CN113999810A - MRC-5 cell recovery culture solution and recovery method - Google Patents
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Abstract
The invention relates to the technical field of cell culture, in particular to an MRC-5 cell recovery culture solution which comprises the following components in parts by volume: 6.5-7.7 parts by volume of a basic culture medium; sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 to 0.3 volume portion; essential amino acid with the mass volume ratio of 3 percent, 0.1 to 0.2 volume portion; 2-3 parts by volume of fetal bovine serum; the MRC-5 cell recovery culture solution can obviously reduce the toxic influence of DMSO (dimethyl sulfoxide) in the preservation solution on MRC-5 cells, MRC-5 cells frozen by the DMSO-containing freezing solution can be directly inoculated to the MRC-5 cell recovery culture solution for culture after being melted, the centrifugal treatment step is omitted, the cell pollution is avoided, the time cost is saved, the cell adherence rate is higher than that of the traditional cell recovery method, the cell quality is high, and the subsequent cell amplification is facilitated.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a recovery culture solution and a recovery method for MRC-5 cells.
Background
Cell freezing has been used extensively in biology as an effective method for preserving cells. Researchers often need to freeze cells and store them for later experimental or clinical use. The traditional low-temperature cryopreservation technology is to add dimethyl sulfoxide (DMSO) and serum into a cell suspension, then place the cell suspension into a cryopreservation tube, slowly freeze the cell suspension in a refrigerator at-80 ℃, place the cryopreservation tube in liquid nitrogen for preservation, enable cells to be temporarily separated from a growth state and preserve the cell characteristics, and rapidly thaw the cells when needed. Moreover, a certain amount of cells are preserved appropriately, so that the cells can be prevented from being lost due to contamination or other accidents of the cultured cells, and the function of preserving the cells is achieved. In addition, some cells can be purchased, hosted, exchanged, and transported in a frozen form.
When the cells are frozen, DMSO with the final concentration of 5% -15% of the protective agent is added, so that the freezing point of the solution can be lowered, water in the cells can permeate out under the condition of slow freezing, and the formation of ice crystals is reduced, thereby avoiding the damage of the cells. The method of 'slow freezing and fast melting' can better ensure the survival of the cells. However, DMSO in the frozen stock solution and residual DMSO in the cell culture medium after recovery often have certain toxic effect on cells in the recovery process, inhibit cell proliferation, and influence the activity of the recovered cells and the development of subsequent tests.
In order to reduce the toxicity of DMSO to cells, the cells are recovered after the traditional cell freezing, the cells are thawed and centrifuged to precipitate the cells, supernatant freezing medium is removed, and then the cells are inoculated with a culture medium for culture. In the research of the applicant, the cell freezing tube is easy to break during the centrifugation process, thereby causing cell pollution and influencing cell expansion. Because the cells grow adherent to the culture medium with antibiotics after recovery, if the bacteria content is not high, the cells can not discover bacterial contamination continuously for several generations, and the existence of the bacterial contamination can be discovered only in the culture solution without antibiotics. This process takes 20-30 days, affecting the overall production or development schedule. In addition, the centrifugation step is also a critical point for cell contamination during the operation. However, the removal of the cryopreservation agent based on centrifugation is simple and easy to operate, the content of DMSO can be significantly reduced, and bacterial contamination is not easily directly attributed to the centrifugation step after the centrifugation step until many treatment steps are found, and related research and reports for cell recovery instead of the centrifugation step are not available at present.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a human embryonic lung fibroblast (MRC-5 cell) recovery culture solution and a recovery method, MRC-5 cells frozen by a frozen stock solution containing DMSO can be directly inoculated into the MRC-5 cell recovery culture solution for culture after being thawed, a centrifugal treatment step is omitted, cell pollution is avoided, time cost is saved, the cell adherence rate is higher than that of the traditional cell recovery method, and the cell quality is high.
Therefore, the invention provides the following technical scheme:
the invention provides an MRC-5 cell recovery culture solution which comprises the following components in parts by volume:
6.5-7.7 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 to 0.3 volume portion;
essential amino acid with the mass volume ratio of 3 percent, 0.1 to 0.2 volume portion;
fetal bovine serum, 2-3 parts by volume.
Optionally, the MRC-5 cell recovery culture solution comprises the following components in parts by volume:
7.7 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 volume part;
3 percent of essential amino acid by mass volume ratio, 0.1 volume portion;
fetal bovine serum, 2 parts by volume.
