CN110904024A

CN110904024A - Method for removing dead cells in suspension cell culture

Info

Publication number: CN110904024A
Application number: CN201911107953.7A
Authority: CN
Inventors: 方松刚; 陈杰
Original assignee: Wuhan Jiyuan High-Tech Co Ltd
Current assignee: Wuhan Jiyuan High-Tech Co Ltd
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2020-03-24

Abstract

The invention mainly relates to a method for removing dead cells in suspension cell culture, and the cells with higher activity are obtained. This patent adopts the gradient centrifugation method to separate dead cell and dead cell piece. The principle of isolating dead cells is: after cell death, cell membrane permeability increases, intracellular water is lost and atrophy, and finally the cell membrane is ruptured. During this process of apoptosis, cells become more dense, and dead cells and fragments of dead cells become denser than normal living cells. The cell mixture is added to a lymphocyte separation medium having a density between that of dead cells and that of live cells, and then centrifuged to separate the dead cells from the live cells. Compared with the traditional centrifugal method, the method for removing dead cells in suspension cells is more thorough in separation, and meanwhile, the damage to living cells is reduced.

Description

Method for removing dead cells in suspension cell culture

Technical Field

The invention mainly relates to living cell purification of suspension cell culture, and belongs to the field of cell culture.

Background

During the discontinuous culture of suspension cells, the whole cell culture cycle can be divided into four stages according to the growth and division conditions of the whole cells: incubation period, logarithmic growth phase, stationary phase, decline period. When the cells enter the logarithmic growth phase, the cells are most actively divided, the cell activity is also the highest, the cell metabolites are increased along with the increase of the division times, the cell density is increased, and a small part of cells show the signs of apoptosis at the later stage of the logarithmic growth phase. After the stable period, the cell density reaches the maximum, and if the fresh culture medium is replaced by supplementing nutrition in time, the number of apoptotic cells is increased, namely, the number of dead cells is increased. Too many dead cells can affect the overall cellular activity and inhibit the growth metabolism of normal cells. Therefore, how to remove dead cells in suspension cell culture is a necessary experimental operation in the suspension cell culture process.

Currently, no commercial product specifically directed to the removal of dead cells has emerged. Most of the traditional methods for removing dead cells adopt a low-speed centrifugation method, which is not thorough in removing the dead cells on one hand, and also can separate and lose some living cells on the other hand. The patent refers to the field of 'separation of cells from blood vessels'. The principle of isolating dead cells is: after cell death, cell membrane permeability increases, intracellular water is lost and atrophy, and finally the cell membrane is ruptured. During this process of apoptosis, cells become more dense, and dead cells and fragments of dead cells become denser than normal living cells. The cell mixture is added to a lymphocyte separation medium having a density between that of dead cells and that of live cells, and then centrifuged to separate the dead cells from the live cells. Compared with the traditional centrifugal method, the method for removing dead cells in suspension cells is more thorough in separation, and meanwhile, the damage to living cells is reduced.

Disclosure of Invention

The invention relates to a method for removing dead cells in suspension cell culture, which aims to separate and remove dead cells and dead cell debris in suspension culture cells to obtain cells with higher activity.

In order to achieve the above object, the present invention proposes the following solutions:

preparing reagents and consumables for use includes: 250ml centrifuge bottles, 50ml centrifuge tubes and ficoll human lymphocyte separation medium.

All cell culture fluid required to separate dead cells was collected in a centrifuge flask, centrifuged at 2300rpm (or 800 g) for 10min, and the supernatant was decanted.

Adding appropriate amount of DPBS solution into the centrifugal flask, and mixing to make the cell concentration at 1-5 x 10⁷Between/ml.

Several 50ml centrifuge tubes were prepared, and 15ml of ficoll human lymphocyte splitting medium was added to each tube.

The cell suspension with the adjusted cell concentration is slowly added into a centrifuge tube filled with the lymph separation liquid, and 30ml of the cell suspension is added into each tube.

After the tube was trimmed, the tube was gently placed in a centrifuge at 2300rpm (or 800 g) for 20 min.

After centrifugation, the upper 30ml of solution in each tube was slowly pipetted into the centrifuge flask, leaving the remaining portion (containing dead cells) to waste.

The cell suspension in the centrifugal bottle is blown and evenly mixed, centrifuged at 2300rpm (or 800 g) for 10min, and the supernatant is poured off.

And (4) the cell sediment in the centrifugal bottle is the separated living cells.

And (3) resuspending and uniformly mixing the cells in the centrifugal bottle by using a culture bottle, and taking 2ml of suspension to detect the proportion of living cells by a trypan blue staining method.

Through trypan blue staining detection, the proportion of the living cells in the finally obtained cell suspension is 99.5%.

Claims

1. A method for removing dead cells in suspension cell culture is characterized in that a density gradient centrifugation method is adopted to remove dead cells and dead cell debris, and a used separation reagent is ficoll human lymph separation liquid.

2. A method for removing dead cells in suspension cell culture is characterized in that the operation process comprises the following steps: collecting all cell culture fluid needing to separate dead cells into a centrifuge bottle, centrifuging at 2300rpm (or 800 g) for 10min, and pouring off supernatant; adding appropriate amount of DPBS solution into the centrifugal flask, and mixing to make the cell concentration at 1-5 x 10⁷Between/ml; preparing a plurality of 50ml centrifuge tubes, and adding 15ml of ficoll human lymphocyte splitting fluid into each tube; slowly adding the cell suspension with the adjusted cell concentration into a centrifuge tube filled with a lymph separation liquid, and adding 30ml of cell suspension into each tube; after the centrifugal tube is balanced, the centrifugal tube is gently placed into a centrifugal machine to be centrifuged at 2300rpm (or 800 g) for 20 min; after centrifugation, the upper 30ml of solution in each tube was slowly aspirated into a centrifuge bottle, leaving the remaining portion (Containing dead cells) are discarded; blowing and uniformly mixing the cell suspension in the centrifugal bottle, centrifuging at 2300rpm (or 800 g) for 10min, and pouring off the supernatant; and (4) the cell sediment in the centrifugal bottle is the separated living cells.