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Efficient capturing, high-content imaging and whole transcriptome analyzing device and method for single cell

A capture device and single-cell technology, applied in the field of single-cell capture and analysis, can solve the problem of not being able to combine high-throughput imaging characterization and whole-transcriptome analysis, and not showing the possibility of RNA sequencing analysis, cell phenotype and genotype Correlation defects and other problems, to achieve the effect of reducing consumption, simple device structure, and long-term effective capture unit

Inactive Publication Date: 2017-11-24
UNIV OF SCI & TECH BEIJING
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the method is limited to rare tumor cells in body fluids, and each individual microwell contains more than one cell, which makes image analysis of single cells difficult and does not demonstrate the possibility of RNA-sequencing analysis
[0005] Therefore, the currently reported methods for single-cell analysis using microfluidic technology cannot achieve the combination of high-throughput imaging characterization of single cells and whole-transcriptome analysis, so there are deficiencies in the study of the correlation between cell phenotype and genotype

Method used

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  • Efficient capturing, high-content imaging and whole transcriptome analyzing device and method for single cell
  • Efficient capturing, high-content imaging and whole transcriptome analyzing device and method for single cell
  • Efficient capturing, high-content imaging and whole transcriptome analyzing device and method for single cell

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Embodiment 1

[0054] Such as Figure 1-2 As shown, this embodiment provides a single cell high-throughput capture device, which is used to capture single cells, and the device includes a microwell array chip and a cell culture plate or a cell culture dish substrate; the microwell The array chip is pasted on the base of the cell culture plate or the base of the cell culture dish, and the surface of the base of the cell culture plate or the base of the cell culture dish is not treated.

[0055] Microwells are distributed on the microwell array chip, the diameter of the microwells is 23 μm±5 μm, the depth is 25±10 μm, and the distance between the centers of any two adjacent microwells is 46 μm±10 μm. Specific dimensions can be guaranteed to meet the special needs of capturing single cells.

[0056] The manufacturing method of the microwell array chip is as follows: using photolithography technology to produce a mold containing a microcolumn array, then pouring polydimethylsiloxane directly on...

Embodiment 2

[0068] This embodiment provides a single cell high-throughput capture method for high-throughput imaging analysis of single cell heterogeneity, using the above-mentioned device, including the following steps:

[0069] Step 1, cell apoptosis induction and staining: use Reversine to induce apoptosis of human lung cancer cells. In this example, four concentration gradients and two time gradients were set respectively: the concentration gradients were 0, 10, 20, 30 μM, and the time gradients were 1, 2h; human lung cancer cells in normal growth state were planted in 24-well plates When it adheres to the wall and covers about 80% of the bottom area, apply different concentrations of drugs, and after 1 h or 2 h, the drugs are removed, and after washing with PBS, the immunofluorescence reagent CellEvent Caspase-3 / Immunofluorescence staining was performed on cells with 7 Red for 30 min. This fluorescent reagent can stain apoptotic cells, and the fluorescence intensity is positively co...

Embodiment 3

[0076] This embodiment provides a method for positioning and picking single cells and performing whole-transcriptome analysis, which is used for high-throughput imaging analysis of single-cell heterogeneity. Using the above-mentioned device, the method includes the following steps:

[0077] Step 1. Prepare a capillary needle with a tip inner diameter of about 30-50 µm;

[0078] Step 2: Take a photo of the single cell area, obtain the bright field image and the fluorescence field image of a certain area, and select and locate the target single cell with the two images;

[0079] Step 3. Insert the capillary needle vertically and observe the position of the needle tip in the eyepiece, such as Figures 7A-7CAs shown, when it is above the target single cell, quickly penetrate it, and the cells are sucked into the capillary needle under the action of capillary force, then take out the capillary needle, insert it into the long rubber tube, and use the mouth from the other end of the ...

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Abstract

The invention belongs to the technical field of single-cell capturing and analysis and in particular relates to an efficient capturing, high-content imaging and whole transcriptome analyzing device and method for a single cell. The device is used for capturing the single cell; the device comprises a polydimethylsiloxane micro-well array chip and a cell culture plate substrate or a cell culture dish substrate; the micro-well array chip is fitted to the cell culture plate substrate or the cell culture dish substrate. The invention provides the device and method, which can be used for simultaneously carrying out single-cell high-throughput imaging analysis, whole transcriptome analysis of the target single cell and deep exploration of heterogeneity between the single cells from a whole transcriptome level of the single cells.

Description

technical field [0001] The invention belongs to the technical field of single cell capture and analysis, and in particular relates to devices and methods for single cell high-efficiency capture, high-content imaging and whole transcriptome analysis. Background technique [0002] High-throughput monitoring of single-cell responses is important for a variety of applications including drug testing, toxicology, and basic cell biology. Recent studies have highlighted the remarkable heterogeneity in the genome, transcriptome and proteome across cancer cell populations, and characterizing this heterogeneity is critical for understanding tumor progression. Single-cell analysis is of great significance to the early diagnosis, treatment, drug screening and research of cell physiology and pathological process of major diseases, and has become one of the research hotspots. [0003] The capillary electrophoresis (CE) separation method developed in the early 1980s is suitable for the det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/533G01N33/574G01N33/50C12M1/00C12N5/00
CPCC12M47/04C12N5/00G01N21/6428G01N21/6452G01N21/6456G01N21/6458G01N33/5011G01N33/533G01N33/574G01N33/57415G01N33/57423G01N2021/6439G01N2021/646
Inventor 赵亮陈甘雨刘杨张学记
Owner UNIV OF SCI & TECH BEIJING
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