Debonding protectant, method for preparing monocell suspension with high survival rate and application of debonding protectant

A protective agent and concentration technology, which is applied in cell dissociation methods, biochemical equipment and methods, pancreatic cells, etc., to achieve the effects of saving economic pressure, improving stability, and shortening the time of experiments

Pending Publication Date: 2020-06-05
CHENGDU DAOSHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve the problem of obtaining single cells with high survival rate in the prior art, this application provides a method for quickly obtaining high survival rate and high single cell rate, and at the same time, it can make the surviving single cells in the prepared single cell suspension It can survive for a long time, which is the most difficult part for the existing single-cell sequencing and primary cell culture

Method used

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  • Debonding protectant, method for preparing monocell suspension with high survival rate and application of debonding protectant
  • Debonding protectant, method for preparing monocell suspension with high survival rate and application of debonding protectant
  • Debonding protectant, method for preparing monocell suspension with high survival rate and application of debonding protectant

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Embodiment 1

[0055] A method for preparing a single-cell suspension with a high survival rate, comprising soaking a target tissue block with a release and protecting agent, and then using an ultrasonic ablation device to ablate the tissue block, specifically comprising the following steps:

[0056] Step S100, take the target tissue block under a sterile environment, and use scissors to cut the tissue block to no more than 2mm 3 small pieces;

[0057] Step S200, placing the target tissue block in a container and washing it with PBS buffer until the PBS buffer is visually clear, wherein the largest external dimension of the tissue block is no more than 2 mm;

[0058] Step S300, placing the target tissue block in a 1.5 ml EP tube, and at the same time, adding the release protection agent described in claim 8 into the EP tube, and placing it at a constant temperature of 37°C for 5 minutes;

[0059] Step S400, adjust the frequency of the ultrasonic ablation instrument to 40kHz, adjust the powe...

Embodiment 2

[0064] In this embodiment, a single-cell suspension is obtained by conventional enzyme digestion method, and the specific steps are as follows:

[0065] Step 1: Place selected enzymes and tissue pieces in EP tubes filled with 1-2 ml of PBS buffer;

[0066] Step 2: Digest the EP tube in Step 1 in an environment with a constant temperature of 37°C for 30 minutes;

[0067] Step 3: Shake and oscillate not less than 5 times intermittently during digestion;

[0068] Step 4: Filter the digested solution with a 400-mesh nylon mesh to remove impurities and tissue pieces, and take the suspension;

[0069] Step 5: Centrifuge the suspension obtained in Step 4, remove the supernatant, and retain the cell pellet; the centrifugation speed is 1500r / min, and the centrifugation time is 5 minutes;

[0070] Step 6: Routine staining with trypan blue, and counting the stained single cell suspension with a hemocytometer.

[0071] The target tissue block in this embodiment is the kidney of an adul...

Embodiment 3

[0073] In this embodiment, a single-cell suspension is obtained by conventional enzyme digestion method, and the specific steps are as follows:

[0074] Step 1: Place selected enzymes and tissue pieces in EP tubes filled with 1-2 ml of PBS buffer;

[0075] Step 2: Digest the EP tube in Step 1 in an environment with a constant temperature of 37°C for 180 minutes;

[0076] Step 3: Shake and oscillate intermittently for no less than 15 times during digestion;

[0077] Step 4: Filter the digested solution with a 400-mesh nylon mesh to remove impurities and tissue pieces, and take the suspension;

[0078] Step 5: Centrifuge the suspension obtained in Step 4, remove the supernatant, and retain the cell pellet; the centrifugation speed is 1500r / min, and the centrifugation time is 5 minutes;

[0079] Step 6: Routine staining with trypan blue, and counting the stained single cell suspension with a hemocytometer. The target tissue block in this embodiment is the kidney of an adult mo...

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Abstract

The application discloses a debonding protectant. The debonding protectant is prepared through mixing the following ingredients: dispase II with a concentration of 14g/L to 58g/L, kanamycin with a concentration of 14g/L to 58g/L, collagenase with the content of 0.8*10<6>U/L to 1.25*10<6>U/L and 0.7g/L-2.2g/L glucose. Simultaneously, the application provides a method for preparing a monocell suspension with high survival rate. Specifically, tissue blocks can be dispersed into single cells in ultrashort time through ablation action of an ultrasonic ablatograph, and cytoactivity is reserved as much as possible, and destruction to the cells is not caused. According to the application, the technical problem in applications of primary cell culture and monocell sequencing that high-activity primary cells are required to be acquired is excellently solved. Meanwhile, consumed time is greatly shortened, the whole dispersion process is shortened into several seconds from several hours required bythe existing enzyme digestion method, the stability is high, the mono-cellularization is good, and the activity of the cells is high.

Description

technical field [0001] The invention relates to the field of reagents for obtaining primary cells, in particular to the field of reagents for obtaining primary living cells through animal tissues, in particular to a release protectant and a method for preparing a single cell suspension with a high survival rate and its application. Background technique [0002] The cultivation of primary cells refers to the cultivation of cells, tissues and organs directly after removal from the body. Therefore, more strictly speaking, it refers to the culture before successful subculture. At this time, the cells maintain the basic properties of the original cells, and if they are normal cells, they still retain the diploid number. But in fact, the cultured cells from the first generation to the tenth generation are generally referred to as primary cell culture. [0003] The most commonly used primary cell cultures are tissue block culture and dispersed cell culture. Tissue block culture i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0676C12N2500/34C12N2501/734C12N2509/00
Inventor 赵伟邱坤廖东升
Owner CHENGDU DAOSHENG BIOTECH CO LTD
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