• Isolation and separation of liver cancer cells from the whole blood using a microfluidic system
  • Naeim Jalali,1 Mahdi Moghimi ,2,*
    1. Department of Mechanical Engineering, Iran University of Science and Technology (IUST), Tehran, Iran
    2. Department of Mechanical Engineering, Iran University of Science and Technology (IUST), Tehran, Iran


  • Introduction: Isolation and separation of cancer cells from the blood are one of the main challenges in the domain of cancer studies. In this paper, a microfluidic microchip has been designed and fabricated which uses filtration capability by reducing the microchip height in several stages and size-based trapping. The geometric shape of this microchip is designed in such a way that at a speed of 90 micrometers/second or less in a microchannel of 6.7 millimeters with a decreasing height of 40 to 5 micrometers, it can trap all CTCs with the least possible damage. Meanwhile, with an increase in flowrate, some cells deform as a result of drag force and escape the trap. Therefore, the results of recent studies show that in microfiltration chips beside the geometric shape, some other factors including the size of the microchannels and flowrate affect cell isolation output dramatically. In the present study, blood was initially lysed using WBC lysis buffer and was later injected into the microchip using a micropump. The results indicated that the capturing in the microchip is accomplished. With its simple design, easy portability, cheap price, lack of need for fluorescent marking, and its least damage to the cells, this microchip is a suitable tool for trapping cells to conduct diagnostic, clinical, and medical examinations.
  • Methods: Isolation and detection of CTCs are highly important in cancer diagnosis. It is very difficult to diagnose high-density CTCs since they are rarely found in the blood, and detecting them in the blood is really hard. Different instruments have recently been manufactured which work based on biochemical and physical methods. Considering the details of recent modern developments in microfluidic methods and the technologies which have detected and isolated CTCs, a new and effective scheme has been designed for capturing cancer cells. Therefore, in the present study, there has been an attempt to trap the cells suspended in fluid flow in the microchannels with changing cross section in the main channel of the microchip. To trap the cancer cells floating in the blood, first, a lysis buffer called WBC Lysis buffer is used which lyses all white and red blood cells simultaneously and does not damage the tissue cells, and then the lysed blood sample containing the cancer cells are injected into the microchip. In this design, micronic channels are used to capture liver cancer cells to reduce height. The geometric design of the microchip allows fixed speed profile so that the cells suspended in the fluid are trapped in different parts of the walls and narrow part of channel holes and stabilize there. The results of experimental tests of lysed blood injection inside this microchip prove size-based trapping of the cells. Therefore, in this study, the researchers have designed and manufactured a microchip which isolates and separates CTCs in the blood according to particle size by means of filtration method. Human liver epithelial cells (HepG2) were obtained from the biotechnology laboratory of Sharif Industrial University. Moreover, the culture medium of Dulbecco’s Modified Eagle’s medium (DMEM)/ F-12 (Cat. No. 11320033) and cow’s embryo serum of (FBS) (Gibco, 10,270–106) were ordered from Thermo Fisher Scientific, USA. The antibiotics (P4333–100 ml) Penicillin-Streptomycin) and (PBS) phosphate buffered saline) were purchased from USA, Sigma-Aldrich. Furthermore, Trypsin (Trypsin-EDTA (0.25%) Gibco (25200-056) was obtained from life technologies. Finally, WBC lysis buffer was obtained from Iran, Mizan. The blood samples (1-3 milliliters) were obtained from the qualified patients (healthy subjects) and were collected in the pipes containing anticlotting EDTA with a proportion of 1:1. All peripheral blood samples were stored at a temperature of 4 Centigrade and were processed for 72 hours. The possibility of isolating CTCs from the blood in the microchip was examined using circulating liver cancer cells. The cell suspension was prepared in the culture medium (DMEM) with different cellular densities in one milliliter. To conduct different experiments, the cell suspension sample was mixed with a healthy blood sample. Blood lysis operation was later carried out using WBC lysis buffer. The lysis operation was conducted several times for the sake of reliability and ensuring that the tissue cell samples were not damaged. The obtained sample was later injected into the microchip. To ensure the accuracy of the results, the experiment was repeated for three times at different time periods.
  • Results: In the first analysis, a cell suspension of (10^4×10) cells in a milliliter was prepared. In this experiment, 10 microliters of the suspension were selected in which there were 1000 living liver cancer cells. This sample of 10 microliters was added to one milliliter of healthy blood and was later mixed with a proportion of 1:1. By means of WBC lysis buffer; the mixture was later transferred into the syringe. In this way, there were only 1000 living cancer cells (called as cell hereafter) in 2 millimeters of lysed blood. The geometric shapes of the cells in the suspension were mainly round or oval and always had an uneven surface with different depths. The suspended cells deform easily under environmental and mechanical forces. In a period of 60 minutes, the lysed cell containing the cell was injected into the microchip at a speed of 35 micrometers/second. After counting the cells, it was found that there were totally 160 cells, and as a result, 100% of the cells were trapped undamaged. To study the effect of increasing flow rate on movement pattern of the cells inside the microchip, in the following experiment the lysed blood with a density of 1000 living cells were injected into the stepped microchip using 2 milliliters syringe at a rate of 150 micrometers/second. The results showed that the cells trapped in the narrow parts of the pores deformed and escaped the space between the surfaces. Meanwhile, a few cells burst under the flow pressure. Therefore, the flow rate was reduced to 100 micrometers/second, but some cells could still escape through the channels with low heights. The cell carcass burst in this stage made the flow viscosity and trapped the living cells among the cell carcass because of the lower flow rate. As a result, at a speed of 100 micrometers/second, the thick cells pass through the traps or explode, reducing cell capture efficiency. The flow rate at this stage is so high that it causes cell deformity and the final explosion of a large number of cells as well as channel deformities.
  • Conclusion: A filtration based microfluidic microchip was designed in the present study in which for appropriate flow rate, CTCs are trapped with the least damage. These microchips were finally built in microchannels with decreasing microchip height in five steps to isolate and detect cancer cells, and microchannel height decreases from 40 to 5 micrometers. This process was followed to trap and capture cancer cells of different sizes. The main parameter in this process is the effect of pressure on allowing cancer cells to pass. To examine the trend of cancer cell capture, the prepared sample was injected into the microchip with a pumping syringe at different rates of 100, and 150 micrometers/second. The injection time and the number of target cells, as well as the total number of injected cells, were recorded to study tumor cell capture efficiency in different cells. The results of the present experiments prove that in microfiltration chips, besides the size and special geometric design of the microchannels, the field and flow rate have dramatic effect on the efficiency of cell isolation since the trapped cells deform under the pressure of much force from the flow and escape the trap (due to their flexible nature), and in case it is not possible to escape, the cells explode under the force of the flow. Considering its simple design, and its use of hydrodynamic principles of the flow as well as the geometric shape of the microchip beside its portability, cheapness, having no need for fluorescent marking, and causing least damage to the cells, this microchip serves as a suitable tool to trap individual cells to conduct diagnostic, clinical, and medical studies.
  • Keywords: Microfluidic chip, Size-based filtration, Liver cancer