CN110272866A - It is a kind of to promote the method and its application for improving cell state - Google Patents
It is a kind of to promote the method and its application for improving cell state Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The present invention relates to a kind of method and its application for promoting improvement cell state, the methods are as follows: transplants mycoplasma positive cell line into immunodeficient mouse body, and raises mouse, then isolate cell line out of Mice Body, obtains mycoplasma negative cells system.Its operating process is more simple also safer relative to the method for currently used removal cell mycoplasma contamination for the method for mycoplasma contamination in the removal cell, this method avoid antibiotic and high-temperature factor simultaneously, injury to cell can be preferably minimized, and process cycle is shorter, also can thoroughly remove mycoplasma contamination and not recur.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of to promote the method and its application for improving cell state.
Background technique
Cell culture is a kind of conventional laboratory techniques of amplification in vitro cell, is widely used at present real in scientific research, clinic
It tests.The successful key of cell culture pollutes first is that controlling, and bacterium, mould, black glue worm, mycoplasma etc. are all to cause to cultivate cell
The microorganism of pollution, wherein mycoplasma contamination is one of cell cultivation process primary pollution source.
Mycoplasma is that one kind does not have cell wall, height pleomorphism, can pass through bacteria filter, can be proliferated with artificial medium culture
Minimum prokaryotic microorganism.Due to that can form Filamentous and branched form, therefore referred to as mycoplasma.Mycoplasma is widely present in
It is mostly not pathogenic in humans and animals body, mainly there are mycoplasma pneumoniae, U. urealyticum, human-like branch former the mycoplasma that people is caused a disease
Body, mycoplasma genitalium etc..Macrophage, lgG and IgM have certain lethal effect to mycoplasma.It is more than in world wide
15% culture cell has mycoplasma contamination, by the cell of mycoplasma contamination, typically no apparent cellular damage, be not easy by
Discover, but can change in the various aspects such as cell metabolism, immune or biochemical characteristic, upgrowth situation and cell survival, thus
Directly affect the stability and reliability of research work, clinical medicine and biological products.
Mycoplasma is smaller than bacterium and fungi on figure, and for general optical microscopy not it is observed that it exists, mycoplasma is difficult
It to discover, while being also difficult to eliminate, mycoplasma can easily pass through standard filter membrane, while also can the most of antibiotic of antagonism.
Influence of the mycoplasma to cell, mainly from the aspect of two: being on the one hand to be directly affected to cell, cell is by mycoplasma contamination
Afterwards, slowly, part cell rounding falls off proliferation from bottle wall, actually latent although the variation of part cell surface is not fairly obvious
Lie prostrate various danger；On the other hand, mycoplasma can inhibit cell DNA, RNA by the arginine in consumption culture medium
Synthesis, reduce the resistance of cell.In short, mycoplasma infection can influence to be various caused by cell: including being metabolized,
Immune or biochemical characteristic, upgrowth situation, the action pathway of enzyme, the composition of cell membrane, chromosome structure, transfection efficiency, Yi Jixi
Various changes such as born of the same parents' survival.
Currently, mycoplasma contamination once occurs for culture cell, most solution is abandoned after inactivation, but if thin
Born of the same parents are of great rarity, and are not available such solution, and the method for currently used removal cell line mycoplasma contamination is main
Have: antibiotic treatment method, heat treatment method, macrophage phagocytosis method.The long processing period of antibiotic treatment method has certain answer
Hair rate, while relative toxicity is larger, it is larger to cell damage.Heat treatment method, it is big to cellular damage, it may cause processed
Metamorphosis occurs for cell, influences cell state.Macrophage phagocytosis method needs that it is dirty to bring muroid virus by mouse peritoneal
A possibility that dye.
CN106635950A discloses a kind of processing method of mycoplasma contamination comprising following steps: obtaining to be processed
Cell；The cell to be processed described in the culture medium culture containing 10 μ g/mL Ciprofloxacins；Timing updates the culture medium, and
And the stand density of the cell to be processed is made to be maintained at 50%-80%；Stop after the case where can't detect mycoplasma contamination
Only use the culture medium.The invention can thoroughly remove mycoplasma contamination, at the same also have stop using Ciprofloxacin after not
The advantage of recurrence.But the process cycle of this method is longer, while the use of antibiotic is more toxic, it is larger to cell damage.
