CN106479891A - Colorectal cancer primitive cell culture kit and its application process - Google Patents
Colorectal cancer primitive cell culture kit and its application process Download PDFInfo
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- CN106479891A CN106479891A CN201611185967.7A CN201611185967A CN106479891A CN 106479891 A CN106479891 A CN 106479891A CN 201611185967 A CN201611185967 A CN 201611185967A CN 106479891 A CN106479891 A CN 106479891A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Abstract
The invention discloses a kind of colorectal cancer primitive cell culture kit and its application process, belong to medical kit technical field.The kit includes the first reagent, the second reagent, the 3rd reagent, the 4th reagent and the 5th reagent, and first reagent, the second reagent, the 3rd reagent, the 4th reagent, the 5th reagent are individually encapsulated.The method applies first reagent, the second reagent, the 3rd reagent, the 4th reagent and the 5th reagent successively, in application process, need not repeat to prepare each reagent, therefore, it is possible to cause operating process to be simplified, so as to, which can not only reduce or even avoid the pollution in application process, additionally it is possible to improve the culture efficiency of colorectal cancer primary cell.
Description
Technical field
The present invention relates to medical kit technical field, more particularly to a kind of colorectal cancer primitive cell culture kit and
Its application process.
Background technology
Colorectal cancer is the malignant tumour for seriously threatening human health, and China incidence of disease to rise most fast alimentary canal pernicious
Tumour, its M ＆ M all come the front three of cancer spectrum.Primitive cell culture technology is to set up colorectal cancer to grind in vitro
Study carefully model, and then the effective means of research colorectal cancer, while be also the steps necessary for isolating and purifying colorectal cancer cell lines, thus right
There is particularly important meaning in the research of colorectal cancer.
But, totally apparently, the success rate for directly carrying out primitive cell culture to colorectal cancer is relatively low, only 9%-30%.
In prior art, the factor of colorectal cancer primitive cell culture success rate is affected mainly to include：
First, in enteron aisle, bacterium is enriched, and specimens of colorectal easily pollutes, and needs Multiple Classes of Antibiotics and disinfectant to locate repeatedly
Reason, sterilization process are very loaded down with trivial details, and effect is difficult to ensure that.
Secondly, the process for specimens of colorectal lacks the flow process of simple and direct, standard, histiocytic separation and digestion process
Lack effective method, cause culture success ratio low.
3rd, the composition of primary culture medium directly determines the height of success rate, and primitive cell culture base needs interpolation many
Hormone, cell factor and other nutriments is planted, to ensure the growing multiplication of tumour cell.Or traditional method composition is not
Foot, cell growth are slow；Formula is complicated, and configuration process is loaded down with trivial details.
Content of the invention
In view of this, the invention provides a kind of colorectal cancer primitive cell culture kit and its application process, which can
The success rate of colorectal cancer primitive cell culture is improved, thus more suitable for practicality.
In order to reach above-mentioned first purpose, the technical scheme of the colorectal cancer primitive cell culture kit that the present invention is provided
As follows：
The colorectal cancer primitive cell culture kit that the present invention is provided include the first reagent, the second reagent, the 3rd reagent, the
Four reagents and the 5th reagent,
Active ingredient in first reagent includes chelated iodine, and the mass concentration of its available iodine is 5000mg/L；
Active ingredient and substance withdrawl syndrome in second reagent includes：EDTA, 0.5mmol/L；NaCl,
137mmol/L；KCl, 2.7mmol/L；Na2HPO4·12H2O, 10mmol/L；K2HPO4, 1.76mmol/L
Active ingredient in 3rd reagent includes NaClO, the span of its weight/mass percentage composition is 8%～
12%；
Active ingredient and mass concentration in 4th reagent includes：Clostridiopetidase A I, 10mg/mL；Clostridiopetidase A II, 10mg/
mL；Clostridiopetidase A IV, 5mg/L；
Active ingredient and content in 5th reagent includes：EGF, 5 μ g；Y27632,1.6mg；Hydrocortisone,
250μg；Cholera toxin, 4.2 μ g；PBS solution：5mL；
First reagent, the second reagent, the 3rd reagent, the 4th reagent, the 5th reagent are individually encapsulated, wherein, institute
It is 200mL: 200mL: 20mL to state the first reagent, the second reagent, the 3rd reagent, the 4th reagent, the volume parts ratio of the 5th reagent:
5mL∶5mL.
