CN114854689A - Culture method of human-derived primary cells - Google Patents
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Abstract
The invention provides a human-derived primary cell culture method, and relates to the technical field of cell culture. In the process of culturing the human-derived primary cells, the invention carries out bacteria identification and drug sensitivity detection on the supernatant of the suspected polluted cell culture solution, and after the cells are cultured to be polluted, the invention selects the antibiotic type and concentration which have no influence on the cell growth according to the drug sensitivity test result to eliminate the bacteria, can reverse the bacterial pollution in early stage, greatly improves the success rate of culturing the primary cells, and has important effect on timely saving the pollution of the precious adherent cells in the cell culture.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a culture method of human-derived primary cells.
Background
Primary culture, also called primary culture, is the first culture performed in vitro after taking tissue cells from a donor. The primary cells are just separated from tissues, the biological characteristics are not changed greatly, the original genetic characteristics are still kept, the gene retention amount is more than 90%, and the in vivo growth characteristics are also most similar and reflected, so the method has more value in experimental researches such as antitumor drug treatment sensitivity tests, cell differentiation, gene expression and the like.
Bacterial contamination is inevitable in cell culture work, and the PCR technology is commonly used for detecting bacterial contamination in cultured cells in the past research, however, the DNA extraction and PCR detection technology of bacterial microorganisms requires higher cost and experimental conditions, and the common laboratory cannot meet the corresponding expenditure and equipment conditions easily. In this context, many laboratories can only choose not to detect, but to discard the cultured cells suspected of bacterial infection directly. However, bacterial contamination of unique, non-replaceable cell lines, particularly rare cell lines such as human colon tumor cells, has been attempted for decontamination and rescue. At present, the conventional pollution prevention methods include an antibiotic sterilization method, a macrophage phagocytosis method, an animal inoculation sterilization method and the like, wherein an antibiotic impact method is commonly used, but which antibiotics can effectively save polluted cells and have small influence on the biological characteristics of the cells? Some researchers use the K-B method to carry out drug sensitivity test to select antibiotics for rescue, but the time from bacterial contamination to irrevocable death of cells is short, the time window for saving the cells for the researchers is short, and the cells are always dead when the identification result is obtained. Therefore, there has also been a study of the early application of broad-spectrum antibiotic treatment in the discovery of bacterial contamination, with the best results of rescue achieved by broad coverage of bacteria with possible cellular infections. However, it is still controversial to use the combination or the single drug.
Therefore, how to discover and identify the bacteria in the polluted cell culture solution as soon as possible and reverse the bacterial pollution in the early stage has important significance for the smooth development of the research work of the medical basic laboratory.
Disclosure of Invention
The invention aims to provide a culture method of human-derived primary cells, which is characterized in that bacteria in a contaminated cell culture solution are identified by adopting MALDI-TOF technology in the primary cell culture process, a Vitek2compact instrument is adopted for drug sensitivity detection, and the type and concentration of antibiotics which do not influence the cell growth are selected according to the drug sensitivity test result to remove the bacteria, so that the aim of early reversing the bacterial contamination is fulfilled, and the success rate of the human-derived primary cell culture is greatly improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a culture method of human-derived primary cells, which comprises the following steps: separating human cells, performing primary cell culture in a complete culture solution without antibiotics, replacing the culture solution according to the growth condition of the cells, taking the suspected contaminated cell culture solution as a sample, inoculating the sample on an agar plate for bacterial culture and purification, performing bacterial identification by using a Vitek MS mass spectrum, performing drug sensitivity detection on the identified bacteria by using a Vitek2compact instrument, and selecting antibiotics according to the drug sensitivity test result to complete the culture of the primary cells.
In the invention, the suspected polluted cell culture solution can be seen under an inverted microscope under a condition that a dot-shaped or rod-shaped unidentified substance and/or irregularly moved violently and/or obviously different from Brownian movement of cell fragments and other impurities before the culture solution is replaced.
