Fig 1.
Image of the transplantation procedure of cell sheets on rat’s liver.
The cell sheet was transplanted onto one site of the liver surface of each rat using a support membrane to facilitate handling. The circle shows the transplanted cell sheet on the liver.
Fig 2.
Vascularisation of HepG2 and MSCs on Matrigel after 14h incubation.
(A) HepG2, (B)HUVECs, (C) BM-MSC, (D) UC-MSC. Cells were seeded into Matrigel in the presence of VEGF. CD31 (green) and Hoechst 33258 staining (blue). x4 magnification.
Fig 3.
Vascularisation of co-culture of HUVEC or MSCs with HepG2 on Matrigel after 14h incubation.
(A) HUVEC+HepG2, (B) UC-MSC+HepG2, (C&D) BM-MSC+HepG2. Cells were seeded into Matrigel in the presence of VEGF. Tubes started to form after 14 hours. CD31 (green) and Hoechst 33258 staining (blue). The presented ratio of the co-culture cells is 1:1. 4x magnification.
Fig 4.
Human VEGF concentration of co-cultured HepG2 with MSCs (UC or BM) after one week of incubation.
HUVECs were used as a positive control. Tow-way NOVA (P = 0.001).
Fig 5.
Urea and albumin concentration of cell culture of HepG2 and stromal cells at day 1 and day 7 day.
Where control is only HepG2 cells, whereas each stromal cells bar represents co-cultured cells of HepG2 and MSC (UC or BM). At day 7 in the culture, the secretion of albumin by the co-culture of HepG2 and UC-SCs significantly increased as compared to untreated HepG2, (P = 0.03). For urea, after 7 days all the cultured cells showed an increase in the urea level within 7 days (P<0.05). Mean± SD. Tow-way ANOVA.
Fig 6.
Fabrication of cell sheets using UPcell dishes.
(A): (a); HepG2, (b); co-culture of HepG2+BM-MSCs, (c); HepG2+UC-MSC. The Mean of the total surface areas of HepG2, HepG2+human UC-SCs and HepG2+human BM-MSCs; 20 mm 2, 10 mm 2, and 19.8 mm 2 respectively. (B); Fluorescent staining of F-actin filaments in fixed cell sheet of HepG2 and stromal cells. Where (a); HepG2 and UC-SC, and (b) HepG2 and BM-SC. x100 magnification. Staining of F- actin (red) indicates the denseness of differentiated tube formation network of HepG2 and UC-MSC in pouch-like cell sheets in vitro.
Fig 7.
Urea and albumin concentration of HepG2 and stromal cell sheets at day 1 and day 7.
The cells were co-cultivated in up-cell dish to form a cell sheet. Where the conditioned medium was collected and HepG2 only cell sheet as control, whereas each stromal cells bar represents co-culture of HepG2 and stromal cells. Albumin or urea secretion by HepG2—UCMSCs, HepG2—BM-MSCs, and HepG2 alone significantly increased at day 7 in culture as compared to day 1, P = 0.009 and P = 0.0004 respectively. Mean±SD. Two-way ANOVA.
Fig 8.
Appearance of rat’s liver after one month after cell sheet transplantation.
Where (A) appearance of tumour generated from HepG2 and BM-SC cell sheet, and a mass (2 cm in size) underneath the skin of the rats. (B) cell sheet of HepG2 alone (C) HepG2 and UC-MSC cell sheet, and (D) attached cell sheet of HepG2 and UC-MSC on the liver without forming any tumour. The average size of the tumour developed after transplantation of only HepG2, HepG2 + BM-MSCs, and HepG2+UC-MSC are; 4.5cm, 4cm and 2.5cm respectively, 5 rats was used in each sample group.
Fig 9.
Histological and immunohistochemial analysis of rat’s liver.
(A): H&E staining of rat’s liver after 4 weeks of cell sheet transplantation. (a) Normal rat liver cells. (b) Morphology of liver after cancer cells transplantation, black arrow shows hepatic cancer, whereas green arrow shows normal hepatocytes. Blue arrow shows inflamed cell and orange arrow represent necrotic HCC cells, x20 magnification. (B): IHC staining of tumour marker GPC3 antibody on rat’s liver after 4 weeks of transplantation. (a) Negative control, normal rat, no transplantation. (b) HepG2 cell sheet, (c) BM-SC and HepG2 cell sheet on the rat with tumour formation, (d) UC-SC and HepG2 cell sheet on the rat with tumour formation. (e) UC-SC and HepG2 cell sheet on the rat without tumour formation. x10 magnification.
Fig 10.
Albumin and urea concentration of rats transplanted with HepG2 and stromal cell sheets within 4 weeks.
Where control is non-transplanted rat, whereas each stromal cells bar represents mixed cells of HepG2 and stromal cells. The highest level was detected on week 4. HepG2 and UC-SCs or HepG2 and BM-SCs secreted higher level of albumin as compared to rats transplanted with HepG2 alone, P<0.05). Tow way anova. W = week. Mean±SD.