Optionally, the basal medium comprises a daily water medium, a DMEM medium or a MEM medium; or
The essential amino acid includes L-glutamine.
The invention provides application of the MRC-5 cell recovery culture solution in cell recovery.
Optionally, use in the resuscitation of MRC-5 cells.
The invention provides a method for recovering MRC-5 cells, which comprises the step of culturing the thawed MRC-5 cells by using the MRC-5 cell recovery culture solution.
Optionally, the culture condition is culture at 37 + -1 deg.C for 16-18 hours.
Optionally, the method comprises the following steps:
thawing the frozen MRC-5 cells for later use;
and (4) inoculating the thawed MRC-5 cells into a MRC-5 cell recovery culture solution for culture.
Optionally, in the MRC-5 cell thawing step, the MRC-5 cell cryopreservation tube taken out of the liquid nitrogen is placed in preheated sterilized water for thawing.
Optionally, the method further comprises the step of performing liquid change culture on the recovered cells, wherein the liquid change culture medium comprises the following components in parts by volume:
8.5-9.2 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 to 0.3 volume portion;
essential amino acid with the mass volume ratio of 3 percent, 0.1 to 0.2 volume portion;
fetal bovine serum, 0.5-1 volume parts.
Optionally, the liquid change medium comprises the following components in parts by volume:
8.7 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 volume part;
3 percent of essential amino acid by mass volume ratio, 0.1 volume portion;
fetal bovine serum, 1 part by volume.
Optionally, the basal medium comprises a daily water medium, a DMEM medium or a MEM medium; or
The essential amino acid includes L-glutamine.
The technical scheme of the invention has the following advantages:
1. the invention provides an MRC-5 cell recovery culture solution which comprises the following components in parts by volume: 6.5-7.7 parts by volume of a basic culture medium; sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 to 0.3 volume portion; essential amino acid with the mass volume ratio of 3 percent, 0.1 to 0.2 volume portion; 2-3 parts by volume of fetal bovine serum; the MRC-5 cell recovery culture solution can obviously reduce the toxic influence of DMSO (dimethyl sulfoxide) in the preservation solution on MRC-5 cells, MRC-5 cells frozen by the DMSO-containing freezing solution can be directly inoculated to the MRC-5 cell recovery culture solution for culture after being melted, the centrifugal treatment step is omitted, the cell pollution is avoided, the time cost is saved, the cell adherence rate is higher than that of the traditional cell recovery method, the cell quality is high, and the subsequent cell amplification is facilitated.
2. The invention provides a recovery method of MRC-5 cells, which comprises the steps of thawing the cryopreserved MRC-5 cells for later use; inoculating the thawed MRC-5 cells into an MRC-5 cell recovery culture solution for culture; the method omits a centrifugal treatment step, avoids cell pollution and saves time and cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the cell adhesion before the liquid change in the MRC-5 cell recovery method of example 7 in Experimental example 1;
FIG. 2 shows the cell adhesion after the liquid change in the MRC-5 cell recovery method of example 7 in Experimental example 1;
FIG. 3 shows the cell adhesion before the liquid change in the MRC-5 cell recovery method of comparative example 1 according to the present invention;
FIG. 4 shows the cell adhesion after the liquid change in the MRC-5 cell recovery method of comparative example 1 in Experimental example 1 according to the present invention;
FIG. 5 is a graph showing the time-to-cell growth number in Experimental example 2 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The daily water culture medium in the following examples is a commercial product, and the components of the daily water culture medium are (9.4 g/L): 6800.0mg of sodium oxide, 400.0mg of potassium oxide, 200.0mg of calcium oxide, 93.5mg of magnesium sulfate, 115.0mg of sodium dihydrogen phosphate, 1000.0mg of glucose, 126.0mg of L-arginine hydrochloride, 31.4mg of L-cystine dihydrochloride monohydrate, 36.0mg of L-tyrosine, 42.0mg of L-histidine hydrochloride monohydrate, 52.0mg of L-isoleucine, 52.0mg of L-leucine, 73.0mg of L-lysine hydrochloride, 15.0mg of L-methionine, 32.0mg of L-phenylalanine, 48.0mg of L-threonine, 10.0mg of L-tryptophan, 46.0mg of L-valine, 75.0mg of succinic acid (succinic acid), sodium succinate (sodium succinate), hexahydrate, 100.0mg of L-methionine tartrate (choline bitartrate), 1.8mg of folic acid, 1.0mg of folic acid, 2.0mg of nicotinamide, 3 mg of vitamin B, 1.0mg of nicotinamide, 1.0mg of calcium pantothenate, pyridoxal hydrochloride 1.0mg, riboflavin (vitamin B2) 0.1mg, thiamine hydrochloride (vitamin B1) 1.0mg, biotin (vitamin B7, coenzyme R) 0.02 mg.