In the invention, the agar plates are blood agar plates, chocolate agar plates and MacConkey plates.
In the invention, the bacterial culture is to use an inoculating loop to dip a sample and respectively inoculate the sample on a blood agar plate, a chocolate agar plate and a Mackanka plate by a partition streak separation method for culture.
In the invention, the sample is inoculated on a blood agar plate and then placed in a 37 ℃ incubator for culture.
In the invention, the sample is inoculated on a chocolate agar plate and a Macconkey plate and then placed with a sample containing 5% CO 2 Culturing in an incubator.
In the invention, the culture time is 18-24 h.
In the invention, the method for sensitive detection of the drug comprises the following steps: preparing bacterial suspension, inserting a drug sensitive card catheter into the bacterial suspension, inputting the information of the drug sensitive card into a Vitek2 system, and performing drug sensitive detection on the identified bacteria by using a Vitek2compact instrument.
According to the preparation method of the bacterial suspension, colonies are selected and dissolved in a 0.3-0.5% NaCl solution, and the turbidity of the bacterial suspension is 0.5-0.63 McF.
In the present invention, the cell is a human colorectal cancer cell.
The invention has the following beneficial effects:
the culture method of the human-derived primary cells uses a Vitek MS bacteria identification technology to identify different quality proteins and obtain a microbial protein quality fingerprint diagram. The Vitek2compact instrument connected with Myla software can continuously monitor the visible light transmittance in the drug sensitive card hole, directly monitor the growth and activity of bacteria to carry out drug sensitivity identification, and the result has higher reliability and stability. According to the invention, after the cell culture is polluted, the antibiotic type and concentration which have no influence on the cell growth are selected to eliminate bacteria according to the drug sensitivity test result, the cell growth state is normal and has no obvious death after 24 hours of culture, the bacterial amount in the culture solution is obviously reduced, the bacterial pollution is reversed in the early stage, the success rate of primary cell culture is greatly improved, compared with the empirical use of broad-spectrum antibiotics, the cell growth is obviously limited even if no bacterial movement is seen, even tumor cell death occurs, and the phenomenon that the fibroblast cannot be exponentially multiplied has obvious advantages. The culture method disclosed by the invention has an important effect on timely saving of precious adherent cell pollution in cell culture, and has an important significance on smoothly developing research work of a medical basic laboratory.
Drawings
FIG. 1 is a flow chart of a method for culturing primary human colorectal cancer cells;
FIG. 2 is a MALDI-TOF peak chart of Achromobacter xylosoxidans;
FIG. 3 is a MALDI-TOF peak plot of Pseudomonas aeruginosa;
FIG. 4 is a graph showing the results of detection of drug sensitivity of Achromobacter xylosoxidans;
FIG. 5 is a graph showing the result of drug sensitivity detection of Pseudomonas aeruginosa;
FIG. 6 is a chart of the 400-fold microscopic cell motility before cefoperazone sulbactam sodium;
figure 7 is a picture of the 400 fold microscopic cell motility before use of cefoperazone sulbactam sodium.
Detailed Description
The invention provides a culture method of human-derived primary cells, which comprises the following steps: separating human cells, performing primary cell culture in a complete culture solution without antibiotics, replacing the culture solution according to the growth condition of the cells, taking the suspected contaminated cell culture solution as a sample, inoculating the sample on an agar plate for bacterial culture and purification, performing bacterial identification by using a Vitek MS mass spectrum, performing drug sensitivity detection on the identified bacteria by using a Vitek2compact instrument, and selecting antibiotics according to the drug sensitivity test result to complete the culture of the primary cells. In the present invention, as a method that can be carried out, the complete medium is prepared from a DMEM high-glucose medium containing 10% fetal bovine serum, both of which are purchased from Gibco. In the present invention, as a practical mode, the primary cell culture process is performed strictly according to the cell sterile operation specification.
