Method for cryopreservation of DC cells and CIK seed cells in blood, prepared cells and application

A seed cell and cryopreservation technology, which is applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of patient injury and inappropriate collection of peripheral blood, so as to improve recovery efficiency and avoid the risk of FBS The effect of using

Inactive Publication Date: 2016-08-10
TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biggest problem with this method is that peripheral blood needs to be collected for each treatment, which brings repeated trauma to the patient, and in some cases, the patient is not suitable for peripheral blood collection

Method used

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  • Method for cryopreservation of DC cells and CIK seed cells in blood, prepared cells and application
  • Method for cryopreservation of DC cells and CIK seed cells in blood, prepared cells and application

Examples

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preparation example Construction

[0023] Preparation of autologous serum:

[0024] Collect 80-120ml of peripheral blood or umbilical cord blood, add physiological saline at a volume ratio of 1:1, mix well, and separate plasma and mononuclear cells through Ficoll lymphocyte separation medium;

[0025] Inactivate the plasma at 55-58°C for 30 minutes, centrifuge the inactivated plasma at 4500g-6000g for 8-12 minutes, filter the supernatant with a 0.22um filter membrane and finally prepare autologous serum;

[0026] Cryopreservation of mononuclear cells:

[0027] 1. After the isolated mononuclear cells were washed once with normal saline containing 1% FBS, the cells were resuspended in immune cell culture medium and counted;

[0028] 2. Prepare DC cell induction medium: the immune cell medium contains FBS with a volume fraction of 5-8%, IL-4 with a final concentration of 50-100IU / ml, and GM-CSF with a final concentration of 500-1500IU / ml;

[0029] 3. Take 1-2*10 according to the cell counting result 7 Inoculate...

Embodiment 1

[0042] Preparation of autologous serum:

[0043]1. Collect 120ml of peripheral blood, add physiological saline at a volume ratio of 1:1, mix well, and separate plasma and mononuclear cells through Ficoll lymphocyte separation medium;

[0044] 2. Inactivate the plasma at 56°C for 30 minutes, centrifuge the inactivated plasma at 4500gg for 12 minutes, filter the supernatant with a 0.22um filter membrane and finally prepare 223ml of autologous serum;

[0045] Cryopreservation of mononuclear cells:

[0046] 1. After the isolated mononuclear cells were washed once with 30ml of normal saline containing 1% FBS, the cells were resuspended in 20ml of immune cell culture medium and counted to obtain 1.98*10 8 cells;

[0047] 2. Prepare DC cell induction medium: the immune cell culture medium contains FBS with a volume fraction of 5%, IL-4 with a final concentration of 100IU / ml, and GM-CSF with a final concentration of 1000IU / ml;

[0048] 3. Take 2*10 according to the cell counting re...

Embodiment 2

[0061] Preparation of autologous serum:

[0062] 1. Collect 80ml of peripheral blood or umbilical cord blood, add physiological saline at a volume ratio of 1:1, mix well, and separate plasma and mononuclear cells through Ficoll lymphocyte separation medium;

[0063] 2. Inactivate the plasma at 55°C for 30 minutes, centrifuge the inactivated plasma at 5000g for 8 minutes, filter the supernatant with a 0.22um filter membrane and finally prepare autologous serum;

[0064] Cryopreservation of mononuclear cells:

[0065] 1. After the isolated mononuclear cells were washed once with 20ml of normal saline containing 1% FBS, the cells were resuspended in immune cell medium and counted to obtain 1.5*10 8 cells;

[0066] 2. Prepare DC cell induction medium: the immune cell culture medium contains FBS with a volume fraction of 6%, IL-4 with a final concentration of 50IU / ml, and GM-CSF with a final concentration of 500IU / ml;

[0067] 3. Take 1*10 according to the cell counting result ...

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Abstract

The invention discloses a method for freezing DC cells and CIK seed cells in blood, and the prepared cells and their application. The mononuclear cells in peripheral blood or umbilical cord blood are frozen in autologous serum after appropriate treatment, and the cell The cryoprotectant makes the survival rate of the cells reach 80%. The resuscitated cells can be used to prepare DC-cells and CIK cells at the same time, and finally prepare DC-CIK cell preparations that can meet the needs of clinical applications. In this way, the requirement of multiple DC-CIK clinical treatments can be realized in a single blood collection, thus providing support for the large-scale development of immune cell therapy.

Description

technical field [0001] The invention relates to the field of biology, in particular to a freezing method for DC cells and CIK seed cells in blood, the prepared cells and the use thereof. Background technique [0002] Immune cell therapy, currently recognized as the fourth medical method against tumors, has been gradually accepted by the mainstream medical community due to its advantages of safety, effectiveness and no side effects. The current immune cell preparation method is mainly to collect peripheral blood from patients, isolate peripheral blood mononuclear cells, and perform in vitro culture and expansion. The biggest problem with this method is that peripheral blood needs to be collected for each treatment, which causes repeated damage to the patient, and in some cases the patient is not suitable for collecting peripheral blood. Therefore, there is a need for a method that can preserve cells for a long time after a single collection and can meet the needs of cell sto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/0784C12N5/0783
CPCA01N1/0221C12N5/0636C12N5/0639C12N2500/84C12N2501/22C12N2501/2302C12N2501/2304C12N2501/24C12N2501/25C12N2501/515C12N2506/11
Inventor 韩洪起鲁振宇刘俊江张冰晶秦臻徐悦
Owner TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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