CN108967417B - A kind of tumor tissues high activity storage transport liquid and preparation method thereof - Google Patents
A kind of tumor tissues high activity storage transport liquid and preparation method thereof Download PDFInfo
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- CN108967417B CN108967417B CN201810901395.0A CN201810901395A CN108967417B CN 108967417 B CN108967417 B CN 108967417B CN 201810901395 A CN201810901395 A CN 201810901395A CN 108967417 B CN108967417 B CN 108967417B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
Abstract
The invention discloses a kind of tumor tissues high activities to save transport liquid and preparation method thereof, ingredient includes: DMEM culture solution and antibiotic, also contain amino acid in preservation transport liquid, growth factor, insulin and a certain amount of sorbierite, tissue morphology can be made to keep preferable, making the preservation liquid of preparation has anticoagulation well, and it can guarantee that the activity of tumor tissues is higher, extend the holding time of tumor tissues in vitro, in the present invention and by controlling the various concrete components and concrete content being divided into, it plays a very good protection to tumor tissues, preservation transport liquid is able to maintain that its morphosis and bioactivity, extend the holding time of tumor tissues in vitro, it can better ensure that the survival rate of tumor tissues originally culture.
Description
Technical field
The present invention relates to field of biotechnology, store transport liquid and its preparation more particularly to a kind of tumor tissues high activity
Method.
Background technique
Storing liquid will keep the bioactivity of cell to meet its application clinically, therefore, how maintain cell
It is crucial that high activity becomes treatment.Before, people use physiological saline or glucose as protection liquid, but due to lacking nutrition, molten
The tension of liquid waits penetrations the reasons such as to have differences, and causes cell to occur vigor in long-term preservation or transport low, a large amount of
Phenomena such as swelling is dead.Then, the composition system that people constantly improve storing liquid is added to KCl, sodium citrate, albumin, dimension
Raw element etc., forms and saves liquid.Although these save liquid and maintain cell activity well in a long time, constituent is multiple
Miscellaneous, process for preparation is cumbersome.
Currently, the external storing liquid of common tumor tissues is phosphate buffered saline solution (Phosphate Buffer
Saline, PBS) and cell culture medium.Wherein the main component of PBS buffer solution includes disodium hydrogen phosphate, sodium dihydrogen phosphate, chlorination
Sodium and potassium chloride etc. have salt balance, adjustable suitable pH buffer function；The main component of cell culture medium includes basis
Culture medium, cow's serum, antibiotic etc. can be used for maintaining the growing multiplication of cell.But it finds in actual use, PBS
Buffer components are simple, but function is limited to, cannot outer tumor tissues of keeping body well bioactivity；Cell culture and master
To be used to that the growing multiplication of cell is maintained to use PBS and cell culture medium not as the optimal selection of tumor tissues storing liquid
When as tumour cell storing liquid, retain the limited activity of tumour cell, so that the success rate for causing tumor tissues to transplant is very low.
To solve the above-mentioned problems, a kind of tumor tissues are disclosed in Chinese patent application 201710175519.7 to protect in vitro
Liquid storage and preparation method thereof, which comprises antibiotic is added in basal medium and obtains solution A；In natural medium
Middle dissolution taurine adds Triton X-100 and obtains B liquid, and the B liquid is added in the A liquid and is mixed,
The tumor tissues storage in vitro liquid is obtained, the storage in vitro liquid prepared by the invention the method is able to maintain that tumor group
It is woven in bioactivity when storage in vitro, with extending tumor tissues the storage in vitro time, improves the success rate of tumour transplatation.
Current oncotherapy is still the huge difficult problem that medical field faces, and the drug being applicable in by different tumour cells is not
Together, more targetedly treatment tumour, doctor need to screen, pair most advantageous to patient when to different tumor patient medications
Drug small and with targeting is acted on, therefore needs to cut the small sample that length is 0.5-1.5 centimetres in the course of surgery
Or micro- sample is taken out in preoperative puncture, is put into and is saved in liquid after flushing, under the conditions of certain temperature, cell is transported in certain time
Mechanism is cultivated, originally culture is carried out to tumour cell, obtains primary tumor cell strain, for screening to the most effective medicine of patient
Object.Therefore need to prepare a kind of tissue preserration transport liquid, it is ensured that cell or tissue kept in long-time transportational process compared with
High activity guarantees that cell has higher survival rate in incubation.
