Unlock instant, AI-driven research and patent intelligence for your innovation.

In vitro cell collection method and its application, in vitro cell material and its application

A cell body and cell technology, applied in the field of biological and medical cell in vitro experiments, can solve problems such as difficult to collect, easy to damage cells, and cell lysis

Inactive Publication Date: 2016-08-17
SICHUAN UNIV
View PDF1 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • In vitro cell collection method and its application, in vitro cell material and its application
  • In vitro cell collection method and its application, in vitro cell material and its application
  • In vitro cell collection method and its application, in vitro cell material and its application

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 Bisothiocyanate: 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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
Login to View More

Abstract

The invention discloses a cell collection method and a cell material. Aiming at the limitation of limited use of the scientific content suggested by the experimental results in existing Transwell migration studies, the present invention firstly provides a method for collecting cells in vitro. In this method, after the Transwell cell migration culture is completed and the culture medium in the upper and lower chambers of the Transwell is removed, the cells in the upper and lower chambers after the Transwell cell migration experiment are digested and collected by using trypsin digestion and combined with mechanical oscillation; In the trypsin cell digestion operation, the washing solution is phosphate buffer solution with pH 7.35-7.45, and the trypsin cell digestion solution contains 0.25% trypsin and 0.01% EDTA. The invention also provides a living cell material with high migration activity and a cell material with low migration activity separated and produced by the same chemokine. The present invention also provides the application of the above collection method and cell material in the analysis test of cell biological behavior. The invention can extend the Transwell migration experiment in vitro to the cell biology behavior and molecular biology research of 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12Q1/02
Inventor 李娟郝晋赵志河王娅婷方善宝
Owner SICHUAN UNIV