CN101851603A discloses a kind of mycoplasma clearing reagent and its application, the group including following weight percents
Point: fluoroquinolone antibiotics 1.3-1.5%；Tetracyclines derivative 0.3-0.5%；Macrolide antibiotics 0.1-
0.3%；Buffer 97.7-98.3%.The mycoplasma clearing reagent of the invention most effective can inhibit and kill mycoplasma, but deposit
As described above the drawbacks of.
It can be seen that the method for removing mycoplasma contamination in the prior art is also very limited, and there are many drawbacks, therefore,
Develop it is a kind of it is novel, safer, small to cell damage, process cycle is short and can thoroughly remove mycoplasma dirt
The method of dye is significantly.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide it is a kind of promoted improve cell state method and its
Using, especially provide it is a kind of removal culture cell in mycoplasma contamination method and its application.This method is relative to common at present
Removal cell mycoplasma contamination method it is safer, while cell damage can be preferably minimized, process cycle is shorter, about
It is 4-5 weeks, and mycoplasma contamination can be thoroughly removed, operating process is relatively simple.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of method for being promoted and improving cell state, the method are as follows: to immunodeficient mouse
Transplanting mycoplasma positive cell line in vivo, and mouse is raised, then cell line is isolated out of Mice Body, it is negative thin to obtain mycoplasma
Born of the same parents system.
Method of the method for mycoplasma contamination relative to currently used removal cell mycoplasma contamination in the removal cell
Its operating process is more simple also safer, while this method avoid antibiotic and high-temperature factor, can be the wound to cell
Evil is preferably minimized, and process cycle is shorter, about 4-5 weeks, also can thoroughly remove mycoplasma contamination and not recur.
Preferably, described method includes following steps:
(1) mycoplasma positive cell line is obtained；
(2) the mycoplasma positive cell line of step (1) is implanted into immunodeficient mouse body and is raised；
(3) cell line is isolated in the Mice Body from step (2) after raising, obtains mycoplasma negative cells system.
Preferably, step (1) described cell line includes tumor cell line and immunocyte system.
Preferably, the tumor cell line includes HepG2, Huh7, MDA-MB-231, U-2OS or MRC-5.
Preferably, the immunocyte includes B cell, T cell or NK cell.
In the present invention, step (1) method for obtaining mycoplasma positive cell line are as follows: use mycoplasma test reagent box
Test sample, the pollution condition of mycoplasma, obtains mycoplasma positive cell line with this in judgement sample.
Concrete mode can be with are as follows: the MycoAlert Mycoplasma Detection produced using LONZA company, the U.S.
Mycoplasma contamination in Kit mycoplasma test reagent box test sample.Concrete operations are as follows: at room temperature, take 50 μ L to be checked
The culture medium supernatant for surveying cell, is added 50 μ L A liquid later, stands reaction 5 minutes, after five minutes in detecting on fluorescence detector
To first time read A value, backward reaction system in, be added 50 μ L B liquid, stand react 10 minutes, detect second after ten minutes
Secondary reading B value.If the ratio (B/A) of B value and A value is greater than 0.9, illustrate have mycoplasma contamination, otherwise, then do not have in the sample
There is mycoplasma contamination.
Preferably, the mode of step (2) described transplanting includes subcutaneous transplantation or tail vein injection.
The mycoplasma positive cell line, which is entered by way of transplanting in immunodeficient mouse body, be proliferated and herein
In the process in successive elimination cell mycoplasma pollution, therefore, in addition to above-mentioned subcutaneous transplantation and tail vein injection mode, other
Field conventional transplant mode can all be realized.Generally, solid tumor cell system uses subcutaneous transplantation mode, immunocyte system and blood
Tumor cell line (such as Leukemia Cell Lines) by the way of tail vein injection.
Preferably, the cell line quantity of step (2) described transplanting is 2 × 105-5×106, such as 2 × 105、4×105、5
×105、6×105、8×105、1×106Or 5 × 106Deng.