The DMSO concentration in the storage solution used in the MRC-5 cell cryopreservation tubes in the examples below was 10% (v/v).
Fetal bovine serum in the following examples was purchased from Gibico.
Example 1
The embodiment provides an MRC-5 cell recovery culture solution, which comprises the following components in percentage by weight:
daily water culture medium, 7.7 ml;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 ml;
0.1ml of L-glutamine with the mass volume ratio of 3 percent;
fetal bovine serum, 2 ml.
Taking a daily water culture medium, 7.5% by mass and volume of sodium bicarbonate, 3% by mass and volume of L-glutamine and fetal calf serum according to the formula, mixing, and preparing the MRC-5 cell recovery culture solution.
Example 2
The embodiment provides an MRC-5 cell recovery culture solution, which comprises the following components in percentage by weight:
DMEM medium, 6.5 ml;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.3 ml;
0.2ml of L-glutamine with the mass volume ratio of 3 percent;
fetal bovine serum, 3 ml.
And (2) taking a DMEM culture medium, 7.5% sodium bicarbonate by mass-volume ratio, 3% L-glutamine by mass-volume ratio and fetal calf serum according to the formula, mixing, and preparing the MRC-5 cell recovery culture solution.
Example 3
The embodiment provides an MRC-5 cell recovery culture solution, which comprises the following components in percentage by weight:
MEM medium, 7 ml;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.3 ml;
0.2ml of L-glutamine with the mass volume ratio of 3 percent;
fetal bovine serum, 2.5 ml.
The preparation method comprises the steps of weighing MEM culture medium, 7.5% sodium bicarbonate by mass-volume ratio, 3% L-glutamine by mass-volume ratio and fetal calf serum according to the formula, mixing, and preparing the MRC-5 cell recovery culture solution.
Example 4
The embodiment provides an MRC-5 cell liquid change culture medium, which comprises the following components in percentage by weight:
8.7ml of daily water culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 ml;
0.1ml of L-glutamine with the mass volume ratio of 3 percent;
fetal bovine serum, 1 ml.
The daily water culture medium, the sodium bicarbonate with the mass volume ratio of 7.5%, the L-glutamine with the mass volume ratio of 3% and the fetal calf serum are measured according to the formula and mixed to prepare the MRC-5 cell liquid changing culture medium.
Example 5
The embodiment provides an MRC-5 cell liquid change culture medium, which comprises the following components in percentage by weight:
DMEM medium, 8.5 ml;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.3 ml;
0.2ml of L-glutamine with the mass volume ratio of 3 percent;
fetal bovine serum, 1 ml.
And (3) measuring a DMEM culture medium, 7.5% sodium bicarbonate by mass-volume ratio, 3% L-glutamine by mass-volume ratio and fetal calf serum according to the formula, mixing, and preparing the MRC-5 cell replacement medium.
Example 6
The embodiment provides an MRC-5 cell liquid change culture medium, which comprises the following components in percentage by weight:
MEM medium, 9.2 ml;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 ml;
0.1ml of L-glutamine with the mass volume ratio of 3 percent;
fetal bovine serum, 0.5 ml.
Measuring MEM culture medium, 7.5% sodium bicarbonate, 3% L-glutamine and fetal calf serum according to the formula, mixing, and preparing the MRC-5 cell replacement medium.
Example 7
The embodiment provides a recovery method of MRC-5 cells, which comprises the following steps:
(1) cell thawing
Before cell recovery, placing sterilized injection water into a 37 ℃ incubator for preheating for 2 hours for later use; and taking out the cryopreservation tube of the MRC-5 cells from the liquid nitrogen tank, quickly putting the tube into sterilized injection water, and shaking to quickly thaw the tube, wherein the process is generally completed within 2-3 minutes.
(2) Cell resuscitation
Taking out MRC-5 cell cryopreservation tube from injection water, spraying 75% alcohol on the surface of the cryopreservation tube for sterilization, placing under laminar flow hood, opening the cryopreservation tube, and sucking cell suspension (2.0 × 10) with 5ml pipette6(1 ml) was added to the MRC-5 cell recovery culture medium of example 1, gently shaken and mixed, and then incubated in an incubator at 37 ℃ for 17 hours.