The colorectal cancer primitive cell culture kit that the present invention is provided can also be applied to the following technical measures to achieve further.
Preferably, the preservation condition of first reagent includes normal temperature, lucifuge.
Preferably, the preservation condition of second reagent includes normal temperature.
Preferably, the preservation condition of the 3rd reagent includes normal temperature, lucifuge.
Preferably, the preservation condition of the 4th reagent preserves 0.5 month～1.5 at a temperature of including 0 DEG C～4 DEG C
Month.
Preferably, the preservation condition of the 5th reagent preserves 0.5 month～1.5 at a temperature of including 0 DEG C～4 DEG C
Month.
Preferably, the colorectal cancer primitive cell culture kit also include the first packaging container, the second packaging container,
3rd packaging container, the 4th packaging container, the 5th packaging container and chuck,
First packaging container is used for encapsulating first reagent, and second packaging container is used for encapsulating described second
Reagent, the 3rd packaging container are used for encapsulating the 3rd reagent, and the 4th packaging container is used for encapsulating the 4th examination
Agent, the 5th packaging container are used for encapsulating the 5th reagent；
The chuck includes chuck body, at least provided with first through hole, the second through hole, threeway in the chuck body
Hole, fourth hole and fifth hole；
First packaging container can be arranged in the first through hole, and second packaging container can be arranged in institute
State in the second through hole, the 3rd packaging container can be arranged in the third through-hole, the 4th packaging container can be worn
In the fourth hole, the 5th packaging container can be arranged in the fifth hole.
Preferably,
First packaging container is coordinated with the first through hole gap；And/or,
Second packaging container is coordinated with second via clearance；And/or,
3rd packaging container is coordinated with the third through-hole gap；And/or,
4th packaging container is coordinated with the fourth hole gap；And/or,
5th packaging container is coordinated with the fifth hole gap.
Preferably, first packaging container and/or the second packaging container and/or the 3rd packaging container and/or the 4th
The bottom surface of packaging container and/or the 5th packaging container is in arc-shaped transition.
Preferably, the colorectal cancer primitive cell culture kit also includes packing box, the packing box includes box body,
The shape of cross section of the box body is adapted with the shape of the chuck.
Preferably, the packing box also includes lid, the upper lid can cover the box body.
Preferably, the upper lid is split-type structural with the box body.
Preferably, hinged between the upper lid and the box body.
Preferably, the box body is provided with the sub- end of a buckle structure, described on cover and be provided with the buckle structure
Female end, the sub- end are adapted with the female end.
Preferably, the shape of the chuck is selected from rectangle or circle.
In order to reach above-mentioned second purpose, the application process of the colorectal cancer primitive cell culture kit that the present invention is provided
Technical scheme as follows：
The application process of the colorectal cancer primitive cell culture kit that the present invention is provided is comprised the following steps：
200mg～500mg colorectal cancer tumor tissues are taken, the colorectal cancer tumor tissues is inserted in superclean bench
One centrifuge tube, obtains the first product；
First reagent is added in first product in superclean bench, the addition of first reagent
Span is 10mL～25mL, and mixes, and obtains the second product；
After the first reagent that superclean bench is exhausted in first centrifuge tube, by first centrifuge tube
Colorectal cancer tumor tissues insert the second centrifuge tube, obtain third product；
Second reagent is added in the third product, and the span of the addition of second reagent is 10mL
～25mL, and mix, obtain the 4th product；
The PBS dilution of the 3rd reagent is configured on superclean bench, wherein, in dilution, the 3rd reagent
Span with the volume ratio of PBS solution is 1: (6～12)；
The tumor tissues taken out on superclean bench in second centrifuge tube are placed in the 3rd centrifuge tube, and to institute
State the PBS dilution that the 3rd reagent described in 10mL～25mL is added in the 3rd centrifuge tube, obtain the 5th product after mixing；
The colorectal cancer tumor tissues taken out on superclean bench in the 3rd centrifuge tube are placed in the 4th centrifuge tube,
10mL～25mL sterile PBS buffer is added in the 4th centrifuge tube, after mixing, be placed in 5- under shaking table 40-60rpm rotating speed
10 minutes, and repeat this step；
After the PBS exhausted on superclean bench in the 4th centrifuge tube, colorectal cancer tumor tissues are inserted
In tissue freezing pipe, the 6th product is obtained；
6th product is shredded by application sterile scissors, obtains the 7th product；
Add in the 7th product after the 4th reagent described in 200 μ L～500 μ L and 37 DEG C of incubators are placed on, for the first time
Incubation 50-60 minute so that after the 7th product digestion, obtain the 8th product；
Contain to 300mL～600mL in the RPMI-1640 culture medium of 8%～15% (wt) hyclone and antibiotic and add
Enter the 5th reagent and mix, obtain hatch culture medium；
The 8th product is moved into after culture dish on superclean bench, the incubation training is added into the culture dish
Foster base, and colorectal cancer primitive cell culture is carried out under the conditions of 37 DEG C in incubator.
