iPS細胞およびHEK293細胞増殖のためのCouette Flowを用いた浮遊培養系の開発

早大学位記番号:新7337早稲田大

The potential of cell sheet technique on the development of hepatocellular carcinoma in rat models.

Hepatocellular carcinoma (HCC) is considered the 3rd leading cause of death by cancer worldwide with the majority of patients were diagnosed in the late stages. Currently, there is no effective therapy. The selection of an animal model that mimics human cancer is essential for the identification of prognostic/predictive markers, candidate genes underlying cancer induction and the examination of factors that may influence the response of cancers to therapeutic agents and regimens. In this study, we developed a HCC nude rat models using cell sheet and examined the effect of human stromal cells (SCs) on the development of the HCC model and on different liver parameters such as albumin and urea.Transplanted cell sheet for HCC rat models was fabricated using thermo-responsive culture dishes. The effect of human umbilical cord mesenchymal stromal cells (UC-MSCs) and human bone marrow mesenchymal stromal cells (BM-MSCs) on the developed tumour was tested. Furthermore, development of tumour and detection of the liver parameter was studied. Additionally, angiogenesis assay was performed using Matrigel.HepG2 cells requires five days to form a complete cell sheet while HepG2 co-cultured with UC-MSCs or BM-MSCs took only three days. The tumour developed within 4 weeks after transplantation of the HCC sheet on the liver of nude rats. Both UC-MSCs and BM-MSCs improved the secretion of liver parameters by increasing the secretion of albumin and urea. Comparatively, the UC-MSCs were more effective than BM-MSCs, but unlike BM-MSCs, UC-MSCs prevented liver tumour formation and the tube formation of HCC.Since this is a novel study to induce liver tumour in rats using hepatocellular carcinoma sheet and stromal cells, the data obtained suggest that cell sheet is a fast and easy technique to develop HCC models as well as UC-MSCs have therapeutic potential for liver diseases. Additionally, the data procured indicates that stromal cells enhanced the fabrication of HepG2 cell sheets. This provides the foundation for future research using stromal cells in preclinical and clinical investigations

Fabrication of cell sheets using UPcell dishes.

<p>(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.</p

Vascularisation of co-culture of HUVEC or MSCs with HepG2 on Matrigel after 14h incubation.

<p>(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.</p

Urea and albumin concentration of cell culture of HepG2 and stromal cells at day 1 and day 7 day.

<p>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.</p

Vascularisation of HepG2 and MSCs on Matrigel after 14h incubation.

<p>(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.</p

Appearance of rat’s liver after one month after cell sheet transplantation.

<p>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.</p

Histological and immunohistochemial analysis of rat’s liver.

<p>(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.</p

Image of the transplantation procedure of cell sheets on rat’s liver.

<p>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.</p