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Integration micro flow control chip used for apoptosis research and application thereof

A microfluidic chip and cell technology, applied in the field of cell apoptosis research, can solve the problems of long processing time, difficulty in real-time analysis of cell apoptosis kinetics, cumbersome operation, etc., and achieve the effect of reducing the consumption of cells and reagents

Inactive Publication Date: 2008-04-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has many limitations, mainly as follows: large demand for cells, long sample pretreatment time, cumbersome operation, mostly offline analysis, it is difficult to capture the relevant information of various processes of cell apoptosis events that occur instantaneously, and it is difficult to realize the analysis of cell apoptosis. Real-time analysis of death kinetics

Method used

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  • Integration micro flow control chip used for apoptosis research and application thereof
  • Integration micro flow control chip used for apoptosis research and application thereof
  • Integration micro flow control chip used for apoptosis research and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] use figure 1The microfluidic chip shown was used to study the morphological changes of apoptosis. After the chip was inoculated with human hepatocellular carcinoma (HepG2), place it at 37°C in 5% CO 2 6 hours in the incubator. After the cells adhered to the wall, add DMEM medium (high sugar) with adriamycin concentrations of 0 μM and 4.0 μM, place the chip in the cell culture incubator for 24 hours, and keep the solution flowing from the concentration gradient generation unit to the cells at a small flow rate. culture unit. Phosphate buffer saline (PBS) was added to wash the cells, and the chip was observed under an optical microscope. Then in 1mg mL -1 acridine orange Cells were labeled with ethidium bromide and immediately viewed under a fluorescence microscope. Such as figure 2 As shown, doxorubicin induces apoptosis of HepG2 cells, and presents typical morphological changes of apoptosis: cells shrink, become round, and the spacing becomes larger; chromatin ...

Embodiment 2

[0036] use figure 1 The microfluidic chip shown was used to study cell membrane changes during cell apoptosis. Chips were inoculated with HepG2 and placed at 37°C, 5% CO 2 6 hours in the incubator. After the cells adhered to the wall, add DMEM medium (high sugar) with adriamycin concentrations of 0 μM and 4.0 μM, place the chip in the cell culture incubator for 24 hours, and keep the solution flowing from the concentration gradient generation unit to the cells at a small flow rate. culture unit. Add PBS solution to wash the cells, add the labeling solution Annexin V / PI, place the chip in the dark for 15 minutes, and finally observe the chip under a fluorescent microscope. As shown in Figure 3, doxorubicin induces characteristic changes in the HepG2 cell membrane: in the early stage of apoptosis, the phosphatidylserine (PS) located on the inner side of the cell membrane is turned outward, showing a green circle (●); in the late stage of apoptosis, the cell membrane is permea...

Embodiment 3

[0038] use figure 1 The microfluidic chip shown was used to study the change of mitochondrial membrane potential (MMP) during apoptosis. Chips were inoculated with HepG2 and placed at 37°C, 5% CO 2 6 hours in the incubator. After the cells adhere to the wall, add DMEM medium (high sugar) with adriamycin concentration of 0 μM and 4.0 μM, place the chip in the cell culture incubator for 24 hours, and keep the solution from the concentration gradient generation unit to the cell culture at a small flow rate. unit. Add PBS solution to wash the cells, add labeling solution Rhodamine-123 (5 μg mL -1 ), the chip was placed in an incubator for 30 minutes, and then PBS solution was added to wash the cells, and finally the chip was placed under a fluorescence microscope for observation. As shown in Figure 4, doxorubicin leads to the leakage of MMP-dependent Rh-123, the fluorescence intensity of HepG2 cells is reduced, and the MMP is dissipated, and the degree is enhanced with the inc...

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Abstract

The invention discloses an integration microfluidic chip used for cell apoptosis research and the application thereof. The chip mainly comprises a concentration gradient generating unit and an array cell culture unit, and each outlet opening of the concentration gradient generating unit positioned in the upstream of the chip is connected with a row of cell culture chambers positioned in the down stream of the chip through a dam-shaped structure. The invention creatively integrates the concentration gradient generation, the culture, the stimulation, the washing, the marking of chip cells and the detection of multi cell responses on the chip, and is applicable to the research of clinical drug-induced tomour cell apoptosis. Compared with the traditional porous plate technology, the invention saves the complex operations for preparing drug solutions with different concentration, greatly simplifies the cell seeding, stimulated, washing and marking operation processes, obviously reduces the consumption of the cells and reagent, and can get a plurality of experiment parameters through operating the invention once.

Description

technical field [0001] The invention relates to cell apoptosis research technology, and in particular provides an integrated microfluidic chip for cell apoptosis research and its application. Background technique [0002] Apoptosis is an active cell death process regulated by genes, which is closely related to the occurrence of many major human diseases, and plays an important role in the occurrence of diseases such as normal embryonic and organ development, immune response, tumor and neurodegeneration. In addition, apoptosis is a complex cell signal transduction process, which is regulated by many signals from inside and outside the cell, and is realized through the transmission of biological signals between cells. This process involves a variety of cell function changes, and produces a variety of apoptosis-related events, such as abnormal cell membrane structure and function, resulting in the loss of two-sided asymmetry of the plasma membrane lipid bilayer, and the loss of...

Claims

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Application Information

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IPC IPC(8): C12M1/00G01N21/64G01N33/48
Inventor 秦建华林炳承叶囡楠刘欣
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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