Such as Chinese patent application discloses a kind of cell-preservation liquid, including following substance: albumin, low molecular weight heparin
Sodium, trehalose and physiological saline.Beneficial effects of the present invention: cell-preservation liquid of the present invention, wherein used white egg
The white viscosity and osmotic pressure that can maintain solution appropriateness, while cellular adhesion can be prevented, maintain cell bio-activity.Heparin sodium
It can prevent fibrin from generating, further prevent cellular adhesion.And trehalose is a kind of non-specific natural protective agent, is had
The effect for protecting biological tissue, cell and large biological molecule can be very good to guarantee cell in the storage and transport of long period
Bioactivity, therefore, cell-preservation liquid of the invention plays excellent maintenance effect to the vigor and form of cell, is suitable for
Long period storage and transport maintain its bioactivity.The cell saved in this application is mesenchymal cell and central memory-type T
Cell, for the storage and transport of tumour cell, there is no any descriptions.
Tumour cell environmental requirement and normal cell different from, tumour cell save liquid disadvantage and are to save liquid to tumour
The bioactivity-protected ideal not enough of cell, saves cell survival rate and is not up to satisfactory effect.In order to adapt to clinic
Needs, develop it is a kind of adapt to for a long time transport and improve tumour cell save motility rate preservation liquid be very necessary.
Summary of the invention
For above-mentioned there are technical problem, the invention proposes a kind of tumor tissues high activities to save transport liquid, to tumour
That organizes is bioactivity-protected preferable, and preservation cell survival rate is higher, adapts to transport for a long time.The present invention also provides one kind
Tumor tissues save the preparation method of transport liquid.
A kind of tumor tissues high activity preservation transport liquid, comprising: DMEM culture solution and antibiotic.
The antibiotic includes gentamicin；The antibiotic further includes one in amphotericin B or nystatin
Kind or two kinds；The main function of antibiotic is restraining and sterilizing bacteria effect, kills the microorganism that tumor tissues may carry, and then prevent
The microorganism infections such as bacterium, fungi, to guarantee the success of tumor cell culture.
The tumor tissues high activity saves transport liquid, further includes amino acid, growth factor and insulin.
The amino acid can be selected from least two in γ-aminobutyric acid, proline and seromycin；
Preferably, the amino acid can be selected from γ-aminobutyric acid and seromycin.
The addition concentration ratio of the γ-aminobutyric acid, seromycin and proline is 1-2: 1: 1-2.
The growth factor may be selected from Porcine HGF, and the Porcine HGF is epithelical cell growth factor
(EGF) and/or fiber mother cell growth factor (FGF).
The tumor tissues high activity saves transport liquid, further includes sorbierite.
γ-aminobutyric acid, alias 4-Aminobutanoicacid, nipecotic acid are a kind of active skull cap components, may participate in a variety of metabolism and live
It is dynamic, physiological activity with higher.
Proline, chemical name are 2-pyrrolidone-5-carboxylic acid, it is a kind of cricoid imino acid, and proline is the non-of human body
Essential amino acid, in vivo, proline are not only ideal osmotic adjustment, and are alternatively arranged as the guarantor of film and enzyme
Substance and free radical scavenger are protected, to play a protective role to growth of the plant under osmotic stress, for potassium ion biology
Another the internal accumulation of important osmotic adjustment in vacuole, proline can play flat to cytoplasm infiltration again
The adjustment effect of weighing apparatus
Seromycin, scientific name " dextrorotation -4- amino -3- tetrahydro isoxazolone ", molecular formula C3H6N2O2, molecular weight
102.09 white or light yellow crystalline powder.Antibiotics, in addition to anti-mycobacterium tuberculosis, to gram-positive bacteria, feminine gender
Bacterium, rickettsia bacterium also have inhibiting effect.
The DMEM culture solution is 84.8-93.5% saving the percent by volume in liquid, be specifically as follows 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92% or 93%.