The concrete mode of subcutaneous transplantation can be with are as follows: by the skin at the back leg groin of mouse after dipping in alcohol with cotton ball soaked in alcohol
It moistens and sterilizes；The cell suspension of 200 μ L is drawn with insulin needle, the 45 degree of subscripts that incline are mapped to the subcutaneous of mouse (cannot be too deeply no
Muscle can be then easily accessible), it is seen that mouse is put back in normal environment and is raised by the lump heaved.
The concrete mode of tail vein injection can be with are as follows: in Biohazard Safety Equipment, anaesthetic is injected into mouse peritoneal, mouse
After anesthesia, mouse tail is gently wiped with cotton ball soaked in alcohol, the cell suspension of 200 μ L is drawn with insulin needle, is infused by tail vein
It injects in Mice Body, if entry point is mouse tail vein, the sense that falls through is had when pushing pin.
Preferably, the time of step (2) described raising is 20-40 days, such as 20 days, 22 days, 25 days, 27 days, 30 days, 32
It, 35 days or 40 days etc..
Preferably, step (2) immunodeficient mouse is NSI immunodeficient mouse.
NSI (NOD-Scid-IL2Rg-/-) mouse is struck under NOD-scid mouse species background using TALEN technology
Except IL2Rc gene, and obtain, it is on the books in the patent document that number of patent application is 201310229629.9.NSI strain
B, T, NK cell are lacked, when such mouse is used as tumor drug screening, treats the test of pathogenic mechanism, to allosome xenogenesis (source of people)
Graft is the mouse model acted on without immunological rejection heteroplastic transplantation completely without immunological rejection.By mycoplasma sun
Property cell line be transplanted in the Strains of Mouse body, separate mycoplasma negative cells can be obtained again after raising a period of time
System.
Preferably, according to the mode of subcutaneous transplantation, step (3) method for isolating cell line out of Mice Body
Are as follows: mouse is put to death and body surface sterilizes, it is sterile to cut transplanted cells system tissue, it is soaked in RPMI complete culture solution, then use pancreas
Protease hydrolyzed obtains mycoplasma negative cells system.
The above-mentioned concrete mode for isolating cell line can be with are as follows: is put to death mouse with cervical dislocation method, by mouse body surface
Disinfection, in Biohazard Safety Equipment, left hand tweezers lift its tumour surrounding skin, and the right hand is cut off with eye scissors, sterile to cut open
Transplanted cells system organizes out, is soaked in RPMI1640 complete culture solution, chooses well-grown tissue block, shredded, and uses
Pancreatin enzymatic hydrolysis, is cleaned with PBS.
Preferably, the mode that the mouse is put to death is cervical dislocation method.
Preferably, described to be shredded tissue with before trypsin digestion.
Preferably, the time of the enzymatic hydrolysis is 3-8min, such as 3min, 4min, 5min, 6min, 7min or 8min etc..
Preferably, according to the mode of intravenous injection, step (3) method for isolating cell line out of Mice Body
Are as follows: mouse is first cooked into deep anesthesia, opens thoracic cavity, exposure heart is pierced into right ventricle with syringe needle, and draw blood can draw 1mL blood
Liquid is carried out the sorting of cell using cell separating liquid and/or cell sorting kit, obtains mycoplasma negative cells system.
Preferably, T cell is separated according to the mode of intravenous injection, step (3) is described to isolate cell out of Mice Body
The method of system are as follows: mouse is first cooked into deep anesthesia, opens thoracic cavity, exposure heart is pierced into right ventricle with syringe needle, and draw blood can inhale
Take 1mL blood.The blood of absorption is mixed with 100 μ L anti-coagulants, then blood and equivalent PBS liquid are mixed well, is inhaled with Pasteur
Pipe is slowly superimposed on Ficoll lymphocyte separation medium liquid level along tube wall, and ratio is Ficoll lymphocyte: (blood+PBS)
=1:2 pays attention to keeping clear interface, acts light and slow, horizontal centrifugal 800g, 20min.It is divided into 3 layers after centrifugation in pipe, upper layer is
Blood plasma and PBS, lower layer are red blood cell and granulocyte, and middle layer Ficoll has one layer of tunica albuginea layer, at intermediate interface with single core
Based on cell, mononuclearcell includes lymphocyte, monocyte and blood platelet etc..With pasteur pipet draw tunica albuginea layer to newly
The PBS liquid of 5 times of volumes is added in Guan Zhong, mixes, and 300g is centrifuged 5min, washs Ficoll.Supernatant is abandoned, cell is counted, it
T cell magnetic bead sorting is carried out with T cell sorting kit afterwards, obtains mycoplasma negative cells system.