In the invention, as an implementation mode, after a sample is purified, a single purified colony coated target plate is picked, 0.5-1 μ l of Vitek MS-CHCA matrix is added for drying at room temperature, and then the bacterium identification is carried out by using Vitek MS mass spectrometry. Preferably, the Vitek MS-CHCA matrix is 0.6-0.95 μ l.
In another embodiment of the present invention, if the colony is bacteria with suspected cell wall, 0.2-0.6 μ l of 25% formic acid is added, dried at room temperature, and then 0.5-1 μ l of Vitek MS-CHCA matrix is added, and then the bacteria identification is performed by using Vitek MS mass spectrometry. Preferably, the 25% formic acid is 0.3-0.5 μ l, and the Vitek MS-CHCA matrix is 0.6-0.95 μ l.
In the invention, as an implementation mode, when the Vitek MS mass spectrum is used for bacteria identification, sample information is input into a mass spectrum workstation, a target plate is placed into a Vitek MS instrument for MS detection, a MALDI-TOF peak diagram is obtained, and the bacteria species are matched through a bacteria spectrum library. In the present invention, the bacterial spectrum library is the bacterial database version V3.0.
In the present invention, the suspected contaminated cell culture solution can be seen under an inverted microscope with a dot-like or rod-like unidentified substance and/or violently irregularly moved and/or significantly different from brownian movement of cell debris and other impurities before the culture solution is replaced. In the present invention, the suspected contaminated cell culture solution can be observed under a microscope with a magnification of 100 times, 200 times or 400 times.
In the present invention, the agar plates are blood agar plates, chocolate agar plates, and mecnkia plates. In the present invention, the blood agar plate, chocolate agar plate and mecnkia plate are well known to those skilled in the art.
In the invention, the bacterial culture is to use an inoculating loop to dip a sample and respectively inoculate the sample on a blood agar plate, a chocolate agar plate and a Macconk plate by a zone-division streak separation method for culture. In the present invention, different agar plates can be used to culture different species of the genus. In the present invention, most of the bacteria can grow in blood agar plates, and chocolate agar plates are mainly used for isolated culture of gram-negative bacteria; the Macconkey plate is mainly used for the isolated culture of intestinal bacteria.
In the present invention, the sample is inoculated on a blood agar plate and then cultured in a 37 ℃ incubator.
In the present invention, the sample is inoculated on chocolate agar plate and Macconkey plate and then placed with 5% CO 2 Culturing in an incubator.
In the invention, the culture time is 18-24 h. Preferably, the culture time is 20-22 h. In the invention, the culture time can ensure the number and concentration of strains, is beneficial to later-stage bacteria identification, and avoids strain death and metabolite accumulation caused by too long culture time.
In the invention, the method for sensitive detection of the drug is as follows: preparing bacterial suspension, inserting a drug sensitive card catheter into the bacterial suspension, inputting the information of the drug sensitive card into a Vitek2 system, and performing drug sensitive detection on the identified bacteria by using a Vitek2compact instrument. In the invention, the drug sensitive card is one or more of gram-negative bacteria drug sensitive card GN 09, gram-negative bacteria drug sensitive card GN67, gram-negative bacteria drug sensitive card N335, gram-positive bacteria drug sensitive card P639 and streptococcus pneumoniae drug sensitive card GP 68 which are selected according to the types of bacteria after colony observation.
In the invention, the preparation method of the bacterial suspension comprises the steps of picking bacterial colonies and dissolving the bacterial colonies into 0.3-0.5% NaCl solution, wherein the specific turbidity of the bacterial suspension is 0.5-0.63 McF. Preferably, colonies are picked and dissolved in 0.35-0.45% NaCl solution.