The concentration of the gentamicin is 5-15mg/L；Preferably, the concentration of the gentamicin is 6-14mg/L；
More preferably, the concentration of the gentamicin is 7-13mg/L；It is further preferred that the concentration of the gentamicin is 8-
12mg/L；Still further preferably, the concentration of the gentamicin is 9-11mg/L；Still further preferably, the celebrating
The concentration of big mycin is 10-11mg/L.
The concentration of the amphotericin B is 0.5-2mg/L；Preferably, the concentration of the amphotericin B is 0.6-
1.9mg/L；More preferably, the concentration of the amphotericin B is 0.7-1.8mg/L；It is further preferred that the both sexes are mould
The concentration of plain B is 0.8-1.7mg/L；Still further preferably, the concentration of the amphotericin B is 0.9-1.6mg/L；Again into
Preferably, the concentration of the amphotericin B is 1.0-1.5mg/L to one step；Still further preferably, the amphotericin B
Concentration be 1.1-1.4mg/L；Still further preferably, the concentration of the amphotericin B is 1.2-1.3mg/L.
The concentration of the nystatin is 50-100mg/L；Preferably, the concentration of the nystatin is 60-
90mg/L；More preferably, the concentration of the nystatin is 70-80mg/L.
The concentration of the γ-aminobutyric acid is 20-50mg/L；Preferably, the concentration of the γ-aminobutyric acid is
25-45mg/L；More preferably, the concentration of the γ-aminobutyric acid is 30-40mg/L；It is further preferred that the γ-
The concentration of aminobutyric acid is 35-40mg/L.
The concentration of the proline is 20-50mg/L；Preferably, the concentration of the proline is 25-45mg/L；Again
Preferably, the concentration of the proline is 30-40mg/L；It is further preferred that the concentration of the proline is 35-
40mg/L。
The concentration of the seromycin is 20-50mg/L；Preferably, the concentration of the seromycin is 25-45mg/
L；More preferably, the concentration of the seromycin is 30-40mg/L.
The concentration of the epithelical cell growth factor (EGF) is 10-20 μ g/L；Preferably, the epidermal cell is raw
The concentration of the long factor (EGF) is 12-18 μ g/L；More preferably, the concentration of the epithelical cell growth factor (EGF) is 13-
17μg/L；It is further preferred that the concentration of the epithelical cell growth factor (EGF) is 14-16 μ g/L；Still further preferably
Ground, the concentration of the epithelical cell growth factor (EGF) are 15-16 μ g/L.
The concentration of the fiber mother cell growth factor (FGF) is 10-20 μ g/L；Preferably, the fiber is female thin
The concentration of the intracellular growth factor (FGF) is 12-18 μ g/L；More preferably, the concentration of the fiber mother cell growth factor (FGF)
For 13-17 μ g/L；It is further preferred that the concentration of the fiber mother cell growth factor (FGF) is 14-16 μ g/L；Again into
Preferably, the concentration of the fiber mother cell growth factor (FGF) is 15-16 μ g/L to one step.
The concentration of the insulin is 1-5mg/L；Preferably, the concentration of the insulin is 2-4mg/L；Further preferably
Ground, the concentration of the insulin are 2-3mg/L.
The concentration of the sorbierite is 10-50g/L；Preferably, the concentration of the sorbierite is 20-40g/L；Further preferably
Ground, the concentration of the sorbierite are 20-30g/L.
The concentration ratio of the insulin and the sorbierite is 1: 2000-40000；Preferably, the insulin
Concentration ratio with the sorbierite is 1: 5000-30000；More preferably, the insulin and the sorbierite is dense
Degree is than being 1: 8000-20000；More preferably, the concentration ratio of the insulin and the sorbierite is 1: 10000-
20000。
Above-mentioned tumor tissues save the preparation method of transport liquid, specifically includes the following steps:
(1) DMEM culture solution is divided into two parts first；
(2) it takes and antibiotic, amino acid and growth factor is added in half DMEM culture solution, obtain culture solution A；
(3) it takes the other half DMEM culture solution that sorbierite and insulin is added, obtains culture solution B；
(4) culture solution B obtained in culture solution A obtained in step (2) and step (3) is uniformly mixed to get height is arrived
Active tumor tissues save transport liquid.
