Preparation method of mesenchymal stem cell expressing human-derived immune stimulating factor LIGHT and prepared MSC-L cell

A stem cell and immune stimulation technology, applied in the field of biological immunotherapy, can solve the problems of cytokine stimulation, low cell density, slow growth, etc., and achieve the effects of high clone formation rate, short doubling time, and easy collection.

Pending Publication Date: 2018-04-17
上海金坤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme digestion, centrifugation and flow cytometry, although the obtained cells will be relatively pure, but the cells may be damaged due to repeated and lengthy operations; in addition, the cell density after sorting is relatively thin, and other cells cannot be obtained. Stimulated by paracrine release of cytokines, growth tends to be slower;

Method used

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  • Preparation method of mesenchymal stem cell expressing human-derived immune stimulating factor LIGHT and prepared MSC-L cell
  • Preparation method of mesenchymal stem cell expressing human-derived immune stimulating factor LIGHT and prepared MSC-L cell
  • Preparation method of mesenchymal stem cell expressing human-derived immune stimulating factor LIGHT and prepared MSC-L cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] This example uses the umbilical cord as the source of MSCs to prepare a method for the preparation of mesenchymal stem cells expressing the human immunostimulatory factor LIGHT, such as figure 1 and figure 2 As shown, the specific steps are as follows:

[0061] (1) Isolating mesenchymal stem cells by an adherent screening method, and performing primary culture and subculture, which includes the following steps:

[0062] a) Rinse the isolated umbilical cord with normal saline until there is no blood clot, immerse it in PBS or normal saline, cut it to 0.25-0.35cm3 fragments, centrifuge at 2500-3000rpm for 1min to obtain a precipitate of umbilical cord tissue, and discard the supernatant;

[0063] b) Add an equal volume of type Ⅱ collagenase to the umbilical cord tissue pellet, pipette evenly, seal it and place it on a shaker at 37°C for 20-40min, pipette it to collect the type Ⅱ collagenase solution containing MSC and transfer it to In the MEM culture medium, centrifug...

Embodiment 2

[0077] This example is a study on the in vivo and in vitro migration characteristics of prepared MSC-L cells.

[0078] In this embodiment, the determination of the expression of LIGHT on the surface of MSC-L cells is carried out, which specifically includes the following steps:

[0079] (1) Collect MSC-L, the total number of cells is less than 10 6 ;

[0080] (2) Centrifuge at 1700rpm for 3min and resuspend with FACS buffer. Then repeat this step one more time.

[0081] (3) Add 50U 2.4G2 and incubate at room temperature for 1-10min. Add LTβR-Ig lug and incubate at room temperature for 15-30min.

[0082] (4) Add 1 ml of FACS buffer, mix gently, centrifuge at 1700 rpm, and discard the supernatant.

[0083] (5) Add anti-mouse IgG2-PE lug and incubate at room temperature for 15-20min.

[0084] (6) Add FACS buffer to wash, and centrifuge at 1700rpm.

[0085] (7) Discard the supernatant. Add 300ulFACS and mix gently. On-board analysis.

[0086] The flow detection results a...

Embodiment 3

[0107] This embodiment is the research on the anti-tumor effect of prepared MSC-L cells, which includes the following experimental steps:

[0108] 1) MSC-L can effectively target and control tumor growth

[0109] 1.1) MSC-L preventive anti-tumor model

[0110] (1) Collect cells when MSCs and MSC-L grow to 70% confluence;

[0111] (2) Wash once with PBS, count, and adjust the cells to an appropriate concentration;

[0112] (3) Mix 200ul of PBS or 200ul of 1×10 6 MSC and MSC-L were subcutaneously inoculated into the left side of BALB / c mice;

[0113] (4) After 2 weeks, inoculate 200u14×10 5 TUBO cells;

[0114] (5) Observe the tumor growth every 2-3 days thereafter.

[0115]1.2) MSC-L therapeutic anti-tumor model

[0116] (1) 200ul of 4×10 5 TUBO cells were inoculated subcutaneously on the left side of the mouse;

[0117] (2) After 7 days, collect the MSCs and MSC-L of the cells grown to 70% confluence, wash, count, and adjust the cells to an appropriate concentration. ...

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Abstract

The invention discloses a preparation method of a mesenchymal stem cell expressing a human-derived immune stimulating factor LIGHT. The preparation method comprises the following steps that the mesenchymal stem cell is separated and subjected to primary culture and secondary culture; and by building up a retroviral and lentiviral vector pMIGR3-hLIGHT and a pCDHEF-hLIGHT plasmid, a human LIGHT geneis transferred into the mesenchymal stem cell to build up an MSC-L cell, and the MSC-L cell is the mesenchymal stem cell expressing the human-derived immune stimulating factor LIGHT. The invention further relates to the MSC-L cell prepared by adopting the method and application of the MSC-L cell. According to the application of the MSC-L cell, an umbilical cord is adopted as an MSC source, the human LIGHT gene is transferred into the MSC, the LIGHT is transferred into a tumor tissue through the MSC, the MSC modified by the LIGHT can change the tolerance state of a host to a tumor by generating strong antitumor cells and immune reaction of tumor-related mesenchymal cells, and accordingly the tumor is controlled effectively. The mesenchymal stem cell can be prepared on a large scale, and used for treatment of various solid malignant tumors and prevention of cancers.

Description

technical field [0001] The invention relates to the technical field of biological immunotherapy, in particular to a method for preparing mesenchymal stem cells expressing human immunostimulatory factor LIGHT, the prepared MSC-L cells and applications thereof. Background technique [0002] In addition to tumor cells, tumor tissue also contains a large amount of tumor stroma, which is often an accomplice to the wanton expansion of tumors. First, the tumor stroma provides a stable microenvironment for tumor progression, acting as a barrier that prevents the effective entry of therapeutic drugs. Moreover, tumor stroma can promote tumor proliferation through direct contact or secretion of cytokines. In addition, stromal cells also suppress host immunity and induce tumor immune tolerance. These findings suggest that the tumor stroma is critical for tumor development. Some researchers have found that only destroying the tumor stroma can control the tumor well, at least keeping t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/28A61P35/00
CPCA61K35/28C07K14/47C12N5/0662C12N15/86C12N2510/00
Inventor 杨选明汪鑫傅阳心叶圣勤徐闻达
Owner 上海金坤生物科技有限公司
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