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Cell in-vitro collection method and application thereof as well as in-vitro cell material and application thereof

A cell body and cell technology, applied in the field of medical cell in vitro experiments and biology, can solve the problems of fragmentation, inability to carry out batch operations, and difficult to carry out.

Inactive Publication Date: 2014-09-03
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the Transwell cell migration experiment, in addition to the cells adhering to the upper and lower bottom surfaces of the Transwell membrane, a large number of cells will be "trapped" in the micropores on the Transwell membrane, which is difficult to collect effectively for subsequent research.
In particular, the area of ​​the Transwell chamber is small, and the conventional chemical digestion method cannot be used for batch operation, and the amount of cell collection is low, which is not enough for subsequent cell biology and molecular biology research
In addition, in the conventional cell digestion method, the cells are detached from their culture plane, and the connection between cells only needs to be destroyed during cell digestion; while in the Transwell migration system, the cells have to pass through a narrow space with a pore size smaller than its diameter. Micropores, when digesting and collecting cells, cells need to be quickly detached from the micropores they are embedded in. This process can easily damage cells, and even lead to cell lysis and fragmentation
Therefore, on the whole, although the in vitro cell research technology has the advantages of simple intervention factors, easy control of experimental conditions, fast economy, good repeatability, and in-depth mechanism research, it is restricted by existing technical conditions, making in vitro cell migration difficult. Experiments usually can only understand the migration ability of cells simply by analyzing the cell migration rate, and researchers hope to further use the results of migration experiments, such as obtaining cells with high migration activity for further research, and comparing cells under in vitro experimental conditions. Studying the biological behavior of migrating and non-migrating cells has always been difficult

Method used

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  • Cell in-vitro collection method and application thereof as well as in-vitro cell material and application thereof
  • Cell in-vitro collection method and application thereof as well as in-vitro cell material and application thereof
  • Cell in-vitro collection method and application thereof as well as in-vitro cell material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Such as Picture 1-1 , as shown in Figures 1-2, the cells that have been migrated and cultured in Transwell in vitro are collected by the method of the present invention.

[0051]In this example, human bone marrow-derived mesenchymal stem cells (human bone marrow mesenchymal stem cells, hereinafter referred to as hMSCs) were used as research objects, and human-derived recombinant PDGF-BB (platelet-derived growth factor) was used as a chemokine to induce cell migration.

[0052] 1. Experimental materials and reagents

[0053] Transwell chamber (i.e. Transwell upper chamber): Coring's Transwell chamber suitable for 6-well cell culture plates, with an area of ​​4.67cm 2 , diameter 24mm; article number: #3428, #354576; membrane material is polycarbonate (Polycarbonate, PC) or polyethylene terephthalate (polyethylene terephthalate, PET), pore size 8.0um.

[0054] Cell culture plate (i.e. Transwell lower chamber): 6-well cell culture plate from Corning Company, Cat. No. CLS3...

Embodiment 2

[0095] Morphological detection experiment of high migratory living cells and low migratory active cells isolated under the same chemokine induction.

[0096] Cell culture medium: DMEM low-glucose medium with 10% fetal bovine serum

[0097] DMEM low-sugar medium: same as Example 1

[0098] Inverted microscope: same as Example 1

[0099] Use the cells in centrifuge tube A and centrifuge tube B collected in Example 1 as materials, inoculate them into cell culture medium respectively, place them in a 37°C, 95% carbon dioxide incubator, and culture them for 24 hours respectively to detect the morphological characteristics of the cells under an inverted microscope ( Figure 2a , Figure 2b ).

Embodiment 3

[0101] Cytoskeleton morphology detection experiment of high migratory living cells and low migratory active cells isolated under the same chemokine induction.

[0102] Tetramethylrhodamine-labeled phalloidin (Phalloidin–Tetramethylrhodamine B isothiocyanate: product of Sigma, Cat. No. P1951

[0103] Inverted microscope: same as Example 1

[0104] Using the cells in centrifuge tube A and centrifuge tube B collected in Example 1 as materials, the cells were stained with tetramethylrhodamine-labeled phalloidin, and the morphology of the cytoskeleton was detected under an inverted microscope ( Figure 3a , Figure 3b ).

[0105] In this example, the phalloidin staining method was operated according to the Sigma product manual.

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Abstract

The invention discloses a cell collection method and a cell material. Aiming at the defect that scientific content for prompting experiment result in the conventional Transwell migration research is limitedly utilized, the invention provides an in-vitro collection method for the first time. According to the method, after the Transwell cell migration culture is completed and culture liquids inside an upper chamber and a lower chamber of Transwell are eliminated, cells in the upper chamber and the lower chamber after the Transwell cell migration experiment are digested and collected by using a method with the combination of trypsinization and mechanical oscillation; in pancreatin cell digestion operation, a phosphate buffer liquid of which the pH value is 7.35-7.45 is used as a washing liquid, and a pancreatin cell digestion liquid contains 0.25% of pancreatin and 0.01 EDTA (Ethylene Diamine Tetraacetic Acid). The invention further provides a high migration living cell material and a low migration living cell material generated from identical chemotactic factor induction through separation. The invention further provides a collection method and an application of the cell material in a cell biological behavior analysis experiment. The Transwell in-vitro migration experiment is expanded to cell biological behavior and molecular biology study on cell migration.

Description

technical field [0001] The invention relates to a method for collecting cells and cell materials, in particular to a method for collecting cells in vitro and cell materials in vitro. It belongs to the technical field of biological and medical cell in vitro experiments. Background technique [0002] Cell migration is the chemotactic movement of cells under the stimulation of exogenous signals. Usually, cells move from low-concentration to high-concentration areas after sensing the concentration gradient of certain substances. In physiological state, cell migration plays an important role in cell foraging, wound healing, embryogenesis, tissue repair, immune response, etc.; in pathological state, cell migration also promotes cancer metastasis, atherosclerosis and The occurrence and development of diseases such as arthritis. Therefore, cell migration is an important research direction in current life sciences. [0003] Transwell migration technique is currently the most widel...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12Q1/02
Inventor 李娟郝晋赵志河王娅婷方善宝
Owner SICHUAN UNIV