In the present invention, the cell line that judgment step (3) is isolated is the method for mycoplasma negative cells system are as follows: will be separated
Obtained cell line is inoculated in the RPMI complete culture solution containing 10%FBS, culture 5-10 days (such as 5 days, 6 days, 7 days, 8
It, 9 days or 10 days etc.) use mycoplasma test reagent box test sample afterwards, the pollution condition of mycoplasma in judgement sample, and carrying out
STR detection verifying cell identity.
The process of STR detection verifying cell identity approximately as:
(1) PCR (PowerPlex is expanded with the more amplification kits of STRTM16HS System)；
(2) PCR product (Applied is detected with 3100 type DNA analysis instrument)；
(3) species identification has been carried out to sample using COX1 gene magnification and electrophoretic techniques.
The method of the verifying cell identity can also use flow cytometry.
As the preferred technical solution of the present invention, described method includes following steps:
(1) cell line to be detected is detected with mycoplasma test reagent box, judges the pollution condition of mycoplasma in cell line, with
This obtains mycoplasma positive cell line；
(2) by the mycoplasma positive cell line of step (1) by subcutaneous transplantation or tail vein injection mode with 2 × 105-5
×106Cell concentration be implanted into NSI immunodeficient mouse body and carry out raising 20-40 days；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, it is sterile to cut transplanted cells system group
It knits, is soaked in RPMI complete culture solution, is shredded, then carry out enzymatic hydrolysis 3-8min with trypsase, it is finally clear with PBS solution
It washes, obtains mycoplasma negative cells system；Or the mouse after raising in step (2) is cooked into deep anesthesia, open thoracic cavity, the exposure heart
It is dirty, it is pierced into right ventricle with syringe needle, draw blood carries out the sorting of cell using cell separating liquid and/or cell sorting kit,
Obtain mycoplasma negative cells system.
In another aspect, the present invention provides a kind of method as described above answering in mycoplasma contamination in removal culture cell
With.
Compared with the existing technology, the invention has the following advantages:
The present invention carries out the removal of mycoplasma in cell with immunodeficient mouse tool, and mycoplasma is dirty in the removal cell
The method of dye is more simple also safer relative to its operating process of the method for currently used removal cell mycoplasma contamination,
Simultaneously this method avoid antibiotic and high-temperature factor, the injury to cell can be preferably minimized, and process cycle is shorter, about
It is 4-5 weeks, also can thoroughly removes mycoplasma contamination and not recur.
Detailed description of the invention
Fig. 1 is the FCM analysis figure of MDA-MB-231 cell line in embodiment 1；
Fig. 2 is the FCM analysis figure of MDA-MB-231 cell line in embodiment 1；
Fig. 3-Fig. 4 is that the STR of MDA-MB-231 cell line in embodiment 1 analyzes result figure；
Fig. 5-Fig. 6 is that the STR of HepG2 cell line in embodiment 2 analyzes result figure；
Fig. 7-Fig. 8 is that the STR of Huh7 cell line in embodiment 3 analyzes result figure；
Fig. 9-Figure 10 is that the STR of U-2OS cell line in embodiment 4 analyzes result figure；
Figure 11-Figure 12 is that the STR of MRC-5 cell line in embodiment 5 analyzes result figure；
Figure 13 is the flow diagram of the method for the invention.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
It is former that following embodiments each provide branch in removal MDA-MB-231, HepG2, Huh7, U-2OS, MRC-5 cell line
The method of body pollution, because being related to the experiment of cell streaming in embodiment 1, therefore the cell line used is MDA-MB-231-GL, this
Cell line is transfected by slow virus carrier, and a fluorescent reporter gene is inserted on MDA-MB-231 cell line genome
Luciferase, luciferase can stablize expression in cell line.