Tumor tissues are stored in the tumor tissues high activity preservation transport liquid the present invention also provides a kind of
Method, comprising the following steps:
S1, liquid is transported firstly, tumor tissues high activity is saved, is filtered with the filter that partial size is 0.2 micron, degerming,
Obtain filtered fluid；
S2, filtered fluid obtained in step S1 is sub-packed in 15 milliliters of aseptic plastic pipe, every pipe contains 6 milli of filtered fluid
It rises, liquid is transported in the preservation after being dispensed；
S3, liquid long-term preservation under -20 degrees celsius is transported into the preservation after dispensing in step S2, is taken the photograph using preceding 4
It thaws, and is stored in 4 degrees celsius until being transported using to get to the preservation of workable tumor tissues under the conditions of family name's degree
Liquid；
S4, by fresh surgical or after puncturing sample sterile PBS flushing, it is put into the guarantor of tumor tissues obtained in step S3
It deposits in transport liquid, cold chain was transported to inspection center in 48 hours, completed the preservation transport of tumor tissues.
Beneficial effects of the present invention:
(1) tumor tissues high activity of the present invention saves transport liquid, used in γ-aminobutyric acid be a kind of day
Right active constituent can protect the activity of tumor tissues well, it is ensured that tumour cell during transportation still can be with
Activity with higher.
(2) present invention joined sorbierite and insulin in the preservation transport liquid of tumor tissues, and research finds sorbierite
Addition with insulin can make tissue morphology that the preservation liquid for preferably making preparation be kept to have anticoagulation well, and can protect
The activity for demonstrate,proving tumor tissues is higher, extends the holding time of tumor tissues in vitro.
(3) it is gentamicin that tumor tissues disclosed by the invention, which save the antibiotic being added in transport liquid, is then added again
One or both of amphotericin B or nystatin.The main function of antibiotic is restraining and sterilizing bacteria effect, kills tumor group
The microorganism that may be carried is knitted, and then prevents the microorganism infections such as bacterium, fungi, to guarantee the success of tumor cell culture.
(4) tumor tissues disclosed by the invention, which save in transport liquid, contains DMEM culture solution, antibiotic, amino acid, growth
The factor and sorbierite and insulin, and by controlling the various concrete components and concrete content being divided into, tumor tissues are risen
To good protective effect, preservation transport liquid is able to maintain that its morphosis and bioactivity, extends tumor tissues in vitro
Holding time, can better ensure that the survival rate of tumor tissues originally culture.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, rather than whole embodiments.Based on the reality in the present invention
Apply example, those of ordinary skill in the art's every other embodiment obtained under the premise of not making creative labor, all
Belong to the scope of protection of the invention.
Breast tumor tissue used in following embodiment and lung cancer tumor tissue are derived from Yinchuan of Ningxia Province, and Shanghai is opened
In 30 breast cancer and 10 patients with lung cancer bodies on the ground such as the members of a family, Beijing and Shanxi, frozen section report turns out to be mammary gland in art
Cancer or lung cancer.
A kind of tumor tissues high activity of embodiment 1-3 saves transport liquid
Table 1
Tumor tissues described in above-described embodiment 1-3 save the preparation method of transport liquid, specifically includes the following steps:
(1) DMEM culture solution is divided into two parts first；
(2) it takes and antibiotic, amino acid and growth factor is added in half DMEM culture solution, obtain culture solution A；
(3) it takes the other half DMEM culture solution that sorbierite and insulin is added, obtains culture solution B；
(4) culture solution B obtained in culture solution A obtained in step (2) and step (3) is uniformly mixed to get height is arrived
Active tumor tissues save transport liquid.