In the present invention, the cell is optionally a human colorectal cancer cell. In the invention, the human colorectal cancer cell material is derived from a fresh human colorectal cancer specimen which is excised by operation.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 isolation and culture of Primary cells
Taking a fresh human large intestine tumor specimen which is removed by operation and has the size of about 0.5 multiplied by 0.5cm, cutting off adipose tissues, immediately putting into PBS liquid added with penicillin and streptomycin, preserving in a sterile and sealed manner, and sending to a laboratory at a low temperature. Irradiating a super clean bench with an ultraviolet lamp for 30min, starting a fan for self-cleaning for 3min, flushing the tumor tissue for 3 times in a sterile super clean bench with sterile PBS (phosphate buffer solution) to remove the supernatant, adding sterile penicillin, streptomycin and a small amount of sterile cell culture solution into a culture dish, and shearing the specimen into tissues with the size of about 0.03-0.08mm by using sterile ophthalmic scissors. Using a human tumor tissue isolation reagent (purchased from Miltenyi Bioted Co., Ltd.), the tissue fragments were digested and disintegrated and loosened by pipetting, and then pipetted into a 6cm dish, 4mL of a DMEM high-sugar medium containing 10% fetal bovine serum was added, and the dish was placed at 37 ℃ in a 5% CO culture 2 Culturing in an incubator. Daily lifeObserving the growth condition of tumor cells, replacing the culture solution according to the growth condition of the cells for 2-3 days, and if the punctiform or rod-shaped unknown substances can be seen under an inverted microscope before replacing the culture solution, and do irregular movement violently, which is obviously different from Brownian movement of cell fragments and other impurities, the culture solution is suspected to be polluted. And (4) reserving culture solution supernatant to a sterile EP tube, and carrying out normal-temperature inspection as soon as possible to carry out bacterial culture identification and drug sensitivity detection.
Example 2 Vitek MS Mass Spectrometry bacterial identification
Inoculating the culture solution with disposable inoculating loop, and respectively inoculating to blood agar plate, chocolate agar plate and Mackanka plate by zone streaking separation method, placing the blood agar plate in a common incubator at 37 deg.C, placing the chocolate agar plate and Mackanka plate in 5% CO 2 The growth of bacterial colonies was observed after 18-24 hours incubation in the incubator, respectively, and purification was performed.
Selecting a single purified colony, coating the colony on a target plate, adding 0.95 mu l of Vitek MS-CHCA matrix, and drying at room temperature; if the bacteria are suspected to have cell walls, 0.45 mu l of 25 percent formic acid is added, 0.95 mu l of Vitek MS-CHCA matrix is added after the bacteria are dried at room temperature, the sample information is input into a mass spectrum workstation, a target plate is placed into a Vitek MS instrument for MS detection, a MALDI-TOF peak diagram is obtained, and the bacteria types of the bacteria are matched to be achromobacter xylosoxidans and pseudomonas aeruginosa through the version V3.0 of a bacteria database.
Example 3 detection of drug sensitivity by Vitek2compact Instrument
The 0.45% NaCl bottle was taken out of the refrigerator and returned to room temperature. A disposable transparent plastic test tube is taken, 3ml of sterile 0.45% NaCl is added into each test tube, the pH value is adjusted to 7.0, and colonies are picked and dissolved in saline water of the test tube. Testing with turbidimeter, and adjusting the turbidity of the bacteria-containing test tube to be in the range of 0.5-0.63 McF.
The achromobacter xylosoxidans and the pseudomonas aeruginosa are gram-negative bacteria, and gram-negative bacteria drug sensitive card N335 is selected to carry out drug sensitive detection on the two strains. Before use, the drug sensitive card is taken out from the refrigerator, the temperature is restored to room temperature, the bacterial suspension test tube and the drug sensitive card are sequentially placed on the card carrying frame, and the catheter of the drug sensitive card is inserted into the bacterial suspension. Inputting the drug sensitive card information into a Vitek2 system, transmitting the strain information identified by the Vitek MS through Myla software, and putting the card carrying frame. The sensitivity of the bacteria to different concentrations of antibiotics was evaluated by measuring the growth of organisms by the amount of light blocked through the well by light transmittance sampling of the same well at 15 minute intervals. Wherein S represents sensitivity, R represents drug resistance, and I represents an intermediary.
