Method for constructing human peripheral blood immune cell bank

An immune cell bank and human peripheral blood technology, applied in the field of cell bank construction, can solve the problems of unsatisfactory survival rate of mononuclear cells, high price of serum-free cryopreservation solution, and unsuitable cryopreservation conditions. Low cost, high activity, easy to use effect

Active Publication Date: 2012-10-31
济南赛尔生物科技股份有限公司
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Problems solved by technology

However, the composition of human peripheral blood mononuclear cells is complex, including both immune cells and non-immune cells. Even immune cells are a combination of various types of cells. These cells have different requirements for cryopreservation conditions. Cryopreservation, because the cryopreservation conditions are not optimal, the survival rate of cryopreserved mononuclear cells is very unsatisfactory when resuscitated, and cannot meet clinical req

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  • Method for constructing human peripheral blood immune cell bank

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Embodiment 1

[0023] Example 1 Construction method of human peripheral blood immune cell bank

[0024] (1) Collect human peripheral blood: perform a physical examination on the donor, and check it according to the blood donation standard, and the blood will be drawn after two days if the standard is met;

[0025] (2) Separate autologous plasma: transfer the extracted peripheral blood to a centrifuge tube, in a centrifuge at 1500 rpm / centrifuge for 10 minutes, take the supernatant, inactivate at 56°C for 30 minutes, and set it at -20°C after completion Store frozen for later use.

[0026] (3) Separate peripheral blood mononuclear cells: Dilute the precipitate after removing autologous plasma in the above steps by volume with normal saline or PBS 1 times, and then add 0.5 times the volume of 4wt% hydroxyethyl starch solution , After mixing, let stand for 30 minutes, separate the supernatant, and add the supernatant to an equal volume of the lymphocyte separation liquid surface, 3000 rpm / separatio...

Embodiment 2

[0032] Example 2 Method for constructing human peripheral blood immune cell bank

[0033] (1) Collect human peripheral blood: perform a physical examination on the donor, and check it in accordance with the blood donation standard. The blood will be drawn after two days if the standard is met;

[0034] (2) Separate autologous plasma: transfer the extracted peripheral blood to a centrifuge tube, separate the heart in a centrifuge at 2500 rpm for 5 minutes, take the supernatant, inactivate at 56°C for 30 minutes, and set it at -20°C after completion Store frozen for later use.

[0035] (3) Separate peripheral blood mononuclear cells: Dilute the precipitate after removing autologous plasma from the above steps by volume with normal saline or PBS 1 times, and then add 1 times the volume of 3wt% hydroxyethyl starch solution , After mixing, let stand for 40 minutes, separate the supernatant, and add the supernatant to an equal volume of the lymphocyte separation liquid surface, 4000 rpm / ...

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Abstract

The invention discloses a method for constructing a human peripheral blood immune cell bank. The method comprises the following steps of: collecting human peripheral blood, separating autologous plasma, separating a peripheral blood mononuclear cell, separating a mononuclear cell by the peripheral blood mononuclear cell and freezing, separating a T lymphocyte by the peripheral blood mononuclear cell and freezing, separating a B lymphocyte by the peripheral blood mononuclear cell and freezing, separating an NK cell by the peripheral blood mononuclear cell and freezing, and encoding and puttingin storage. According to the invention, immune cells of health or young people are separated and are respectively independently frozen, and relative numbers are stored and put into storage, so that the stored human immune cells have the characteristics of high activity, high purity and convenience for use, and fetal calf serum can be replaced by the human autologous plasma, so that introduction of a foreign protein can be avoided. Meanwhile, the method, provided by the invention, has the advantages of low cost, low requirements on laboratory conditions, and wide application.

Description

Technical field [0001] The invention relates to a method for constructing a cell bank, in particular to a method for constructing a human peripheral blood immune cell bank. Background technique [0002] Immune cells refer to all cells related to immune response, mainly including T cells, B cells, killer cells, natural killer cells, monocytes, etc. In recent years, tumor and virus immunotherapy technology has been slowly applied to the clinic. Such as tumor vaccines and adoptive immunotherapy, these methods mainly use autologous immune cells to be stimulated, cultured, expanded and then infused in vitro to improve the immunity of patients. At present, such cells mainly include DC, CIK, NK, and DC-CIK. Wait. [0003] These immune cell treatments are mainly taken from the patient’s own peripheral blood, which is achieved by obtaining peripheral blood mononuclear cells and then undergoing a series of operations. However, the general population receiving immune cell treatment is mainl...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/02C12N5/0786C12N5/0783C12N5/0781
Inventor 王肇光薛书奎蔡平平田甜邢晓李财新陈莉
Owner 济南赛尔生物科技股份有限公司
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