Preparation method and application of human TSCMs (T memory stem cells)
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A cell and blood cell separation machine technology, applied in the field of in vitro expansion of human peripheral blood TSCM cells, can solve the problems of not being able to meet one-time reinfusion, and the amount of cultured cells is small
Inactive Publication Date: 2016-05-04
SHENZHEN HORNETCORN BIOTECH
View PDF0 Cites 10 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Problems solved by technology
[0003] The existing TSCM culture methods in foreign countries have a small amount of cultured cells, which cannot meet the amount required for one reinfusion
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
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0012] In Example 1, PBMCs were stimulated with rhIL-7, rhIL-15, rhIL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody, and IM-12 was used to block stem cell differentiation to obtain TSCM cells. The specific operation includes the following steps:
[0013] 1. Under sterile conditions, collect peripheral venous blood from the patient with a blood cell separator or a 50mL syringe, and obtain mononuclear cells by density gradient centrifugation of Ficoll-diatrizoate glucosamine. The specific steps are: 1500 rpm, centrifuge for 10 minutes, absorb the upper plasma layer, inactivate at 56°C for 30 minutes and centrifuge for later use, dilute the precipitated blood cells with normal saline at a ratio of 1:1, human lymphocyte separation medium and diluted blood at a ratio of 1 Add the ratio of 2 to the centrifuge tube, centrifuge at 2000 rpm for 20 minutes, centrifuge the blood that has been left for a long time for 30 minutes, carefully absorb the buffy coat, wash ...
Embodiment 2
[0016] In Example 2, the morphology, viability and uniformity of the cultured TSCM cells were detected. The specific operation includes the following steps:
[0017] 1. Morphological observation. After 24 hours of cell culture, it can be seen that TSCM cells sink to the bottom of the culture dish and tend to colonize. After 48 hours, the colonies begin to grow larger.
[0018] 2. Viability detection, take the cultured cells, centrifuge at 1500 rpm for 5 minutes, discard the medium, stain with 100ng / mLPI containing 5% fetal bovine serum and 0.1% sodium azide for 15 minutes, 1500 rpm Centrifuge / centrifuge for 5 minutes, discard the supernatant, resuspend in normal saline, and detect with a flow cytometer.
[0019] 3. Homogeneity test, take the cells on day 0, 7, 14 and 21 respectively, wash them twice with normal saline containing 5% fetal bovine serum and 0.1% sodium azide, and adjust the cell density to 1×10 6 cells / mL, add 50 μL cell suspension to each detection tube, add f...
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
Property
Measurement
Unit
concentration
aaaaa
aaaaa
Login to view more
Abstract
The invention discloses a preparation method of human TSCMs (T memory stem cells), which comprises the following steps: (1) collecting peripheral venous blood with a blood cell separator or injector under aseptic conditions, and carrying out Ficoll-Hypaque density gradient centrifugation to obtain mononuclear cells; (2) putting the separated PBMCs (peripheral blood mononuclear cells) in a culture medium containing irritants and cell factors, regulating the cell density to (0.5-2)*10<6>/mL, transferring into a cell culture plate, culture bottle or culture bag, and culturing in an incubator; (3) after culturing the cells for 48-72 hours, replacing half of the culture solution, supplementing the equivalent culture solution to keep the cell density at (0.5-2)*10<6>/mL; and according to the cell growth state, harvesting the cells in the 10th-21st day. An anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody are utilized as the irritants to activate the TSCMs, the cell factors rh IL-7, rh IL-15 and rh IL-2 are combined for irritation, and IM-12 is utilized to stop the stem cell differentiation, so that the cultured TSCMs have the common characteristics of both memory cells and stem cells, thereby providing a new optional scheme for clinical adoptive immunity cellular therapy.
Description
technical field [0001] The invention belongs to the technical field of cellular immunology, in particular to a method and application for expanding human peripheral blood TSCM cells in vitro. Background technique [0002] In recent years, with the development of immunology technology, non-specific immune cell therapy has been accepted by more and more people in clinic because of its certain effect. At present, the existing immune cell therapy includes CIK, CTL and other cells, which have a short survival time in the body, and the therapeutic effect varies greatly with individual differences and different preparation methods. The ability of immune cells to self-renew, maintain homeostasis, and kill tumors will weaken with differentiation, and finally become terminally differentiated cells, showing a state of aging. Most of the CIK, CTL and other cells on the market tend to be terminally differentiated cells. TSCM cells can exist in the body for a long time through self-re...
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
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more