Cell cryopreservation and resuscitation method and prepared cell preparation
A cryopreservation and cell technology, applied in the field of cell preparations, can solve the problems of increased cryopreservation cost, low induction efficiency, unsatisfactory cells, etc., so as to improve survival rate and shelf life, avoid high cryopreservation costs, and avoid cells damage effect
Inactive Publication Date: 2015-08-26
UNION STEMCELL & GENE ENG
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Problems solved by technology
However, these methods also have their own defects: first, most of the mononuclear cells collected each time are frozen as a single cell type, and it is impossible to collect mononuclear cells at one time to meet the requirements for DC and CIK in DC-CIK immune cell therapy. Requirements for the number of cells; secondly, the direct induction of DC cells from frozen mononuclear cells has the problem of low induction efficiency, while the direct freezing of mature DC cells induced by freezing will lead to cell lysis, and the destruction of cells cannot meet the clinical requirements for DC cells. Thirdly, the mononuclear cells collected each time have the characteristics of continuous proliferation during the induction process of CIK, which makes it far beyond the clinical 10 9 The number of cells is required. If the cost of CIK cell preparation is to be saved, some mononuclear cells will be wasted. If the collected mononuclear cells are directly induced by CIK cells and frozen, it will increase the cost of freezing and occupy the frozen space of the liquid nitrogen tank. Storage space is too close to cause waste, which is not conducive to large-scale storage services
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Embodiment 1
[0032] 1. Use lymphocyte separation medium to separate peripheral blood mononuclear cells (PBMC) from fresh cells collected by a peripheral blood apheresis machine:
[0033] 1) Use a peripheral blood apheresis machine to collect cells 6*10 9 Cells were diluted with normal saline to a volume of 300ml and added heparin sodium injection to a final concentration of 20u / ml.
[0034] 2) Add 75ml of Ficoll lymphocyte separation solution to each of two 250ml centrifuge tubes, divide the 300ml PBMC suspension into two parts and add them to the upper layer of Ficoll in the centrifuge tubes respectively (operate gently to maintain obvious stratification). Centrifuge in a centrifuge, the parameters are: 500g, 19 degrees, 23 minutes, start and brake 1 (the centrifuge used in this patent is the LEGEND RT+ type desktop centrifuge produced by THERMO company, all relevant parameters refer to the centrifuge manual) .
[0035] 3) After the centrifugation, take out the centrifuge tube gently to...
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The invention discloses a cell cryopreservation and resuscitation method and a prepared cell preparation. The cell cryopreservation and resuscitation method comprises the steps of: freezing a part of mononuclear cells in -196 DEG C liquid nitrogen respectively via a mode of a mononuclear cell and an initially induced immature DC cell, resuscitating the frozen mononuclear cells and directly inducing the mononuclear cells to form CIK, resuscitating the frozen immature DC cells and inducing the cells into matured DC cells. According to the cell cryopreservation and resuscitation method and the prepared cell preparation, waste of mononuclear cells collected at each time can be avoided, indexes such as activity and number of cells are guaranteed, clinical application and large-scale storage service of DC-CIK cell therapy also can be satisfied.
Description
technical field [0001] The invention relates to a method for freezing and resuscitating cells and a cell preparation prepared therefrom. Background technique [0002] Driven by the rapid development of molecular biology and bioengineering technology, biological therapy based on immune cell therapy has received increasing attention, and has gradually become the fourth largest treatment mode in modern malignant tumor treatment after radiotherapy, chemotherapy and surgery. . At present, DC-CIK immune cell therapy is the most effective in clinical application, and it is also the mainstream of cell therapy in China. In order to solve the problem that patients need multiple blood collections in immune cell therapy, and to ensure the course of treatment, clinically, most of them adopt the method of freezing peripheral blood mononuclear cells and then inducing immune cells, or directly CIK freezing immune cells, to a certain extent meet the clinical needs. However, these methods ...
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Login to View More IPC IPC(8): C12N5/078C12N5/0784C12N5/0783A01N1/02
Inventor 陈晓波洪敬欣韩洪起武文杰靳霞王雪莲张宇光韩俊领
Owner UNION STEMCELL & GENE ENG