The application process of the colorectal cancer primitive cell culture kit that the present invention is provided can also be entered using following technical measures
One step is realized.
Preferably, the method for the mixing includes reverse and/or application shaking table.
Preferably, the 8th product is moved into after culture dish on the superclean bench, also include to stand 5min～
The step of 10min.
Preferably, the 6th product is shredded by application sterile scissors, during obtaining the 7th product, the described 6th produces
It is pasty state that the mark shredded by thing is the 6th product, and the agglomerate being wherein visible by naked eyes.
In application process, the first reagent of application has carried out one to the colorectal cancer primitive cell culture kit that the present invention is provided
Secondary sterilization, afterwards, all of operation is operated under aseptic environment, equivalent to provide a kind of two steps sterilization,
Which not only simplify the handling process of specimens of colorectal, additionally it is possible to reduce the use of antibiotic, therefore, it is possible to substantially carry out zero
Pollution；Also, the digestion method that the kit is applied in application process be to the 7th product in add 200 μ L～500 μ L
37 DEG C of incubators are placed on after four reagents, for the first time incubation 40-60 minute so that the 7th product digestion, separate with simple mechanical
Compare, colorectal cancer cells loss in this course can be reduced, and the pollution in operating process can be reduced, and improve
Digestive efficiency.Additionally, the kit by the first reagent being applied to during colorectal cancer primitive cell culture, the second reagent,
Three reagents, the 4th reagent and the 5th reagent are individually encapsulated, and in application process, need not repeat to prepare each reagent, therefore
Enable to operating process to be simplified, so as to improve the culture efficiency of colorectal cancer primary cell.
Description of the drawings
By reading the detailed description of hereafter preferred embodiment, various other advantages and benefit are common for this area
Technical staff will be clear from understanding.Accompanying drawing is only used for illustrating the purpose of preferred embodiment, and is not considered as to the present invention
Restriction.And in whole accompanying drawing, it is denoted by the same reference numerals identical part.In the accompanying drawings：
Fig. 1 is the box body structure schematic diagram (figure of the colorectal cancer primitive cell culture kit that the embodiment of the present invention one is provided
In, each reagent is not shown)；
Fig. 2 is that the structure of the chuck that applies in the colorectal cancer primitive cell culture kit that the embodiment of the present invention one is provided is shown
It is intended to；
Fig. 3 is the box body structure schematic diagram (figure of the colorectal cancer primitive cell culture kit that the embodiment of the present invention two is provided
In, each reagent is not shown)；
Fig. 4 is that the structure of the chuck that applies in the colorectal cancer primitive cell culture kit that the embodiment of the present invention two is provided is shown
It is intended to；
Fig. 5 is application in the colorectal cancer primitive cell culture kit that the embodiment of the present invention one or embodiment two are provided
First packaging container and/or the second packaging container and/or the 3rd packaging container and/or the 4th packaging container and/or the 5th encapsulation
The structural representation of container；
Fig. 6 is the application process steps flow chart of the colorectal cancer primitive cell culture kit that the embodiment of the present invention three is provided
Figure.
Specific embodiment
The present invention is to solve the problems, such as prior art, provides a kind of colorectal cancer primitive cell culture kit and its answers
With method, which can improve the success rate of colorectal cancer primitive cell culture, thus more suitable for practicality.
For further illustrating that the present invention is to reach technological means and effect that predetermined goal of the invention is taken, below in conjunction with
Accompanying drawing and preferred embodiment, to according to colorectal cancer primary cell kit proposed by the present invention and its application process, which is specifically real
Mode, structure, feature and its effect is applied, after describing in detail such as.In the following description, different " embodiment " or " embodiment "
Referred to is not necessarily same embodiment.Additionally, special characteristic in one or more embodiments, structure or feature can be by any conjunctions
Conformal formula combination.
