CN113293136B

CN113293136B - Method for resuscitating cells and application

Info

Publication number: CN113293136B
Application number: CN202110566729.5A
Authority: CN
Inventors: 万谦; 彭文珍; 王锐峰
Original assignee: Chengdu New Life Hope Medical Laboratory Co ltd
Current assignee: Chengdu New Life Hope Medical Laboratory Co ltd
Priority date: 2021-05-24
Filing date: 2021-05-24
Publication date: 2023-06-13
Anticipated expiration: 2041-05-24
Also published as: CN113293136A

Abstract

The invention discloses a method for resuscitating cells and application thereof, wherein the method comprises the following steps: s1, thawing and thawing a cryopreservation tube storing cells to be resuscitated to obtain cell liquid; s2, adding PBS into the cell liquid obtained in the step S1, and uniformly mixing to obtain a cell mixed solution, wherein the volume ratio of the PBS to the cell liquid is 4:1; s3, adding the uniformly mixed cell mixed solution into one hole of a six-hole plate, placing the mixed solution into a cell incubator for standing for a few minutes, and sucking and removing supernatant; s4, adding culture solution into the hole for sucking the supernatant to resuspend the cell sediment, and placing the cell sediment into a cell culture box for culture, wherein the volume of the cell liquid in the cell mixed solution of the culture solution and the added hole is 5:1. The invention can realize cell recovery without using a centrifuge or washing cells, has shorter recovery time, and can be suitable for occasions without the centrifuge, with less cell number and urgent time.

Description

Method for resuscitating cells and application

Technical Field

The invention relates to the field of biotechnology, in particular to a method for resuscitating cells and application thereof.

Background

Cryopreservation and resuscitating cells are important basic procedures in cell biology experiments. Generally, the cell cryopreservation solution is a cell culture solution containing 10% (v/v) dimethyl sulfoxide (DMSO), so that the conventional resuscitating method needs to wash cells to remove dimethyl sulfoxide (DMSO) before resuscitating the cells, and the conventional resuscitating method mainly adopts a centrifugal machine to centrifuge to remove supernatant, so that the conventional resuscitating method needs to use the centrifugal machine to wash the cells. However, traditional resuscitation methods are not well suited for some applications:

(1) Where a suitable centrifuge is temporarily not available, such as during a maintenance period when the centrifuge is malfunctioning;

(2) The number of cells is small, and more cells are lost when the conventional washing operation is performed;

(3) Time is urgent, where there is not enough time to wash the cells.

Disclosure of Invention

The invention aims to provide a method for recovering cells, which can realize cell recovery without using a centrifuge or washing the cells, has shorter recovery time and can be suitable for occasions without the centrifuge, with small cell quantity and urgent time.

The invention is realized by the following technical scheme:

a method of resuscitating a cell comprising the steps of:

s1, thawing and thawing a cryopreservation tube storing cells to be resuscitated to obtain cell liquid;

s2, adding PBS into the cell liquid obtained in the step S1, and uniformly mixing to obtain a cell mixed solution, wherein the volume ratio of the PBS to the cell liquid is 4:1;

s3, adding the uniformly mixed cell mixed solution into one hole of a six-hole plate, placing the mixed solution into a cell incubator for standing for a few minutes, and sucking and removing supernatant;

s4, adding culture solution into the hole for sucking the supernatant to resuspend the cell sediment, and placing the cell sediment into a cell culture box for culture, wherein the volume of the cell liquid in the cell mixed solution of the culture solution and the added hole is 5:1.

Cell cryopreservation solutions generally contain 10% dimethyl sulfoxide (DMSO), which is added to prevent ice crystals from forming in the contents of cells during cell cryopreservation, thereby damaging the cells, so DMSO is a cell cryopreservation protectant; however, DMSO has some cytotoxicity and must be removed in resuscitating cells, so conventional cell resuscitators use centrifugation to remove supernatant to remove DMSO.

