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A kind of cell cryopreservation medium and application thereof for in vitro cell culture

A culture medium and cell technology, applied in the field of biomedicine, can solve problems such as only reaching 50%

Active Publication Date: 2020-03-31
北京健坤禾润科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for immune cells that are difficult to survive in vitro, especially lymphocytes, they are more sensitive to damage in cell cryopreservation, and the survival rate of cells after cryopreservation often only reaches 50%.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, using the cell cryopreservation medium containing fibrinogen to freeze mammalian lymphocytes

[0028] 1. Preparation of mouse spleen lymphocytes

[0029] Sacrifice BALB / c mice by cervical dislocation, soak in 75% ethanol solution for 5 minutes, then aseptically take the spleen of the mice on the ultra-clean workbench, and grind it with the inner core of a 5ml syringe while adding phosphate buffered saline (PBS) dropwise Prepare the spleen cell suspension, and use a 40 μm pore size sterile nylon filter to filter the cells to prepare a single cell suspension; centrifuge at 250g for 5 minutes, discard the supernatant, then add 10ml red blood cell lysate to lyse the red blood cells, and then centrifuge and culture at RPMI 1640 Base washing to obtain mouse spleen lymphocytes.

[0030] 2. Preparation of cell cryopreservation solution

[0031] This embodiment provides 7 kinds of cell freezing solutions, these 7 kinds of cell freezing solutions are respectively ...

Embodiment 2

[0048] Embodiment 2, the influence of the cell cryopreservation liquid containing fibrinogen on the survival rate of cryopreservation cells at different times

[0049] 1. Preparation of mouse spleen lymphocytes

[0050] Sacrifice BALB / c mice by cervical dislocation, soak in 75% ethanol solution for 5 minutes, then aseptically take the spleen of the mice on the ultra-clean workbench, and grind it with the inner core of a 5ml syringe while adding phosphate buffered saline (PBS) dropwise Prepare the spleen cell suspension, and use a 40 μm pore size sterile nylon filter to filter the cells to prepare a single cell suspension; centrifuge at 250g for 5 minutes, discard the supernatant, then add 10ml red blood cell lysate to lyse the red blood cells, and then centrifuge and culture at RPMI 1640 Base washing to obtain mouse spleen lymphocytes.

[0051] 2. Preparation of cell cryopreservation solution

[0052] This embodiment provides two kinds of cell cryopreservation solutions, whi...

Embodiment 3

[0064] Embodiment 3, the effect of the cell cryopreservation solution prepared by various protein components on the survival rate of frozen cells

[0065] 1. Preparation of mouse spleen lymphocytes

[0066] Sacrifice BALB / c mice by cervical dislocation, soak in 75% ethanol solution for 5 minutes, then aseptically take the spleen of the mice on the ultra-clean workbench, and grind it with the inner core of a 5ml syringe while adding phosphate buffered saline (PBS) dropwise Prepare the spleen cell suspension, and use a 40 μm pore size sterile nylon filter to filter the cells to prepare a single cell suspension; centrifuge at 250g for 5 minutes, discard the supernatant, then add 10ml red blood cell lysate to lyse the red blood cells, and then centrifuge and culture at RPMI 1640 Base washing to obtain mouse spleen lymphocytes.

[0067] 2. Preparation of cell cryopreservation solution

[0068] This embodiment provides 9 kinds of cell freezing solutions, which are respectively con...

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PUM

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Abstract

The invention discloses a cell cryopreservation culture medium for cell in-vitro culture and application thereof. The cell cryopreservation culture medium comprises fetal calf serum, dimethyl sulfoxide and fibrinogen. Experiments prove that fibrinogen is added into an existing cell cryopreservation solution, the cell cryopreservation injury can be remarkably reduced, and the survival rate of cryopreservation cells can be remarkably improved, for example, the cryopreservation survival rate of lymphocyte can be remarkably improved. The cell cryopreservation culture medium can be used for in-vitro culture of mammalian cells such as mammalian lymphocyte, and can be applied to medicine lead compound screening, drug effect evaluation and recombinant protein drug production.

Description

technical field [0001] The invention relates to a cell cryopreservation medium for cell in vitro culture and application thereof in the field of biomedicine. Background technique [0002] In drug development and production, in vitro cell culture is a necessary technology for drug lead compound screening, drug efficacy evaluation and production of recombinant protein drugs. In the in vitro cell culture technology system, cell cryopreservation is an essential method for long-term preservation of cells. [0003] At present, the commonly used cell cryopreservation solution is bovine serum containing 5%-15% glycerol or dimethyl sulfoxide. The cryopreservation and resuscitation operation of freezing and quick thawing can reduce the formation of ice crystals and cell damage in cells, and achieve better cell cryopreservation effect (American Journal of Physiology, 1984, 247, C125-C142). Generally, the survival rate of frozen cells can reach more than 70-80%. However, immune cells...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02C12N5/0783C12N5/0781
Inventor 曹秀娟
Owner 北京健坤禾润科技有限公司