By tumor tissues be stored in the tumor tissues high activity save method in transport liquid the following steps are included:
S1, liquid is transported firstly, tumor tissues high activity is saved, is filtered with the filter that partial size is 0.2 micron, degerming,
Obtain filtered fluid；
S2, filtered fluid obtained in step S1 is sub-packed in 15 milliliters of aseptic plastic pipe, every pipe contains 6 milli of filtered fluid
It rises, liquid is transported in the preservation after being dispensed；
S3, liquid long-term preservation under -20 degrees celsius is transported into the preservation after dispensing in step S2, is taken the photograph using preceding 4
It thaws, and is stored in 4 degrees celsius until being transported using to get to the preservation of workable tumor tissues under the conditions of family name's degree
Liquid；
S4, by fresh surgical or after puncturing sample sterile PBS flushing, it is put into the guarantor of tumor tissues obtained in step S3
It deposits in transport liquid, cold chain was transported to inspection center in 48 hours, completed the preservation transport of tumor tissues.
Test example
By the PBS buffer solution of same volume, DMEM cell culture fluid and the present invention according to embodiment 1-3 disclose content at
The tumor tissues preservation transport liquid of preparation is divided to be divided into 5 groups of every group of 100 pipes as preservation transport liquid preservation transport tumor tissues, transport
Time is 6 hours, the weight such as tumor tissues saved.
The tumor tissues transported are 5 parts from same human breast carcinoma tumor tissues, cultivate 2-3 after transporting to destination
The culture situation of tumour cell is observed after week, and its survival rate is compared, and specific data see the table below 2
It should be pointed out that other conditions are all the same other than the above-mentioned difference pointed out.
Table 2 utilizes the various transports for saving transport liquid and carrying out breast tumor tissue
As shown in Table 2, breast tumor tissue holding time in saving transport liquid is 6 hours.As a result utilizing PBS
Culture 2-3 weeks is carried out after the transport of buffer, breast cancer tumor cells strain survival rate is 54%, is transported using DMEM cell culture fluid
Culture 2-3 weeks is carried out after defeated, the survival rate of breast cancer tumor cells strain is 65%, and three kinds of preservations provided by the invention is utilized to transport
Culture 2-3 weeks is carried out after infusion transport, the survival rate of breast cancer tumor cells strain is to be up to 98%, 100%, 99% respectively, because
The survival rate of the different preservation transport liquid of this tri- kinds of component content of embodiment 1-3 provided by the invention is all remarkably higher than first two guarantor
Liquid storage.
In order to further prove the transport preservation provided by the invention for saving transport liquid and being suitable for kinds of tumors tissue, by it
For the transport of lung cancer tumor tissue, by the PBS buffer solution of same volume, DMEM cell culture fluid and the present invention according to embodiment
The tumor tissues preservation transport liquid that 1-3 discloses the ingredient preparation of content is divided into 5 groups of every group of 100 pipes as preservation transport liquid preservation fortune
Defeated tumor tissues, haulage time are 6 hours, the weight such as tumor tissues saved.
The tumor tissues transported are 5 parts from same human lung cancer tumor tissues, are cultivated 2-3 weeks after transporting to destination
The culture situation of tumour cell is observed afterwards, and its survival rate is compared, and specific data see the table below 3
It should be pointed out that other conditions are all the same other than the above-mentioned difference pointed out.
Table 3 utilizes the various transports for saving transport liquid and carrying out lung cancer tumor tissue
As shown in Table 3, lung cancer tumor group be woven in preservation transport liquid in the holding time be 6 hours.As a result slow using PBS
Culture 2-3 weeks is carried out after fliud flushing transport, lung cancer tumor cell strain survival rate is 50%, is transported using DMEM cell culture fluid laggard
Row culture 2-3 weeks, the survival rate of lung cancer tumor cell strain is 58%, and three kinds of preservations provided by the invention transport liquid is utilized to transport
After carry out culture 2-3 week, the survival rate of lung cancer tumor cell strain respectively up to 95%, 93%, 95% is therefore provided by the invention
The survival rate of the different preservation transport liquid of tri- kinds of component contents of embodiment 1-3 is all remarkably higher than first two and saves liquid.
Preservation transport liquid prepared by the present invention, which can be apparent from, by several groups of data provided in table 2-3 can be used for a variety of swell
The preservation of tumor tissue is transported, and can guarantee cell 6 hours breast carcinoma cell strains survival rate 98% or more, lung cancer is thin
The survival rate of born of the same parents' strain is 95% or more.