The applicant has found through long-term research and experiments that:

(1) The cultured cells naturally sink when standing, particularly some cultured cells growing on an adherence, such as human cervical cancer cells Hela cells, and more than 80% of the suspended cells sink to the bottom of the culture container when standing for 5 minutes;

(2) After 5ml of culture solution is added to one of the six-well plate, the culture solution is sucked and discarded by a pipette, and the residual culture solution in the culture well is only about 0.08ml;

(3) When the DMSO concentration is less than 0.1%, the toxicity to the cells is negligible, and normal culture of the cells is not disturbed, and is generally considered to be free of cytotoxicity.

The inventive idea of the method according to the invention is based on the finding that:

the cell liquid is not subjected to centrifugal precipitation to remove supernatant liquid, cell washing is carried out to remove DMSO, the mixed liquid of the cell liquid and PBS is directly added into one hole of a six-hole plate, the supernatant liquid is sucked by standing, the residual amount of DMSO in the hole after the supernatant liquid is sucked is less than 0.1%, and then the mixed liquid is directly added into culture solution for formula resuscitation.

In the art, it is generally believed that DMSO can be removed only by centrifugation washing, and the present invention overcomes the technical bias by allowing the cell mixture solution to stand in culture wells for precipitation and then blotting the supernatant to replace centrifugation washing to remove DMS.

The invention adopts a centrifugal machine, does not carry out cell washing, and is particularly applicable to the following occasions:

(3) Time is urgent, where there is not enough time to wash the cells.

Further, the volume of the cell liquid was 1ml, and the volumes of PBS and culture liquid were 4ml and 5ml, respectively.

The applicant found through experiments that:

after 5ml of the cell mixture solution was added to one of the six-well plates, and after the cell mixture solution was removed by pipetting, the cell mixture solution remaining in this culture well was only about 0.08ml, whereby the DMSO residue in the resuscitation method of the present invention was calculated as: 0.08 ml/5 ml×10%/5 ml=0.032%, DMSO residual is much less than 0.1%, and has no toxicity to Hela cells.

That is, the supernatant can be removed by allowing the cell mixture solution to stand in the culture well and then sucking it to dry, instead of centrifugation washing.

In the art, the ordinary skilled person is not concerned about the study of DMSO concentration and toxicity, and less one has knowledge of this limit of 0.1%. How to reduce DMSO concentration below 0.1%, the skilled person generally knows only about using conventional centrifugation, and the skilled person does not specifically examine whether the suction-discard method can be used, and the present application finds that the residue of the original solution after the suction-discard treatment is "0.08 ml/5 ml=1.6%", further calculates the final concentration of the culture solution containing DMSO in the culture solution "(1.6% x 10%)/5 ml=0.032%", further compares the found "0.032% <0.1%", and finally concludes that the cells can be recovered by the suction-discard method, which is not found by the skilled person.

In examining how much PBS and culture solution was used for the pipetting and pipetting operations, the applicant has examined to precipitate cells with the addition of 1ml PBS and to re-suspend the cell pellet with 3 ml culture solution, but, by calculation, the final concentration of DMSO was 0.13%, exceeding the limit of 0.1%, which is not preferable, the end of the present application is "precipitate cells with 4ml PBS and re-suspend the cell pellet with 5ml culture solution", such that the final concentration of DMSO is 0.032%, less than the limit of 0.1%.

Further, the thawing condition in the step S1 is a water bath at 37 ℃.

Further, the supernatant was gently pipetted off in step S3.

Further, the standing time in step S3 is 5 to 6 minutes.

Further, the formula of the culture solution in step S4 is:

DMEM high sugar medium +10% fetal bovine serum +100U/ml penicillin +100 μg/ml streptomycin.

Further, the culture conditions in step S4 are: 37 ℃,5% carbon dioxide.

Further, the cells to be resuscitated in step S3 include Hela cells.

Use of a method for resuscitating cells, including Hela cells, in cell resuscitating.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention overcomes the technical bias, and the supernatant liquid can be sucked after standing and precipitating in the culture hole through the cell mixed solution instead of centrifugal washing to remove DMS, so that the invention can be suitable for occasions without a centrifugal machine, with small cell quantity and urgent time, and shortens the recovery time.

