ABSTRACT
The contractile force of hepatic stellate cells plays a very important role in liver damage, hepatitis and fibrosis. In this paper, a method based on polydimethylsiloxane (PDMS) thin micropillar arrays is proposed to measure the contractile force of human hepatic stellate cell line LX-2, which enables dynamic measurement of the subcellular distribution of force magnitude and direction. First, thin micropillar arrays on glass bottom dish were fabricated using two-step casting process in order to meet the working distance requirement of 100× objective lens. After hydrophilic treatment and protein imprint, cells were seeded on the micropillar arrays. LX-2 cells, which were quiesced by growth in serum-free medium, were activated by adding fetal bovine serum (FBS). The deflections of the micropillars were achieved by image processing technique, and then the contractile force of cells exerted on the micropillars was calculated according to mechanical simulation results, and was analyzed under both quiescent and activated conditions. The experimental results show that the average traction force of quiescent cells is about 20 nN, while the contractile force of activated cells increased to 110 nN upon adding FBS. This method can quantify the contractile force of LX-2 cell on subcellular scale in both quiescent and activated states, which may benefit pathology study and drug screen for chronic liver diseases resulted from liver fibrosis.
Subject(s)
Humans , Cell Line , Hepatic Stellate Cells , Cell Biology , Image Processing, Computer-Assisted , Mechanical PhenomenaABSTRACT
A microfluidic chip with micropillar arrays for three-dimensional (3D) cell culture was designed and validated.The chip consisted of a polydimethylsiloxane (PDMS) channel plate and a glass cover plate.One cell culture chamber composed of two rows of micropillar arrays and two lateral channels for transporting the culture medium were integrated on the PDMS channel plate.The spacing between micropillars directly affects the chip performance, which is critical for the design of the chip.In this work, the spacing between micropillars was optimized by numerical simulation and experimental validation.With the optimized microfluidic chip, the mixture of cells and extracellular matrix mimics could be steadily injected into the cell culture chamber, the nutrients in the culture medium from the lateral channels could quickly diffuse into the chamber, and the cell metabolites could also timely diffuse out of the chamber.To test the stability of the microenvironment in the microfluidic chip, neural stem cells were three-dimensionally cultured.