A kind of tumor tissues high activity of embodiment 4-6 saves transport liquid
Table 4
Preparation method: in addition to ingredient is different, other operations and step are identical as embodiment 1-3.
Transportation resources: identical as embodiment 1-3.
Test example
By the PBS buffer solution of same volume, DMEM cell culture fluid and the present invention according to embodiment 4-6 disclose content at
The tumor tissues preservation transport liquid of preparation is divided to be divided into 5 groups of every group of 100 pipes as preservation transport liquid preservation transport tumor tissues, transport
Time is 12 hours, the weight such as tumor tissues saved.
Used tumor tissues are 5 parts from same human breast carcinoma tumor tissues, cultivate 2-3 after transporting to destination
The culture situation of tumour cell is observed after week, and its survival rate is compared, and specific data see the table below 5
Table 5 utilizes the various transports for saving transport liquid and carrying out breast tumor tissue
Ibid as shown in Table 5, breast cancer tumour holding time in saving transport liquid is 12 hours.As a result utilizing
Culture 2-3 weeks is carried out after the transport of PBS buffer solution, the survival rate of breast cancer tumor cells strain is 48%, is trained using DMEM cell
Culture 2-3 weeks is carried out after nutrient solution transport, the survival rate of breast cancer tumor cells strain is 52%, and utilizes three kinds provided by the invention
Culture 2-3 weeks is carried out after saving transport liquid transport, the survival rate of breast cancer tumor cells strain is up to 95%, 97%, 96% respectively,
Therefore the survival rate of the different preservation transport liquid of tri- kinds of component contents of embodiment 4-6 provided by the invention is all remarkably higher than first two
Save liquid.
In order to further prove the transport preservation provided by the invention for saving transport liquid and being suitable for kinds of tumors tissue, by it
For the transport of lung cancer tumor tissue, by the PBS buffer solution of same volume, DMEM cell culture fluid and the present invention according to embodiment
The tumor tissues preservation transport liquid that 4-6 discloses the ingredient preparation of content is divided into 5 groups of every group of 100 pipes as preservation transport liquid preservation fortune
Defeated tumor tissues, haulage time are 12 hours, the weight such as tumor tissues saved.
The tumor tissues transported are 5 parts from same human lung cancer tumor tissues, are cultivated 2-3 weeks after transporting to destination
The culture situation of tumour cell is observed afterwards, and its survival rate is compared, and specific data see the table below 6
It should be pointed out that other conditions are all the same other than the above-mentioned difference pointed out.
Table 6 utilizes the various transports for saving transport liquid and carrying out lung cancer tumor tissue
As shown in Table 6, lung cancer tumor holding time in saving transport liquid is 12 hours.As a result being buffered using PBS
Culture 2-3 weeks is carried out after the transport of liquid, the survival rate of lung cancer tumor cell strain is 36%, after the transport of DMEM cell culture fluid
Culture 2-3 weeks is carried out, lung cancer tumor cell strain survival rate is 41%, and three kinds of preservations provided by the invention transport liquid is utilized to transport
After carry out culture 2-3 week, lung cancer tumor cell strain survival rate respectively up to 93%, 94%, 93%, therefore reality provided by the invention
The survival rate for applying the different preservation transport liquid of tri- kinds of component contents of a 4-6 is all remarkably higher than first two and saves liquid.
Preservation transport liquid prepared by the present invention, which can be apparent from, by several groups of data provided in table 5-6 can be used for a variety of swell
The preservation of oncocyte is transported, and can guarantee cell 12 hours breast carcinoma cell strains survival rate 95% or more, lung cancer
The survival rate of cell strain is 93% or more.
A kind of tumor tissues high activity of embodiment 7-9 saves transport liquid
Table 7
Preparation method: in addition to ingredient is different, other operations and step are identical as embodiment 1-3.
Transportation resources: identical as embodiment 1-3.
Test example
By the PBS buffer solution of same volume, DMEM cell culture fluid and the present invention according to embodiment 7-9 disclose content at
The tumor tissues preservation transport liquid of preparation is divided to be divided into 5 groups of every group of 100 pipes as preservation transport liquid preservation transport tumor tissues, transport
Time is 48 hours, the weight such as tumor tissues saved.