2. The invention has simple operation and easy realization, and is beneficial to reducing the cost of cell resuscitation.

Drawings

The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:

FIG. 1 is a graph of resuscitation effects using a conventional resuscitation method;

fig. 2 is a graph of resuscitation effects using the resuscitation method of the present invention.

Detailed Description

For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.

Example 1:

a method of resuscitating a cell comprising the steps of:

s1, taking out a cryopreservation tube of Hela cells from a liquid nitrogen tank, and rapidly thawing and thawing the Hela cells in a water bath at 37 ℃ to obtain 1ml of cell liquid;

s2, adding 4ml of PBS into the cell liquid obtained in the step S1, and uniformly mixing to obtain a cell mixed solution;

s3, adding the well-mixed cell solution into one of six-hole plates (Corning, USA), placing the mixture into a cell incubator (Thermo, model 3111, USA) for standing for 5 minutes, and then gently sucking out the supernatant by using a pipette;

s4, adding 5ml of culture solution to the supernatant-sucking hole to resuspend the cell pellet, placing the cell pellet in a cell incubator (Thermo, model 3111, USA) for culturing, and observing and photographing under an inverted microscope (Leica, DMi 3000B, germany) after 72 hours, and the result is shown in FIG. 2.

In this embodiment, the formula of the culture solution in step S4 is:

DMEM high sugar culture medium+10% fetal bovine serum+100U/ml penicillin+100 _μ g/ml streptomycin.

The culture conditions in step S4 are: 37 ℃,5% carbon dioxide.

Comparative example 1:

traditional resuscitation methods: the cryopreservation tube of Hela cells was removed from the liquid nitrogen tank, thawed rapidly in a 37℃water bath to give 1ml of cell liquid, added with 4ml of PBS, mixed well, centrifuged at 200 Xg for 5 minutes in a centrifuge (Eppendorf, 5810R, germany), the supernatant was discarded, 5ml of culture medium was added to resuspend the cell pellet, one of the wells of a six-well plate (Corning, USA), cultured in a cell incubator (Thermo, model 3111, USA), and after 72 hours the photograph was observed under an inverted microscope (Leica, DMi 3000B, germany), as shown in FIG. 1.

The culture solution and the culture conditions of this comparative example were the same as in example 1.

As can be seen from a comparison of fig. 1 and 2:

the resuscitation effect of the resuscitation method of example 1 is substantially identical to, even better than, the resuscitation effect of the conventional resuscitation method of comparative example 1, and example 1 also has the following advantages over comparative example 1:

a centrifugal machine is not used, so that the equipment cost is saved; can be suitable for resuscitation with less cell number; the whole resuscitation process is short in time and can be suitable for time-urgent occasions.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A method of resuscitating a cell comprising the steps of:

s3, adding the uniformly mixed cell mixed solution into one hole of a six-hole plate, placing the mixture into a cell incubator for standing for 5-6 minutes, and absorbing and removing supernatant;

s4, adding a culture solution into the hole for sucking the supernatant to resuspend the cell sediment, and placing the cell sediment into a cell culture box for culture, wherein the volume ratio of the culture solution to the cell liquid in the cell mixed solution in the hole is 5:1;

the cells to be resuscitated in the step S1 are Hela cells;

the formula of the culture solution in the step S4 is as follows:

2. The method of claim 1, wherein the cellular fluid has a volume of 1ml and the PBS and culture fluid have volumes of 4ml and 5ml, respectively.

3. The method according to claim 1, wherein the thawing conditions in step S1 are a 37 ℃ water bath.

4. The method according to claim 1, wherein the supernatant is gently pipetted off in step S3.

5. The method of claim 1, wherein the culturing conditions in step S4 are: 37 ℃,5% carbon dioxide.

6. Use of a method of resuscitating cells according to any one of claims 1 to 5, wherein the cells comprise Hela cells.