Embodiment 1
The present embodiment provides it is a kind of removal MDA-MB-231-GL cell line in mycoplasma contamination method, specifically include with
Lower step:
(1) MDA-MB-231-GL cell line to be detected is detected with mycoplasma test reagent box, judges mycoplasma in cell line
Pollution condition, the results are shown in Table 6, B/A > 0.9, obtain mycoplasma positive MDA-MB-231-GL cell line；
(2) by the skin wet at the back leg groin of NSI immunodeficient mouse and disinfection after dipping in alcohol with cotton ball soaked in alcohol,
The mycoplasma positive MDA-MB-231-GL cell line of the step of drawing 200 μ L with insulin needle (1), with 2 × 105Cell concentration
The 45 degree of subscripts that incline are mapped to the subcutaneous of mouse, it is seen that mouse is put back in normal environment and raised 20 days by the lump heaved；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, in Biohazard Safety Equipment, left hand
Lift its tumour surrounding skin with tweezers, the right hand is cut off with eye scissors, sterile to cut transplanting MDA-MB-231-GL cell line
Tissue, is soaked in RPMI complete culture solution, chooses well-grown tissue and is shredded, then is digested with trypsase
3min is finally cleaned with PBS solution, obtains mycoplasma negative cells system.
The isolated MDA-MB-231-GL cell line of step (3) is inoculated in the RPMI containing 10%FBS to cultivate completely
In liquid, mycoplasma test reagent box test sample is used after culture 5 days, the pollution condition of mycoplasma in judgement sample, as a result such as table 7
It is shown, illustrate that isolated cell line is mycoplasma negative cells system.And carry out the experiment of cell stream formula and STR detection verifying carefully
Born of the same parents' identity.
Cell streaming experimental result is as depicted in figs. 1 and 2: Fig. 1 is that cell living is drawn door, Fig. 2 by cell granulations degree
It is to detect MDA-MB-231-GL target cell system in living cells, divides out of Mice Body from flow cytometry level Preliminary Identification
The cell separated out is MDA-MB-231-GL cell line.
For STR testing result as shown in table 1, Fig. 3 and Fig. 4, the result of Fig. 3 summarizes such as table 1, shows on locus without three equipotentials
Gene and tetra-allelic, and without the pollution of other Human cell lines；STR testing result is inputted into ATCC database and DSMZ
Database is compared that (its concrete operations is all made of the conventional use of STR detection mode of those skilled in the art and process
Detected), cell is MDA-MB-231 cell line as the result is shown；Sample is human cell line to Fig. 4 as the result is shown, and without other
The pollution of Species Cell system.
Table 1
Embodiment 2
The present embodiment provides a kind of methods of mycoplasma contamination in removal HepG2 cell line, specifically includes the following steps:
(1) HepG2 cell line to be detected is detected with mycoplasma test reagent box, judges the pollution feelings of mycoplasma in cell line
Condition, the results are shown in Table 6, B/A > 0.9, obtains mycoplasma positive HepG2 cell line；
(2) by the skin wet at the back leg groin of NSI immunodeficient mouse and disinfection after dipping in alcohol with cotton ball soaked in alcohol,
The mycoplasma positive HepG2 cell line of the step of drawing 200 μ L with insulin needle (1), with 2 × 105Cell concentration incline 45 degree of angles
It is injected into the subcutaneous of mouse, it is seen that mouse is put back in normal environment and raised 40 days by the lump heaved；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, in Biohazard Safety Equipment, left hand
Lift its tumour surrounding skin with tweezers, the right hand is cut off with eye scissors, sterile to cut transplanting HepG2 cell line tissue, leaching
It steeps in RPMI complete culture solution, chooses well-grown tissue and shredded, then carry out enzymatic hydrolysis 8min with trypsase, finally
It is cleaned with PBS solution, obtains mycoplasma negative cells system.