(3) Cell exchange liquid
Culturing for 17 hours in a 37 ℃ incubator, taking out, observing the anchorage condition of cells under a microscope, wherein the anchorage rate is more than or equal to 90%, then placing a culture bottle under a laminar flow hood, sucking the cell culture supernatant, adding the liquid change culture medium (preheated at 37 ℃) of the example 4, and continuing culturing at 37 ℃.
Example 8
The embodiment provides a recovery method of MRC-5 cells, which comprises the following steps:
(1) cell thawing
Before cell recovery, placing sterilized injection water into a 37 ℃ incubator for preheating for 2 hours for later use; and taking out the cryopreservation tube of the MRC-5 cells from the liquid nitrogen tank, quickly putting the tube into sterilized injection water, and shaking to quickly thaw the tube, wherein the process is generally completed within 2-3 minutes.
(2) Cell resuscitation
Taking out MRC-5 cell cryopreservation tube from injection water, spraying 75% alcohol on the surface of the cryopreservation tube for sterilization, placing under laminar flow hood, opening the cryopreservation tube, and sucking cell suspension (2.0 × 10) with 5ml pipette6(1 ml) was added to the MRC-5 cell recovery culture medium of example 2, gently shaken and mixed, and then incubated in an incubator at 37. + -. 1 ℃ for 16 hours.
(3) Cell exchange liquid
Culturing for 16 hours in an incubator at 37 ℃, taking out, observing the anchorage condition of cells under a microscope, wherein the anchorage rate is more than or equal to 90%, then placing a culture bottle into laminar flow, absorbing cell culture supernatant, adding the liquid change culture medium (preheated at 37 ℃) of example 5, and continuing culturing at 37 ℃.
Example 9
The embodiment provides a recovery method of MRC-5 cells, which comprises the following steps:
(1) cell thawing
Before cell recovery, placing sterilized injection water into a 37 ℃ incubator for preheating for 2 hours for later use; and taking out the cryopreservation tube of the MRC-5 cells from the liquid nitrogen tank, quickly putting the tube into sterilized injection water, and shaking to quickly thaw the tube, wherein the process is generally completed within 2-3 minutes.
(2) Cell resuscitation
Taking out MRC-5 cell cryopreservation tube from injection water, spraying 75% alcohol on the surface of the cryopreservation tube for sterilization, placing under laminar flow hood, opening the cryopreservation tube, and sucking cell suspension (2.0 × 10) with 5ml pipette6(1 ml) was added to the MRC-5 cell recovery culture medium of example 3, gently shaken and mixed, and then incubated in an incubator at 37. + -. 1 ℃ for 18 hours.
(3) Cell exchange liquid
Culturing for 18 hours in a 37 ℃ incubator, taking out, observing the anchorage condition of cells under a microscope, wherein the anchorage rate is more than or equal to 90%, then placing a culture bottle under a laminar flow hood, sucking the cell culture supernatant, adding the liquid change culture medium (preheated at 37 ℃) of the example 6, and continuing culturing at 37 ℃.
Comparative example 1
The embodiment provides a traditional MRC-5 cell recovery method, which comprises the following steps:
(1) cell thawing
Before cell recovery, placing sterilized injection water into a 37 ℃ incubator for preheating for 2 hours for later use; and taking out the cryopreservation tube of the MRC-5 cells from the liquid nitrogen tank, quickly putting the tube into sterilized injection water, and shaking to quickly thaw the tube, wherein the process is generally completed within 2-3 minutes.
(2) Centrifuging to precipitate cells
The cryopreserved tube of MRC-5 cells was removed from the injection water and centrifuged to pellet the cells (2000 rpm, 5 min).
(3) Cell resuscitation
Spraying 75% alcohol on the surface of the freezing tube for disinfection, placing the freezing tube under a laminar flow hood, opening the freezing tube, sucking and removing supernatant, inoculating the precipitated cells into a common MRC-5 cell recovery culture medium (adopting the liquid change culture medium in example 4), and placing the cell in an incubator at 37 ℃ for culture for 17 hours.
(4) Cell exchange liquid
Culturing at 37 deg.C for 17 hr, taking out, observing cell adherence condition under microscope with adherence rate not less than 90%, placing culture flask into laminar flow, removing cell culture supernatant, adding culture solution for changing solution (adopting the medium for changing solution in example 4) (preheating at 37 deg.C), and culturing at 37 deg.C.
