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Application of live cell sensor in cryopreservation of plant cells

A technology for plant cells and cryopreservation, which is applied in the field of cell detection and can solve problems such as cryopreservation of cells that have not yet been applied

Active Publication Date: 2018-05-29
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the living cell sensor is often used in the fermentation process to measure the amount of cells in the fermentation broth, but has not been applied to cell cryopreservation

Method used

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  • Application of live cell sensor in cryopreservation of plant cells
  • Application of live cell sensor in cryopreservation of plant cells
  • Application of live cell sensor in cryopreservation of plant cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1, the linear relationship between the capacitance value of living cells and the cell concentration

[0082] The cells in the logarithmic growth phase were used as the initial cells, and the cells were diluted 2, 4, 6, and 8 times respectively, and the capacitance value was measured by a living cell sensor.

[0083] The result is as figure 1 ,From figure 1 It can be seen that the capacitance value of living cells has a good linear relationship with the cell concentration. The cell capacitance value in the logarithmic growth phase is about 100pF / cm, and only the medium solution has a basic capacitance value of about 7pF / cm. After the cells are completely killed, the capacitance value is not much different from the basic capacitance value of the medium. This was also confirmed in subsequent experiments.

[0084] From figure 1 From the results, it can be concluded that the capacitance value of living cells can well characterize the concentration of living ce...

Embodiment 2

[0085] Embodiment 2, the impact of different types of cryoprotectants on cell cryopreservation

[0086] Glycerin, DMSO, sucrose, sorbitol, etc. have been used as cryoprotectants. Among them, glycerin and DMSO are osmotic cryoprotectants, and sucrose and sorbitol are non-permeable cryoprotectants. This example discusses how to choose a cryoprotectant and what concentration to choose to obtain the most excellent protective effect.

[0087] Different concentrations of DMSO (5%, 10%) were mixed with sucrose (5%, 10%) and sorbitol (5%, 10%) as cryoprotectants for research, and the cells were treated with liquid containing 0.35M sucrose After the culture medium was pretreated for 18 hours, the pretreated cells were centrifuged, and after the supernatant was removed, a cryoprotectant was added and incubated at 0-4° C. and 110 rpm for 45 minutes. The incubated cells were first cooled at -20°C for 40 minutes, and then placed in a -80°C ultra-low temperature refrigerator for 24 hours,...

Embodiment 3

[0100] Embodiment 3, the impact of different types of pretreatment agents on cell cryopreservation

[0101] The type of pretreatment agent and the concentration of pretreatment agent are different, which also affect the cryopreservation of cells. When the concentration of pretreatment agent is too high, although the dehydration effect is better, the damage of high concentration pretreatment agent to cells is also relatively large. On the basis of the previous experiments, sucrose 0.2M, 0.35M, 0.5M and trehalose 0.2M, 0.35M, 0.5M were selected for investigation, and the control was the cells that were directly frozen without pretreatment. Freezing time: after 40 minutes at -20°C, store at -80°C for 24 hours; freezing medium: cell growth medium (liquid medium) + 10% DMSO + 10% sucrose; then resuscitate Determination.

[0102] The experimental results are shown in Table 3.

[0103] Table 3. The effect of the type and concentration of pretreatment agent on the activity of cells ...

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Abstract

The present invention relates to application of a live cell sensor in cryopreservation of plant cells. For the first time, the live cell sensor is used as a method to quickly evaluate the cryopreservation effect on the cells, and the cryopreservation effect of a cryoprotectant on the cells can be evaluated by changes in capacitance values of the living cells before and after cell freezing and resuscitation. By use of a living cell electrode, the concentration of the living cells can be quickly and accurately characterized. By combining of the capacitance values of the living cells with detection results of 2,3,4-triphenyltetrazolium chloride (TTC), the cryopreservation effect of a cryopreservation method on the cells can be evaluated rapidly and quantitatively, and inefficiency and no quantification of the preservation methods of the plant cells in the prior art can be overcome. Compared with the traditional methods, the comprehensive score of the cryopreservation of the cells can be improved by 5 times or more.

Description

technical field [0001] The invention belongs to the field of cell detection, and more specifically, the invention relates to the application of a living cell sensor in low-temperature preservation of plant cells. Background technique [0002] The conservation of existing plant cell lines is mainly through the following two methods. The first is subcultured storage in growth media, and the other is cryopreservation of cells. Preserving cells through long-term subculture has the disadvantages of time-consuming, labor-intensive, expensive, and loss of plant cell characteristics. When the cells are under ultra-low temperature conditions, they will stop a series of internal metabolic activities, and can maintain the characteristics of the cells themselves, greatly reducing the risk of cell characteristics disappearing. In the cryopreservation of plant cells, the cryopreservation method plays a decisive role in the successful preservation of cells. Due to the large amount of wa...

Claims

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Application Information

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IPC IPC(8): A01N1/02G01N27/22
CPCA01N1/0221G01N27/22
Inventor 郭美锦李佳瑞王泽建陈雨霞庄英萍宋云飞黄帅
Owner EAST CHINA UNIV OF SCI & TECH
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