The terms "and/or", only a kind of incidence relation of description affiliated partner, represents there may be three kinds of passes
System, for example, A and/or B, specifically it is interpreted as：A and B can be included simultaneously, can be with individualism A, it is also possible to individualism
B, can possess above-mentioned three kinds of any one situations.
The colorectal cancer primitive cell culture kit that the present invention is provided include the first reagent, the second reagent, the 3rd reagent, the
Four reagents and the 5th reagent.
Active ingredient in first reagent includes chelated iodine, and the mass concentration of its available iodine is 5000mg/L；
Active ingredient and substance withdrawl syndrome in second reagent includes：EDTA, 0.5mmol/L；NaCl, 137mmol/L；
KCl, 2.7mmol/L；Na2HPO4·12H2O, 10mmol/L；K2HPO4, 1.76mmol/L
Active ingredient in 3rd reagent includes NaClO, and the span of its weight/mass percentage composition is 8%～12%；
Active ingredient and mass concentration in 4th reagent includes：Clostridiopetidase A I, 10mg/mL；Clostridiopetidase A II, 10mg/mL；
Clostridiopetidase A IV, 5mg/L；
Active ingredient and content in 5th reagent includes：EGF, 5 μ g；Y27632 (producer Abcam；Article No.：
ab120129；Lot number：GR195774-32), 1.6mg；Hydrocortisone, 250 μ g；Cholera toxin, 4.2 μ g；PBS solution：5mL；
First reagent, the second reagent, the 3rd reagent, the 4th reagent, the 5th reagent are individually encapsulated, wherein, the first examination
Agent, the second reagent, the 3rd reagent, the 4th reagent, the volume parts ratio of the 5th reagent are 200mL: 200mL: 20mL: 5mL: 5mL.
Wherein, the preservation condition of the first reagent includes normal temperature, lucifuge.
Wherein, the preservation condition of the second reagent includes normal temperature.
Wherein, the preservation condition of the 3rd reagent includes normal temperature, lucifuge.
Wherein, preserve 0.5 month～2 months at a temperature of the preservation condition of the 4th reagent includes 0 DEG C～4 DEG C.
Wherein, preserve 0.5 month～2 months at a temperature of the preservation condition of the 5th reagent includes 0 DEG C～4 DEG C.
In the present embodiment, colorectal cancer primitive cell culture kit also include the first packaging container (not shown),
Two packaging container (not shown)s, the 3rd packaging container (not shown), the 4th packaging container (not shown), the 5th
Packaging container (not shown) and chuck (being numbered 101 in accompanying drawing 1 and accompanying drawing 2, be numbered 201 in accompanying drawing 3 and accompanying drawing 4).
First packaging container (not shown) is used for encapsulating the first reagent, and the second packaging container (not shown) is used for encapsulation second
Reagent, the 3rd packaging container (not shown) are used for encapsulating the 3rd reagent, and the 4th packaging container (not shown) is used for sealing
The 4th reagent is filled, the 5th packaging container (not shown) is used for encapsulating the 5th reagent.In the enforcement shown in accompanying drawing 1 and accompanying drawing 2
In example one, chuck 101 includes chuck body 102, in chuck body 102 at least provided with first through hole 103, the second through hole 104,
Three through holes 105, fourth hole 106 and fifth hole 107；First packaging container (not shown) can be arranged in first through hole
In 103, the second packaging container (not shown) can be arranged in the second through hole 104, and the 3rd packaging container (do not show by figure
Go out) can be arranged in third through-hole 105, the 4th packaging container (not shown) can be arranged in fourth hole 106, the
Five packaging container (not shown)s can be arranged in fifth hole 107.In the embodiment two shown in accompanying drawing 3 and accompanying drawing 4,
Chuck 201 includes chuck body 202, at least provided with first through hole 203, the second through hole 204, third through-hole in chuck body 202
205th, fourth hole 206 and fifth hole 207；First packaging container (not shown) can be arranged in first through hole 203,
Second packaging container (not shown) can be arranged in the second through hole 204, and the 3rd packaging container (not shown) can
It is arranged in third through-hole 205, the 4th packaging container (not shown) can be arranged in fourth hole 206, the 5th encapsulation
Container (not shown) can be arranged in fifth hole 207.