Experimental example 1 cell anchorage rate experiment
This example revives 6 MRC-5 cell cryopreservation tubes (the cell concentration of each cell cryopreservation tube before cell cryopreservation is 2.0X 10)6One/ml) of the cells, recovering 3 freezing tubes by the method of comparative example 1, namely inoculating the cells into MRC-5 liquid changing culture medium for culture after centrifuging to remove protective agent, and collecting the other 3 freezing tubesThe cells are recovered by the method of example 7, that is, the thawed cells are directly inoculated into the MRC-5 cell recovery culture solution of the invention for culture.
(1) After culturing the cells (3 cells each) obtained by the two recovery methods in the cell recovery step for 16-18 hours, washing with PBS to remove supernatant dead cells and nonadherent cells respectively, and counting the adherent cells.
Calculating the anchorage rate of the cells: cell adherence rate = number of cells after adherence/number of cells at resuscitation × 100%
(2) And observing the cell adherence condition of the cells in the two recovery methods under a microscope and taking pictures for recording before and after liquid change respectively.
The experimental results are as follows:
(1) the results of cell anchorage rate are shown in table 1 below.
TABLE 1 results of cell adherence rate
As can be seen from the above table 1, the cell adherence rate of the MRC-5 cell recovery method of the invention is higher than that of the conventional cell recovery method, and in the MRC-5 cell recovery method of the invention, cells can adhere to the wall rapidly, which indicates that the cell state is good, and the cells with more superiority are screened to complete cell amplification, thereby enhancing the cell quality.
(2) The cell adherence conditions before and after liquid change are shown in figures 1-4, and the observation in the figures can be visualized, the cell adherence rate of the MRC-5 cell recovery method is higher than that of the traditional cell recovery method, and the cell quality is strong.
Experimental example 2 cell growth Rate test
The cells in the two recovery modes (cells after liquid change culture) were passaged 1 time (trypsinized and split into bottles of T25 cells) along with the cells in Experimental example 1, and the number of cells on day 0 was counted after digestion, and thereafter 1 bottle of cells was digested every 2 days and counted for 12 consecutive days. The time was plotted against the number of cell proliferations, and the results are shown in Table 2 below and FIG. 5.
TABLE 2 cell count
As can be seen from Table 2 above and FIG. 5, the improved cell recovery method of the present invention has a better cell proliferation rate than the conventional cell recovery method.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. The MRC-5 cell recovery culture solution is characterized by comprising the following components in parts by volume:
6.5-7.7 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 to 0.3 volume portion;
essential amino acid with the mass volume ratio of 3 percent, 0.1 to 0.2 volume portion;
fetal bovine serum, 2-3 parts by volume.
2. The MRC-5 cell recovery culture fluid of claim 1, comprising the following components in parts by volume:
7.7 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 volume part;
3 percent of essential amino acid by mass volume ratio, 0.1 volume portion;
fetal bovine serum, 2 parts by volume.
3. The MRC-5 cell recovery culture medium according to claim 1 or 2,
the basic culture medium comprises a daily water culture medium, a DMEM culture medium or an MEM culture medium; or
The essential amino acid includes L-glutamine.
4. Use of the MRC-5 cell recovery medium of any one of claims 1 to 3 for cell recovery.
5. Use according to claim 4, in the resuscitation of MRC-5 cells.
6. A method for recovering MRC-5 cells, comprising culturing the thawed MRC-5 cells in the MRC-5 cell recovery culture medium according to any one of claims 1 to 3.
7. The method for resuscitating MRC-5 cells of claim 6, wherein the culture condition is 37 ± 1 ℃ for 16-18 hours.
8. The method for resuscitation of MRC-5 cells according to claim 6 or 7, comprising the steps of:
thawing the frozen MRC-5 cells for later use;
and (4) inoculating the thawed MRC-5 cells into a MRC-5 cell recovery culture solution for culture.
9. The method for resuscitating MRC-5 cells as claimed in claim 8, wherein the MRC-5 cell thawing step comprises thawing a cryopreserved tube of MRC-5 cells, which is removed from liquid nitrogen, in preheated sterilized water.
10. The method for recovering MRC-5 cells according to claim 8, further comprising the step of subjecting the recovered cells to a liquid change culture, wherein the liquid change culture comprises the following components in parts by volume:
8.5-9.2 parts by volume of a basic culture medium;
sodium bicarbonate with the mass volume ratio of 7.5 percent and 0.2 to 0.3 volume portion;
essential amino acid with the mass volume ratio of 3 percent, 0.1 to 0.2 volume portion;
fetal bovine serum, 0.5-1 volume parts.
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