The isolated HepG2 cell line of step (3) is inoculated in the RPMI complete culture solution containing 10%FBS, is trained
Mycoplasma test reagent box test sample is used after supporting 10 days, the pollution condition of mycoplasma in judgement sample, the results are shown in Table 7,
Illustrate that isolated cell line is mycoplasma negative cells system.And carry out STR detection verifying cell identity.
For STR testing result as shown in table 2, Fig. 5 and Fig. 6, the result of Fig. 5 summarizes such as table 2, shows on locus without three equipotentials
Gene and tetra-allelic, and without the pollution of other Human cell lines；STR testing result is inputted into ATCC database and DSMZ
Database is compared that (its concrete operations is all made of the conventional use of STR detection mode of those skilled in the art and process
Detected), cell is HepG2 cell line as the result is shown；Sample is human cell line to Fig. 6 as the result is shown, and without other species
The pollution of cell line.
Table 2
Embodiment 3
The present embodiment provides a kind of methods of mycoplasma contamination in removal Huh7 cell line, specifically includes the following steps:
(1) Huh7 cell line to be detected is detected with mycoplasma test reagent box, judges the pollution feelings of mycoplasma in cell line
Condition, the results are shown in Table 6, B/A > 0.9, obtains mycoplasma positive Huh7 cell line；
(2) by the skin wet at the back leg groin of NSI immunodeficient mouse and disinfection after dipping in alcohol with cotton ball soaked in alcohol,
The mycoplasma positive Huh7 cell line of the step of drawing 200 μ L with insulin needle (1), with 2 × 106Cell concentration incline 45 degree of subscripts
It is mapped to the subcutaneous of mouse, it is seen that mouse is put back in normal environment and raised 30 days by the lump heaved；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, in Biohazard Safety Equipment, left hand
Lifting its tumour surrounding skin with tweezers, the right hand is cut off with eye scissors, and it is sterile to cut transplanting Huh7 cell line tissue, it impregnates
In RPMI complete culture solution, chooses well-grown tissue and shredded, then carry out enzymatic hydrolysis 5min with trypsase, finally use
PBS solution cleaning, obtains mycoplasma negative cells system.
The isolated Huh7 cell line of step (3) is inoculated in the RPMI complete culture solution containing 10%FBS, is cultivated
Mycoplasma test reagent box test sample is used after 7 days, the pollution condition of mycoplasma in judgement sample, the results are shown in Table 7, explanation
Isolated cell line is mycoplasma negative cells system.And carry out STR detection verifying cell identity.
For STR testing result as shown in table 3, Fig. 7 and Fig. 8, the result of Fig. 7 summarizes such as table 3, shows on locus without three equipotentials
Gene and tetra-allelic, and without the pollution of other Human cell lines；STR testing result is inputted into ATCC database and DSMZ
Database is compared that (its concrete operations is all made of the conventional use of STR detection mode of those skilled in the art and process
Detected), cell is Huh7 cell line as the result is shown；Sample is human cell line to Fig. 8 as the result is shown, and thin without other species
The pollution of born of the same parents system.
Table 3
Embodiment 4
The present embodiment provides a kind of methods of mycoplasma contamination in removal U-2OS cell line, specifically includes the following steps:
(1) U-2OS cell line to be detected is detected with mycoplasma test reagent box, judges the pollution feelings of mycoplasma in cell line
Condition, the results are shown in Table 6, B/A > 0.9, obtains mycoplasma positive U-2OS cell line；
(2) by the skin wet at the back leg groin of NSI immunodeficient mouse and disinfection after dipping in alcohol with cotton ball soaked in alcohol,
The mycoplasma positive Huh7 cell line of the step of drawing 200 μ L with insulin needle (1), with 5 × 105Cell concentration incline 45 degree of subscripts
It is mapped to the subcutaneous of mouse, it is seen that mouse is put back in normal environment and raised 35 days by the lump heaved；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, in Biohazard Safety Equipment, left hand
Lift its tumour surrounding skin with tweezers, the right hand is cut off with eye scissors, sterile to cut transplanting U-2OS cell line tissue, leaching
It steeps in RPMI complete culture solution, chooses well-grown tissue and shredded, then carry out enzymatic hydrolysis 5min with trypsase, finally
It is cleaned with PBS solution, obtains mycoplasma negative cells system.
