71 / 2019-08-28 21:59:10

The Model of Liver Acinar Microfluidics Chip with Three-Vascular Structure

liver,hepatic acinar,microfluidics chip

全体主题 > Design and fabrication of biological systems

Introduction
Nowadays, drug development is often carried out with animals, which means the high cost and the long research period. The drugs tested in animals may not work on human body. Therefore, it is urgent for the pharmaceutical industry to construct a liver model mimicking the microenvironment in vitro.
Hepatic acinar, which is established by hepatic vascular perfusion method based on the relationship between hepatocytes and intrahepatic microcirculation blood flow, is considered to be the smallest structural unit and the smallest functional unit of the liver. Thus constructing a liver acinar model can be more responsive to pathological change in liver.
In this study, we designed and fabricated a microfluidics chip with three-vascular structure to mimic liver acinar. Then we determine the best formulation of the bioink and proper printing parameters. Also, we construct a liver chip based on two sets of blood circulation systems of hepatic lobules. In the end, we performed perfusion experiments to verify the function of the chip.
Materials and methods
A. Cell Preparation & Determination of bioink’s formulation
L02 cells were cultured and harvested before printing. Hexagonal grid structure was printed by these four different hydrogel (4% GelMa, 4% GelMa+1mg/ml Collagen, 4% GelMa+2mg/ml Collagen, 2mg/ml Collagen) mixed with cells was prepared. CCK8 was used to assess the proliferation of cells in these four different bioink.
B. Measurement of simulation parameters
The concentration of glucose and glutamine in the media was measured daily using Glucose Oxidase Method, GOD (APPLYGEN) and CES124Ge ELISA KIT For Glutamine (USCN), and the rate of glucose and glutamine absorption per cell was obtained by combining the above cell number curve determined by CCK8 kit. Four kinds of hydrogels were prepared and their permeation rate was measured. Thin material transfer module in chemical reaction engineering in COMSOL Multiphysics 5.2 is used to conduct modelling.
C. Fabrication of microfluidics chip & Perfusion experiment
The microfluidics chip was fabricated by micro-nano machining technology. The scaffold was printed in the chip. The media was perfused from the inlet of the chip for seven days. Every day we would sample the exudates at different positions of the chip to check the albumin and urea levels.
Results
4% GelMa solution and 4% GelMa+1mg/ml collagen solution can be constructed into regular hexagonal shapes at a temperature of 23 degrees, a needle size of 30g and a pressure of 11 Mpa (Fig.1). Since collagen is completely opposite to GelMa's thermal sensitivity, 4% GelMa and 2mg/ml Collagen are difficult to mix evenly. 2mg/ml Collagen is not printable.

Fig.1 the printing structure of 4%GelMa and 4%GelMa+1mg/ml Col
In order to obtain the optimal hexagon structure, we changed the line width (Fig.2).

Fig.2 Hexagon structure printed with different line widths; (A) line width=1.2; (B)(D) line width=1.3; (C) line width=1.4
When the line width is 1.2, two adjacent hydrogels blend with each other. And 1.3 is the best line width which means the scaffold doesn't beach together and the pores aren't too big.
The liver chip (Fig.3) has one medium inlet and two media outlets. The hexagonal structure in the middle is the cell culture area, with square columns on the sides. The gaps between the adjacent columns gradually increases from both sides to the inboard, with a maximum of no more than 12 μm.

Fig.3 Liver Chip (left)
The medium flows into the cell culture area from the inlet and through the gap of the square column. The concentration gradient of nutrients like the hepatic acinar is achieved by the digestion of cells and diffusion of hydrogel (Fig.4).

Fig.4 Three zones of hepatic acinus (left); The concentration simulation of liver chip (right)
Conclusions
The model is designed to control the concentration in different areas of hepatic lobule by simulating microenvironment of hepatic acinar in vivo to study liver injury. The central target area of hepatic acinus is most susceptible to drug toxicity, which means it can also be used for drug screening.