-laden gelatin/alginate/fibrin hydrogel cultured in a plate; (D) a grid ASC-laden gelatin/alginate/fibrin construct after being
cultured for 1 month; (E) a multicellular construct after 3 weeks culture, containing both ASCs encapsulated in the gelatin/alginate/fibrin hydrogel
before epidermal growth factor (EGF) engagement and relatively integrated pancreatic islets seeding in the predefined channels (immunostaining
with anti-insulin in green); (F) some envelopes of the islets were broken after 1 month of culture; (G) immunostaining of the 3D construct with mAbs
for CD31+ cells (i.e., mature endothelial cells (ECs) from the ASC differentiation after 3 days of culture with EGF added in the culture medium) in
green, having a fully confluent layer of ECs (i.e., endothelium) on the surface of the predefined channels; (H) a vertical image of the 3D construct
showing the fully confluent endothelium (formed from ECs) and the predefined go-through channels; (I) immunostaining of the 3D construct with
mAbs for CD34+ cells (i.e., ECs) in green and pyridine (PI) for cell nuclei (nucleus) in red; (J) immunostaining of the 3D construct with mAbs
for CD34+ ECs in green and pyridine (PI) for cell nuclei (nucleus) in red after 3 days of culture without EGF added in the culture medium; (K)
immunostaining of the 3D construct with mAbs for CD31+ ECs in green and pyridine (PI) for cell nuclei (nucleus) in red after 3 days of culture with
EGF added in the culture medium; (L) a control of (K), immunostaining of the 3D construct with mAbs for CD31+ ECs differentiated from the ASCs
in green and pyridine (PI) for cell nuclei (nucleus) in red after 3 days of culture without EGF added in the culture medium; (M) immunostaining of
the 3D construct with mAbs for CD31+ cells in green and oil red O staining for adipocytes in red, showing both the heterogeneous tissues coming
from the ASC differentiation after a cocktail growth factor engagement, that is, on the surface of the channels the endothelium coming from the
ASCs differentiation after being treated with EGF for 3 days, deep inside the gelatin/alginate/fibrin hydrogel the adipose tissue coming from the ASC
differentiation after being subsequently treated with insulin, dexamethasone, and isobutylmethylxanthine (IBMX) for another 3 days; (N) a control
of (M) showing all the ASCs in the 3D construct differentiated into target adipose tissue after 3 days of treatment with insulin, dexamethasone, and
IBMX, but no EGF; (O) immunostaining of two-dimensional (2D) cultured ASCs with mAbs for CD31+ ECs in green and pyridine (PI) for cell
nuclei (nucleus) in red after 3 days of culture with EGF added in the culture medium; (P) immunostaining of 2D cultured ASCs with mAbs for
CD31+ ECs in green and pyridine (PI) for cell nuclei (nucleus) in red after 3 days of culture without EGF added in the culture medium. Reprinted
from Biomaterials, Vol 31 Issue 14, Xu M, Wang X, Yan Y, et al., A Cell-Assembly Derived Physiological 3D Model of the Metabolic Syndrome,
Based on Adipose-Derived Stromal Cells and a Gelatin/Alginate/Fibrinogen Matrix, 3868-3877., Copyright (2010), with permission from Elsevier.

246 International Journal of Bioprinting (2022)–Volume 8, Issue 3