According to the results of drug sensitivity detection, as shown in fig. 4-5, achromobacter xylosoxidans is sensitive to ticarcillin/clavulanic acid, piperacillin/tazobactam, cefoperazone/sulbactam, ceftazidime, imipenem, meropenem, doxycycline, sulfamethoxazole, minocycline and tigecycline; pseudomonas aeruginosa is sensitive to piperacillin/tazobactam, cefoperazone/sulbactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, tobramycin, amikacin, ciclisaxin, polymyxin.
EXAMPLE 4 antibacterial agent
And integrating bacterium identification and drug sensitivity results by using Myla software to form a report. According to the reported bacterial and sensitive antibiotic drug species, the use of cefoperazone sodium and sulbactam sodium has minimal impact on the biological properties of primary cells compared with the advantages and disadvantages of each sensitive antibiotic. The method specifically comprises the following steps: 1.5g of cefoperazone sodium and sulbactam sodium for injection (1.0 g based on cefoperazone and 0.5g based on sulbactam) are taken and dissolved into 50 mL0.9% sodium chloride solution, 250 mu L of the solution is added when 100mL of complete culture solution is prepared, and the working concentration of the cefoperazone sodium and sulbactam sodium is determined to be 74.8 mu g/mL according to the specification of the medicine revised by 21 days 6 and 2019 and the working concentration of the streptomycin used in the conventional cell culture.
The results show that more unidentified substances in a dot or rod shape among cells can be seen before the cefoperazone sulbactam sodium is used, and the movement can be seen under a microscope, which is shown in figure 6; the amount of unidentified material in the form of dots or rods in the visual field was significantly reduced after the administration of cefoperazone sulbactam sodium, see figure 7.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for culturing human-derived primary cells is characterized by comprising the following steps: separating human cells, performing primary cell culture in a complete culture solution without antibiotics, replacing the culture solution according to the growth condition of the cells, taking the suspected contaminated cell culture solution as a sample, inoculating the sample on an agar plate for bacterial culture and purification, performing bacterial identification by using a Vitek MS mass spectrum, performing drug sensitivity detection on the identified bacteria by using a Vitek2compact instrument, and selecting antibiotics according to the drug sensitivity test result to complete the culture of the primary cells.
2. The method according to claim 1, wherein the suspected contaminated cell culture solution is observed under an inverted microscope with a dot-like or rod-like unidentified substance and/or is vigorously irregularly moved and/or is significantly different from brownian movement of cell debris and other impurities before the culture solution is replaced.
3. The culture method according to claim 1, wherein the agar plate is a blood agar plate, a chocolate agar plate, or a Macconk plate.
4. The culture method according to claim 1, wherein the bacterial culture is carried out by taking a sample with an inoculating loop and inoculating the sample on a blood agar plate, a chocolate agar plate and a Macconk plate respectively by a zone-division streaking method.
5. The culture method according to claim 4, wherein the sample is inoculated on a blood agar plate and cultured in an incubator at 37 ℃.
6. The culture method according to claim 4, wherein the sample is inoculated on a chocolate agar plate and a Macconyya plate and then placed thereon with 5% CO 2 Culturing in an incubator.
7. The method according to claim 5 or 6, wherein the cultivation time is 18 to 24 hours.
8. The culture method according to claim 1, wherein the drug-sensitive detection method comprises: preparing bacterial suspension, inserting a drug sensitive card catheter into the bacterial suspension, inputting the information of the drug sensitive card into a Vitek2 system, and performing drug sensitive detection on identified bacteria by using a Vitek2compact instrument.
9. The method according to claim 1, wherein the bacterial suspension is prepared by picking colonies and dissolving the colonies in a 0.3-0.5% NaCl solution, and the turbidity of the bacterial suspension is 0.5-0.63 McF.
10. The method of claim 1, wherein the cells are human colorectal cancer cells.
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