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. 2016 Jun 15;8(6):2562-74.
eCollection 2016.

Growing a whole porcine liver organ ex situ for six hours without red blood cells or hemoglobin

Jing Dong  1 , Lingling Xia  2 , Hefang Shen  1 , Congwen Bian  3 , Sujin Bao  4 , Min Zhang  5 , Yiqi Du  6 , Yan Dai  1 , Lijuan Zhao  1 , Yuanhong Xu  5 , Qiru Xiong  3 , Jianjian Xu  7 , Lili Xu  8
Affiliations

Growing a whole porcine liver organ ex situ for six hours without red blood cells or hemoglobin

Jing Dong et al. Am J Transl Res. .

Abstract

Liver transplantation is an effective approach to end-stage liver disease. Shortage of donor liver and increased waiting time for liver transplantation necessitate the development of an organ culture system by which livers can be cultured and maintained ex situ for a prolonged period of time. The aim of this work is to test whether cell culture condition in vitro could be used to culture whole livers ex situ without the use of erythrocytes. Twelve castrated male land race/farm young porcine livers were exposed to 30 min warm ischemia and 30 min cold perfusion. Livers were isolated and connected to an Ex situ liver culture system using a standard culture medium RPMI1640 supplied with 10% of fetal bovine serum and sufficient dissolved oxygen under a normothermic condition for 6 hours. Metabolic biomarkers, bile and urea production, hepatic cell viability and histology analysis of biopsies were examined and newly proliferated hepatic cells labeled by BrdU were analyzed after 6 hours ex situ culture. The results from biochemical assays and histology analysis indicate that livers after the organ culture still maintain the full function.

Conclusions: Our data demonstrate that the liver culture system established in this work can be used to culture whole livers ex situ in the absence of erythrocytes.

Keywords: 3D culture; BrdU histology analysis; BrdU proliferation assay; Ex situ liver culture; organ growing; oxygen carrier free; warm ischemia; without erythrocytes.

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Figures

Figure 1
Figure 1
Hepatic cells cultured in vitro and calibration of dissolved oxygen in cell culture and in Ex situ liver culture. Cell shrinking was induced by the absence of FBS. A. Representation of hepatic cells cultured in 0% FBS after 24 hours. B. Hepatic cells cultured with 10% of FBS. The black arrows point to the shrunken cells. C. Analyzed results of shrinking cells from the cell cultures. D. The hepatic cell culture medium contains 5.8 mg/L of dissolved oxygen. E. Dissolved oxygen during inflow of the hepatic artery and portal vein; outflow of the hepatic vein was monitored at 2, 4, and 6 hours during ESLC. F. Oxygen consumption of liver tissue is shown during ESLC. G. Representative figure of the devices provide dissolved oxygen at various mmHg values. Cell were accounted 25 fields for each sample with microscope and data are represented as means ± SEM (n = 3 independent experiments; *P<0.05).
Figure 2
Figure 2
A diagram of ESLC system. ESLC consisted of an organ chamber, two pumps (one centrifugal pump and one roller pump), an oxygenator at the top to maintain oxygen via the hepatic artery/portal vein, and three heat exchangers to maintain the physiological temperature. Two flow meters were installed to monitor the inflow of the hepatic artery and the portal vein. Two online dissolved oxygen meters were also installed for inflow and outflow. Bile production was collected by draining externally (A). The actual ESLC system is shown in (B).
Figure 3
Figure 3
Biochemical marker changes in liver organ culture medium. A. Enzymes released; B. Blood urea nitrogen produced during ESLC; C. Albumin production monitored in the medium during ESLC. Data are represented as means ± SEM (n = 3 independent experiments).
Figure 4
Figure 4
Bile production and composition were measured at 2, 4, and 6 h. A. The volume of bile produced. B. GGT and LDH released in the medium; C. Total bilirubin in the bile produced; D. Total bile acids (TBA) and HCO3; E. Representative samples of produced bile collected every 2 hours during ESLC.
Figure 5
Figure 5
Hepatocyte viability and albumin production from hepatocytes isolated before and after ESLC. A piece of the liver tissue enzymatically digested and Trypan blue stained before ESLC (A) and after ESLC (B). (C) Trypan blue positive for cell control was compared in the testing experiment. (D) Viability of heptatic cells as assayed using trypan blue before and after ESLC. (E) Albumin production per million cells isolated before and after ESLC cultured in Petri dishes. These data are shown as the mean ± STDEV (n = 5).
Figure 6
Figure 6
BrdU hepatocyte proliferative assay in ESLC. The representative tissue containing BrdU-positive cells taken after 6 hours of ESLC is shown in (A), and two newly proliferated neighboring positive cells (black box with broken lines) was enlarged and inserted on the right side. (B) showed the experimental negative control and (C) is the analyzed number of BrdU-positive hepatocytes in comparison with control. These data are shown as the mean ± STDEV (n = 3).
Figure 7
Figure 7
HE staining of the liver and bile duct biopsies following 6 hours of ESLC. A. HE staining of the liver tissues before ESLC (left column) and 6 hours after ESLC (right column). The top panel shows the hepatic architecture of the liver tissue (magnification ×200), while the bottom row shows enlargement of the center vein with a completely enclosed endothelial layer. B. Histology of the bile duct and peribiliary glands after 6 hours of ESLC. The top row shows the morphology of the common bile duct (upper left) and peribiliary glands (upper right). The bottom row shows an enlarged common bile duct (lower left) and enlarged peribiliary glands (lower right).
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