The isolated U-2OS cell line of step (3) is inoculated in the RPMI complete culture solution containing 10%FBS, is trained
Mycoplasma test reagent box test sample is used after supporting 5 days, the pollution condition of mycoplasma in judgement sample, the results are shown in Table 7, says
Bright isolated cell line is mycoplasma negative cells system.And carry out STR detection verifying cell identity.
For STR testing result as shown in table 4, Fig. 9 and Figure 10, the result of Fig. 9 summarizes such as table 4, shows on locus without third
Position gene and tetra-allelic, and without the pollution of other Human cell lines；By STR testing result input ATCC database and
DSMZ database be compared (its concrete operations be all made of the conventional use of STR detection mode of those skilled in the art and
Process is detected), cell is U-2OS cell line as the result is shown；Sample is human cell line to Figure 10 as the result is shown, and without other
The pollution of Species Cell system.
Table 4
Embodiment 5
The present embodiment provides a kind of methods of mycoplasma contamination in removal MRC-5 cell line, specifically includes the following steps:
(1) MRC-5 cell line to be detected is detected with mycoplasma test reagent box, judges the pollution feelings of mycoplasma in cell line
Condition, the results are shown in Table 6, B/A > 0.9, obtains mycoplasma positive MRC-5 cell line；
(2) by the skin wet at the back leg groin of NSI immunodeficient mouse and disinfection after dipping in alcohol with cotton ball soaked in alcohol,
The mycoplasma positive MRC-5 cell line of the step of drawing 200 μ L with insulin needle (1), with 5 × 105Cell concentration incline 45 degree of angles
It is injected into the subcutaneous of mouse, it is seen that mouse is put back in normal environment and raised 35 days by the lump heaved；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, in Biohazard Safety Equipment, left hand
Lift its tumour surrounding skin with tweezers, the right hand is cut off with eye scissors, sterile to cut transplanting MRC-5 cell line tissue, leaching
It steeps in RPMI complete culture solution, chooses well-grown tissue and shredded, then carry out enzymatic hydrolysis 5min with trypsase, finally
It is cleaned with PBS solution, obtains mycoplasma negative cells system.
The isolated MRC-5 cell line of step (3) is inoculated in the RPMI complete culture solution containing 10%FBS, is trained
Mycoplasma test reagent box test sample is used after supporting 5 days, the pollution condition of mycoplasma in judgement sample, the results are shown in Table 7, says
Bright isolated cell line is mycoplasma negative cells system.And carry out STR detection verifying cell identity.
For STR testing result as shown in table 5, Figure 11 and Figure 12, the result of Figure 11 summarizes such as table 5, shows on locus without three
Allele and tetra-allelic, and without the pollution of other Human cell lines；By STR testing result input ATCC database and
DSMZ database be compared (its concrete operations be all made of the conventional use of STR detection mode of those skilled in the art and
Process is detected), cell is MRC-5 cell line as the result is shown；Sample is human cell line to Figure 12 as the result is shown, and without other
The pollution of Species Cell system.
Table 5
Table 6
Table 7
The side of a kind of promotion of the invention improves the Applicant declares that the present invention is explained by the above embodiments cell state
Method and its application, but the present invention is not limited to the above embodiments, that is, does not mean that the present invention must rely on above-described embodiment
It can implement.It should be clear to those skilled in the art, any improvement in the present invention, to each raw material of product of the present invention
Addition, selection of concrete mode of equivalence replacement and auxiliary element etc., all fall within protection scope of the present invention and the open scope it
It is interior.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
Claims (10)
1. a kind of promote the method for improving cell state, which is characterized in that the method are as follows: transplanted into immunodeficient mouse body
Mycoplasma positive cell line, and mouse is raised, then cell line is isolated out of Mice Body, obtain mycoplasma negative cells system.
