3D Printing of a Vascularized Mini‐Liver Based on the Size‐Dependent Functional Enhancements of Cell Spheroids for Rescue of Liver Failure

Abstract The emerging stem cell‐derived hepatocyte‐like cells (HLCs) are the alternative cell sources of hepatocytes for treatment of highly lethal acute liver failure (ALF). However, the hostile local environment and the immature cell differentiation may compromise their therapeutic efficacy. To this end, human adipose‐derived mesenchymal stromal/stem cells (hASCs) are engineered into different‐sized multicellular spheroids and co‐cultured with 3D coaxially and hexagonally patterned human umbilical vein endothelial cells (HUVECs) in a liver lobule‐like manner to enhance their hepatic differentiation efficiency. It is found that small‐sized hASC spheroids, with a diameter of ≈50 µm, show superior pro‐angiogenic effects and hepatic differentiation compared to the other counterparts. The size‐dependent functional enhancements are mediated by the Wnt signaling pathway. Meanwhile, co‐culture of hASCs with HUVECs, at a HUVECs/hASCs seeding density ratio of 2:1, distinctly promotes hepatic differentiation and vascularization both in vitro and in vivo, especially when endothelial cells are patterned into hollow hexagons. After subcutaneous implantation, the mini‐liver, consisting of HLC spheroids and 3D‐printed interconnected vasculatures, can effectively improve liver regeneration in two ALF animal models through amelioration of local oxidative stress and inflammation, reduction of liver necrosis, as well as increase of cell proliferation, thereby showing great promise for clinical translation.


Figure S1 .
Figure S1.Degradation and permeability study of inks.(a) Optical images and (b) weight loss quantification of SPI and HVI in 20 μg mL -1 collagenase II at 37 ℃ at different time points (day 0, day 1, day 2, day 3, and day 7).The red dashed lines highlight the remaining hydrogels.All data are normalized to the initial weight and presented as mean ± SEM, n = 3. (c) The cumulative releasing profiles of rhodamine B from different inks.All data are normalized to the total value and presented as mean ± SEM, n = 5.SPI: stuffing parenchyma ink; HVI: hexagonal vasculature ink.

Figure S2 .
Figure S2.The representative images showing the perfusion of rhodamine B into the 3Dprinted hollow hexagon at different time points.

Figure S3 .
Figure S3.The pro-angiogenic potential of different hydrogels.(a-b) Fluorescent images of calcein AM-stained HUVECs on different substrates (Matrigel, SPI/HVI 1:1 mixture, or HVI) 4 h after cell seeding and total length of the formed meshes.All data are normalized to the value of the "Matrigel" group and presented as mean ± SEM, n = 3. ***p < 0.001, and not significant (ns) p > 0.05.Scale bar: 200 μm.(c) The relative cell proliferation of HUVECs cultured in different hydrogels.All data are normalized to the value of the "HVI" group on day 1 and presented as mean ± SEM, n = 3. SPI: stuffing parenchyma ink; HVI: hexagonal vasculature ink.

Figure S4 .
Figure S4.Characterization of hASC spheroids.(a) A schematic image illustrates the procedures for characterization of different-sized hASC spheroids.(b) Cell viability of hASCs cultured on different substrates at 24 h.Green: live cells; Red: dead cells.Scale bar: 100 μm.(c) Size quantification of hASC spheroids.Data are presented as mean ± SEM. n = 5.(d) The relative mRNA expression of representative genes in hASC spheroids with different sizes.Data are normalized to the value of "S spheroid" group and presented as mean ± SEM. n = 3. 0.01 < *p < 0.05, 0.001 < **p < 0.01, and ***p < 0.001, not significant (ns) p > 0.05.

Figure S8 .
Figure S8.Characterization of hASC spheroids.(a) A schematic image illustrates the procedures for the characterization of different-sized hASC spheroids (XS spheroid, S spheroid, XL spheroid).(b) Optical microscopy images of formed hASC spheroids with distinct sizes using a seeding density of 10k cells per piece of microwell array in 24 h.Scale bar: 200 μm (inset scale bar: 50 μm).(c) Size quantification of hASC spheroids.Data are presented as mean ± SEM. n = 5.(d) The relative mRNA expression of representative genes in hASC spheroids with different sizes.Data are normalized to the value of "S spheroid" group and presented as mean ± SEM. n = 3. 0.01 < *p < 0.05, 0.001 < **p < 0.01, and ***p < 0.001, not significant (ns) p > 0.05.

