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Serum replacement for immune cell suspending culture

A serum substitute and immune cell technology, applied in the direction of cell culture active agent, blood/immune system cells, culture process, etc., can solve the problems of strong pertinence, low cell yield, poor storage and application of medium, and achieve The ingredients are clear, the protein content is low, and the effect of avoiding repeated freezing and thawing

Inactive Publication Date: 2017-10-31
康思葆(北京)生物技术有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] Although the serum-free medium has many advantages that the traditional serum-containing medium cannot match, it still has the following deficiencies to be improved: a. Cells are susceptible to certain mechanical and chemical factors in the serum-free medium. The preservation and application of the medium are not as convenient as the traditional synthetic medium; b. Strong pertinence, different types of cells, even different cell lines or cell strains have different requirements for the nutrients in the medium; c. Use serum-free medium for original The cell yield is low when the generation cells are separated; d. At present, most of the insulin and transferrin necessary for serum-free medium are still derived from animals, which has not eliminated the potential safety hazards brought by serum; e. The cost is high

Method used

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  • Serum replacement for immune cell suspending culture
  • Serum replacement for immune cell suspending culture
  • Serum replacement for immune cell suspending culture

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Preparation of Serum Substitute for Immune Cell Suspension Culture

[0030] Serum replacement formula A of the present invention is shown in Table 1.

[0031] Table 1: Formulation of the Serum Replacement of the Invention (Formulation A)

[0032]

[0033] The preparation method of the serum substitute is as follows:

[0034] 1. Prepare iron-saturated human transferrin solution:

[0035] (1) 40mg FeCl 3 Dissolved in 20ml of 1mM HCl solution, aliquoted and stored at -20°C to obtain No. 1 storage solution;

[0036] (2) Take 200 mg of human transferrin, dissolve it in 20 ml of RPMI1640 medium, add 300 μl of No. 1 stock solution and stir evenly to obtain an iron-saturated human transferrin solution (10 mg / ml), store at -30 °C save.

[0037] 2. Preparation of cholesterol solution: Weigh 58 mg of cholesterol powder, add it to 20 ml of RPMI1640 medium, shake it at 4°C for 1 hour with an ultrasonic oscillator to make it completely emulsified, and then obtain a...

Embodiment 2

[0042] Embodiment 2. Preparation of the serum-free medium comprising the serum substitute of the present invention

[0043] 1. Prepare commercially available medium according to the instructions to obtain RPMI 1640 basal medium (RPMI Medium 1640 medium, Beijing Oriental Huahui Biomedical Technology Co., Ltd.). The formulation of RPMI 1640 basal medium is shown in Table 2.

[0044] 2. The formulation of the serum-free medium containing the serum substitute of the present invention: the serum-free medium (medium A) of the present invention comprises 90% of RPMI 1640 basal medium and 10% of serum substitute A.

[0045] 3. Preparation of serum-free medium (medium A): before use, slowly add 100ml serum substitute A to 800ml RPMI 1640 basal medium, stir well, adjust the pH to 7.2 with 5% NaHCO3, and then add RPMI1640 The volume of the culture medium was supplemented to 1000 ml to obtain a serum-free medium (medium A) containing the serum substitute A of the present invention.

[0...

Embodiment 3

[0048] Example 3. Evaluation of the proliferation ability of CIK cells cultured in serum-free medium A

[0049] Peripheral blood mononuclear cells (PBMC) were isolated and purified according to the methods reported in the literature using peripheral blood from healthy people, and induced in vitro to prepare CIK cells. The experiment is divided into 4 groups (Table 3), using respectively: 1) serum-free medium medium A (medium A) of the present invention, 2) complete medium (RPMI 1640+10%FBS), 3) imported serum-free Medium (X-VIVO 15 (LONZA)) and 4) domestic serum-free medium for cell culture.

[0050] After PBMC purification, the cell number was adjusted to 5 × 10 6 / ml, inoculated into T175 culture flasks. From the first day of induction, the medium was changed in half every 3 days, and the final concentration of cytokines in the medium was kept constant. At the same time, samples were taken and counted by the trypan blue exclusion method until the end of the 21st day of cu...

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Abstract

The invention relates to a serum replacement for immune cell suspending culture. The serum replacement is prepared from medicinal grade human albumin, recombinant human transferrin, recombinant human insulin, lipid, trace elements, hormones, shearing force protector, resveratrol, D-glucosamine hydrochloride, R-848 and C3. The serum replacement can be used for preparing a serum-free culture medium conveniently by a certain process before being used with a basic culture medium, and the serum-free culture medium can be used for suspending culture of immune cells. The serum replacement has the advantages that (1) the content of protein ingredients can be furthest reduced due to replaceable small-molecular compound in the serum replacement, ingredients are definite, and uniformity is strong; (2) the serum replacement can be prepared by a conventional method, and stored at negative 80-negative 20 DEG C; and (3) the serum replacement can be subpackaged and stored, and can be conveniently mixed with a basic culture medium before use, so that repeated freezing and unfreezing of the culture medium can be avoided.

Description

technical field [0001] The invention relates to a serum substitute for immune cell suspension culture, which belongs to the technical field of cell engineering and biomedicine. Background technique [0002] Somatic cell therapy refers to the treatment method of using human autologous, allogeneic or heterogeneous (non-human) somatic cells, which are reinfused (or implanted) into the human body after extracorporeal manipulation. This in vitro operation includes cell passage, expansion, screening, and drugs or other treatments that can change the biological behavior of cells. The somatic cells after in vitro operation can be used for the treatment of diseases, and can also be used for the diagnosis and prevention of diseases. There are many different types of somatic cell therapy, including in vivo reinfusion of in vitro activated mononuclear cells such as lymphokine activated killer cells (Lymphokine Activated Killer Cells, LAK), tumor infiltrating lymphocytes (Tumor Infiltrat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/02
CPCC12N5/0638C12N5/0646C12N2500/10C12N2500/24C12N2500/36C12N2500/90C12N2501/30C12N2501/33C12N2501/999C12N2501/998
Inventor 吴海涛赵侃周丽丽
Owner 康思葆(北京)生物技术有限公司
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