2. the method as described in claim 1, which is characterized in that described method includes following steps:
(1) mycoplasma positive cell line is obtained；
(2) the mycoplasma positive cell line of step (1) is implanted into immunodeficient mouse body and is raised；
(3) cell line is isolated in the Mice Body from step (2) after raising, obtains mycoplasma negative cells system.
3. method according to claim 2, which is characterized in that step (1) described cell line includes tumor cell line and is immunized
Cell line；
Preferably, the tumor cell line includes HepG2, Huh7, MDA-MB-231, U-2OS or MRC-5；
Preferably, the immunocyte includes B cell, T cell or NK cell.
4. method as claimed in claim 2 or claim 3, which is characterized in that step (1) side for obtaining mycoplasma positive cell line
Method are as follows: use mycoplasma test reagent box test sample, the pollution condition of mycoplasma in judgement sample obtains the mycoplasma positive with this
Cell line.
5. the method as described in any one of claim 2-4, which is characterized in that the mode of step (2) described transplanting includes skin
Lower transplanting or tail vein injection；
Preferably, the cell line quantity of step (2) described transplanting is 2 × 105-5×106；
Preferably, step (2) immunodeficient mouse is NSI immunodeficient mouse.
6. the method as described in any one of claim 2-5, which is characterized in that the time of step (2) described raising is 20-40
It.
7. the method as described in any one of claim 2-6, which is characterized in that step (3) is described to be isolated out of Mice Body
The method of cell line are as follows: mouse is put to death and body surface sterilizes, it is sterile to cut transplanted cells system tissue, it is soaked in RPMI and cultivates completely
In liquid, then with trypsin digestion, mycoplasma negative cells system is obtained；
Preferably, the mode that the mouse is put to death is cervical dislocation method；
Preferably, described to be shredded tissue with before trypsin digestion；
Preferably, the time of the enzymatic hydrolysis is 3-8min；
Preferably, step (3) method that cell line is isolated out of Mice Body are as follows: mouse is first cooked into deep anesthesia, opens chest
Chamber, exposure heart are pierced into right ventricle with syringe needle, and draw blood is carried out thin using cell separating liquid and/or cell sorting kit
The sorting of born of the same parents obtains mycoplasma negative cells system.
8. the method as described in any one of claim 2-7, which is characterized in that the cell line that judgment step (3) is isolated is
The method of mycoplasma negative cells system are as follows: isolated cell line is inoculated in the RPMI complete culture solution containing 10%FBS
In, mycoplasma test reagent box test sample is used after culture 5-10 days, the pollution condition of mycoplasma in judgement sample, and carry out
STR detection verifying cell identity.
9. the method as described in any one of claim 2-7, which is characterized in that described method includes following steps:
(1) cell line to be detected is detected with mycoplasma test reagent box, judges the pollution condition of mycoplasma in cell line, is obtained with this
Obtain mycoplasma positive cell line；
(2) by the mycoplasma positive cell line of step (1) by subcutaneous transplantation or tail vein injection mode with 2 × 105-5×106
Cell concentration be implanted into NSI immunodeficient mouse body and carry out raising 20-40 days；
(3) the mouse cervical dislocation method after raising in step (2) is put to death and body surface sterilizes, it is sterile to cut transplanted cells system tissue,
It is soaked in RPMI complete culture solution, is shredded, then carry out enzymatic hydrolysis 3-8min with trypsase, finally cleaned with PBS solution,
Obtain mycoplasma negative cells system；Or the mouse after raising in step (2) is cooked into deep anesthesia, thoracic cavity is opened, exposure heart is used
Syringe needle is pierced into right ventricle, and draw blood is carried out the sorting of cell using cell separating liquid and/or cell sorting kit, obtained
Mycoplasma negative cells system.
10. application of the method as claimed in any one of claims 1-9 wherein in removal culture cell in mycoplasma contamination.
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| CN111705040A (en) * | 2020-06-17 | 2020-09-25 | 国药集团动物保健股份有限公司 | Method for removing mycoplasma in pseudorabies virus liquid |
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| CN103409468A (en) * | 2013-03-20 | 2013-11-27 | 中国科学院广州生物医药与健康研究院 | Method for establishing immunodeficiency mouse model |
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