The tumor tissues transported are 5 parts from same human breast carcinoma tumor tissues, cultivate 2-3 after transporting to destination
The culture situation of tumour cell is observed after week, and its survival rate is compared, and specific data see the table below 8
Table 8 utilizes the various transports for saving transport liquid and carrying out breast tumor tissue
As shown in Table 8, breast cancer tumour holding time in saving transport liquid is within 48 hours.As a result utilizing
Culture 2-3 weeks is carried out after the transport of PBS buffer solution, the survival rate of breast cancer tumor cells strain is 24%, is trained using DMEM cell
Culture 2-3 weeks is carried out after nutrient solution transport, the survival rate of breast cancer tumor cells strain is 36%, and utilizes three kinds provided by the invention
Culture 2-3 weeks is carried out after saving transport liquid transport, the survival rate of breast cancer tumor cells strain is up to 94%, 96%, 95% respectively,
Therefore the survival rate of the different preservation transport liquid of tri- kinds of component contents of embodiment 7-9 provided by the invention is all remarkably higher than first two
Save liquid.
In order to further prove the transport preservation provided by the invention for saving transport liquid and being suitable for kinds of tumors tissue, by it
For the transport of lung cancer tumor cell, by the PBS buffer solution of same volume, DMEM cell culture fluid and the present invention according to embodiment
The tumor tissues preservation transport liquid that 7-9 discloses the ingredient preparation of content is divided into 5 groups of every group of 100 pipes as preservation transport liquid preservation fortune
Defeated tumor tissues, haulage time are 48 hours, the weight such as tumor tissues saved.
The tumor tissues transported are 5 parts from same human lung cancer tumor tissues, are cultivated 2-3 weeks after transporting to destination
The culture situation of tumour cell is observed afterwards, and its survival rate is compared, and specific data see the table below 9
It should be pointed out that other conditions are all the same other than the above-mentioned difference pointed out.
Table 9 utilizes the various transports for saving transport liquid and carrying out lung cancer tumor tissue
As shown in Table 9, lung cancer tumor group be woven in preservation transport liquid in the holding time be 48 hours within.As a result utilizing
Culture 2-3 weeks is carried out after the transport of PBS buffer solution, the survival rate of lung cancer tumor cell strain is 22%, utilizes DMEM cell culture
Culture 2-3 weeks is carried out after liquid transport, the survival rate of lung cancer tumor cell strain is 34%, and utilizes three kinds provided by the invention preservations
Culture 2-3 weeks is carried out after transport liquid transport, the survival rate of lung cancer tumor cell strain is up to 92%, 90%, 92%, therefore this respectively
The survival rate for inventing the different preservation transport liquid of the tri- kinds of component contents of embodiment 7-9 provided is all remarkably higher than first two preservation
Liquid.
Preservation transport liquid prepared by the present invention, which can be apparent from, by several groups of data provided in table 8-9 can be used for a variety of swell
The preservation of oncocyte is transported, and can guarantee cell 12 hours breast carcinoma cell strains survival rate 94% or more, lung cancer
The survival rate of cell strain is 92% or more.
It can be seen that from above-mentioned testing result and save transport liquid using tumor tissues provided by the invention, it is thin to carry out tumour
The preservation of born of the same parents, even if transport (within 48 hours) tumour cell can still keep very high survival rate for a long time.
A kind of tumor tissues high activity of comparative example 1-3 saves transport liquid
Table 10
Above-mentioned comparative example 1-3 and the difference of embodiment 7 are: without adding gentamicin, comparison in comparative example 1
Without adding γ-aminobutyric acid, seromycin and proline in embodiment 2, the concentration of sorbierite and insulin in comparative example 3
Than being 50000: 1, γ-aminobutyric acid in comparative example 4, the concentration ratio of seromycin and proline is 4: 1: 12.
Test example
The same volume present invention is disclosed to the tumor group of the ingredient preparation of content according to embodiment 7 and comparative example 1-4
It knits preservation transport liquid and is divided into 5 groups of every group of 100 pipes as transport liquid is saved and save transport tumor tissues, haulage time is 48 hours,
The weight such as tumor tissues saved.