Figure S10 .
Figure S10.Pro-angiogenic effects of different-sized hASC spheroids.(a) A schematic image illustrates the procedures for studying the proangiogenic effects of hASC spheroids-derived secretome.(b) Migration of HUVECs cultured with different-sized hASC spheroids-derived supernatant media for 24 h.(c) The quantification of the remaining area.All data are normalized to the value of "S spheroid" group and presented as mean ± SEM, n = 4.(d) Tube formation of HUVECs cultured with different hASC supernatant media at 4 h and 8 h.(e) The quantification of the formed tubes at 8 h.All data are normalized to the value of "S spheroid" group and presented as mean ± SEM, n = 3. 0.01 < *p < 0.05, 0.001 < **p < 0.01, and ***p < 0.001, not significant (ns) p > 0.05.Scale bar: 500 μm.

Figure S15 .
Figure S15.GO analysis of the significantly expressed genes between cell spheroids and single cells.n = 3, p < 0.05.

Figure S16 .
Figure S16.The characterization of Wnt signaling pathway-related molecules.(a) The relative mRNA expression levels of WNT5A, WNT5B, WNT9A, WNT16, and CTNNB1 genes in hASC spheroids with different sizes.Data are normalized to the value of "L spheroid" group.(b) The Wnt5b level in the supernatant of hASC spheroids with different sizes.All data are presented as mean ± SEM, n = 3. L spheroid: cell spheroid with a diameter of 150 m; S spheroid: cell spheroid with a diameter of 50 m.

Figure S17 .
Figure S17.Wnt/β-catenin pathway mediates the enhancements of pro-angiogenic potential and hepatic differentiation in the small-sized hASC spheroids (S spheroid).(a) The optical images and (b) mean mesh size quantification of HUVECs formed tubes on the Matrigel with different medium formulae at 8 h.Scale bar: 200 μm.CM: S spheroid-derived conditioned medium; FM: fresh medium.All data are normalized to the value of "CM/DMSO" group.(c) The mRNA expression of hepatocyte-related genes in S spheroid after hepatic induction.All data are normalized to the value of "DMSO" group.(d) Immunofluorescent staining of albumin in the differentiated hASC spheroids.Green: Alexa Fluor 488-labelled albumin; Blue: DAPIlabelled cell nuclei.Scale bar: 50 μm.(e) The canonical Wnt/β-catenin signaling pathway in S spheroid.All data are presented as mean ± SEM, n = 3. 0.01 < *p < 0.05, 0.001 < **p < 0.01, ***p < 0.001, and not significant (ns) p > 0.05.

Figure S25 .
Figure S25.The western blots of GAPDH and iNOS in the mouse livers with different treatments on day 1 and their quantification by image J. GAPDH was used as the reference.All data are normalized to the value of GAPDH and presented as mean ± SEM, n = 5. 0.01 < *p < 0.05, 0.001 < **p < 0.01, and ***p < 0.001, not significant (ns) p > 0.05.

Figure S27 .
Figure S27.Characterization of APAP-challenged mice.(a) The relative weight change of Balb/c mice with different treatments.All data are normalized to the value at 0 h and presented as mean ± SEM, n = 3.(b) The gross images of mouse livers with different treatments.(c) Immunofluorescent staining of iNOS in the mouse livers with different treatments.Green: Alexa Fluor 488-labelled iNOS; Blue: DAPI-labelled cell nuclei.Scale bar: 200 μm."Normal":normal mice without challenging of acetaminophen (APAP); "ALF": APAP-challenged mice at 16 h without treatment; "Sham": APAP-challenged mice without treatment at 88 h; "Single HLCs": APAP-challenged mice with subcutaneous implantation of single hASCs-derived hepatocyte-like cells (HLCs) in porcine liver-derived decellularized extracellular matrix (PLdECM) hydrogel at 88 h; "Single HLCs+3D printed HUVECs": APAP-challenged mice with subcutaneous implantation of single HLCs in PLdECM hydrogel and 3D printed HUVECs at 88 h; "HLC spheroids+3D printed HUVECs": APAP-challenged mice with subcutaneous implantation of HLC spheroids in PLdECM hydrogel and 3D printed HUVECs at 88 h.The dosage of HLCs was kept consistent at 1 × 10 6 cells/mouse.