The tumor tissues transported are 5 parts from same human breast carcinoma tumor tissues, cultivate 2-3 after transporting to destination
The culture situation of tumour cell is observed after week, and its survival rate is compared, and specific data see the table below 11
Table 11 utilizes the various transports for saving transport liquid and carrying out breast cancer tumor cells
As shown in Table 11, breast tumor tissue holding time in saving transport liquid is within 48 hours.As a result
Culture 2-3 weeks, breast cancer are carried out after saving the transport of transport liquid using the tumor tissues of the ingredient preparation of the open content of embodiment 7
The survival rate of tumor cell line is 92%, is cultivated after the three kinds of preservations transport liquid transport provided using comparative example 1-4
2-3 weeks, the survival rate of breast cancer tumor cells strain was respectively 70%, 72%, 81%, 85%, therefore tri- kinds of comparative example 1-4
The survival rate of the different preservation transport liquid of component content is substantially less than the tumor tissues of the ingredient preparation of the open content of embodiment 7
Save transport liquid.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (4)
1. a kind of tumor tissues high activity saves transport liquid, it is characterised in that: the tumor tissues high activity saves transport liquid packet
Containing following component:
DMEM nutrient solution volume percentage is 84.8-93.5%；
Antibiotic 1.2-100mg/L；
Amino acid 30-80mg/L；
Growth factor 15-35 μ g/L；
Insulin 1-5mg/L；
It further includes the sorbierite that concentration is 10-50g/L that the tumor tissues high activity, which saves transport liquid,；
The antibiotic includes gentamicin；The antibiotic also include one of amphotericin B and nystatin or
Two kinds；
The growth factor is Porcine HGF, and the Porcine HGF is epithelical cell growth factor (EGF) and/or fibre
It ties up mother cell growth factor (FGF)；
The amino acid is concentration than for the γ-aminobutyric acid of 1-2:1:1-2, seromycin and proline.
2. tumor tissues high activity according to claim 1 saves transport liquid, it is characterised in that: the DMEM culture solution
It is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% or 93% saving the percent by volume in liquid.
3. the preparation method that tumor tissues high activity according to claim 1 or 2 saves transport liquid, it is characterised in that: tool
Body the following steps are included:
(1) DMEM culture solution is divided into two parts first；
(2) it takes and antibiotic, amino acid and growth factor is added in half DMEM culture solution, obtain culture solution A；
(3) it takes the other half DMEM culture solution that sorbierite and insulin is added, obtains culture solution B；
(4) culture solution B obtained in culture solution A obtained in step (2) and step (3) is uniformly mixed to get high activity is arrived
Tumor tissues save transport liquid.
4. tumor tissues high activity according to claim 1 or 2 saves transport liquid, it is characterised in that: protect tumor tissues
Be stored in the tumor tissues high activity save method in transport liquid the following steps are included:
S1, liquid is transported firstly, tumor tissues high activity is saved, is filtered with the filter that partial size is 0.2 micron, degerming obtains
Filtered fluid；
S2, filtered fluid obtained in step S1 being sub-packed in 15 milliliters of aseptic plastic pipe, every pipe contains 6 milliliters of filtered fluid,
Liquid is transported in preservation after being dispensed；
S3, the preservation transport liquid in step S2 after packing is saved under -20 degrees celsius, using preceding in 4 degrees celsius
Lower defrosting, and be stored in 4 degrees celsius until use saves transport liquid to get to workable tumor tissues；
S4, by fresh surgical or after puncturing sample sterile PBS flushing, tumor tissues obtained in step S3 is put into and save fortune
In infusion, cold chain was transported to inspection center in 48 hours, completed the preservation transport of tumor tissues.
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| CN111838134A (en) * | 2020-07-31 | 2020-10-30 | 江苏莱森生物科技研究院有限公司 | Tumor tissue transfer liquid and application |
| CN112586495B (en) * | 2020-12-31 | 2022-05-17 | 广州市达瑞生物技术股份有限公司 | Tumor tissue preservation solution and application |
| CN112841168B (en) * | 2020-12-31 | 2021-12-10 | 创芯国际生物科技(广州)有限公司 | Tissue preservation solution and application thereof |
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