Wherein, the first packaging container (not shown) and first through hole (the 103 of embodiment one, the 203 of embodiment two)
Gap coordinates；And/or, the second packaging container (not shown) and the second through hole (the 104 of embodiment one, embodiment two
204) gap coordinates；And/or, the 3rd packaging container (not shown) and third through-hole (the 105 of embodiment one, embodiment two
205) gap coordinate；And/or, the 4th packaging container (not shown) and fourth hole (the 106 of embodiment one, embodiment
The 206 of two) gap cooperation；And/or, (implement by the 107 of embodiment one with fifth hole for the 5th packaging container (not shown)
The 207 of example two) gap cooperation.In this case, the residue due to each packaging container (not shown) in each through hole is living
Dynamic space is less, each packaging container (not shown) can be avoided to topple over, it is to avoid the loss of each reagent or pollution.
Referring to accompanying drawing 5, the first packaging container and/or the second packaging container and/or the 3rd packaging container and/or the 4th are encapsulated
The bottom surface of container and/or the 5th packaging container is in arc-shaped transition, and in the present embodiment, label 301 generally represents each packaging container,
Label 302 represents the arc-shaped transition for being arranged at each packaging container bottom surface, in this case, can avoid due to packaging container
The dead angle of bottom and hold the deposition of matter in the reagent that causes, each reagent stable homogeneous being further ensured that in each packaging container, drop
Low operating error.
Wherein, also include packing box (108 in embodiment one, 208 in embodiment two), packing box is (in embodiment one
108,208 in embodiment two) include box body (111 in embodiment one, 211 in embodiment two), box body (embodiment one
In 111,211 in embodiment two) shape of cross section and chuck (101 in embodiment one, 201 in embodiment two)
Shape is adapted, and in this case, ensure that chuck (101 in embodiment one, 201 in embodiment two) in box body
In (109 in embodiment one, 209 in embodiment two) with side wall (111 in embodiment one, 211 in embodiment two) it
Between not run-off the straight, further avoid each packaging container (not shown) from toppling over, it is to avoid the loss of each reagent or
Pollution.
Wherein, packing box (108 in embodiment one, 208 in embodiment two) also includes lid (in embodiment one
112,212 in embodiment two), upper lid (112 in embodiment one, 212 in embodiment two) can cover box body and (implement
In example one 109,209 in embodiment two), the colorectal cancer primary cell of present invention offer in this case, can be provided
Cultivate reagent box in transport or carrying process, lose by each packaging container (not shown).
Wherein, referring to accompanying drawing 3, in the colorectal cancer primitive cell culture kit 208 that the embodiment of the present invention two is provided, make
For a kind of fit system between upper lid 212 and box body 209, can be split-type structural between upper lid 212 and box body 209.?
Under the premise of this, can also realize by the way of threaded or chimeric cooperation coordinating between upper lid 212 and box body 209.
Wherein, referring to accompanying drawing 1, in the colorectal cancer primitive cell culture kit 108 that the embodiment of the present invention one is provided, make
For upper lid 112 and box body 109 between another fit system, hinged between upper lid 112 and box body 109.In the present embodiment, on
Lid 112 is realized hinged with box body 109 by the wherein a lines of its side wall along 116, and upper lid 112 can be with the edge 116 as revolving
Turn central rotation, 112 be turned on or off of lid in realization, now, the material of the box body can be the material such as paper, plastics；Have
When, in order that this is hinged more rationally, the accessories such as hinge can also be set in this place.
In the present embodiment, one end of upper lid 112 is additionally provided with lid edge 113, now, when upper lid 110 covers box body 109,
The lid edge 113 can be attached on the side wall 111 of box body 109, can further ensure that box body 109 is capped 110 and covers comprehensively
Lid, so as to ensure the packaging effect of box body 109.
Wherein, box body (the 109 of embodiment one, the 209 of embodiment two) is provided with the sub- end (embodiment one of a buckle structure
114, the 214 of embodiment two), the female end that upper lid (the 110 of embodiment one, the 210 of embodiment two) are provided with buckle structure is (real
Apply the 115 of example one, the 215 of embodiment two), sub- end (the 114 of embodiment one, the 214 of embodiment two) and female end be (embodiment one
115, the 215 of embodiment two) it is adapted.Now, lid (the 110 of embodiment one, the 210 of embodiment two) can be avoided from box body
(109 of embodiment one, the 209 of embodiment two) are upper to lose, and reduces the latent of each packaging container (not shown) loss further
In risk.
Wherein, the shape of chuck is selected from rectangle (embodiment one) or circular (embodiment two).
The application process of the colorectal cancer primitive cell culture kit provided referring to accompanying drawing 6, the present invention is comprised the following steps：
Step S1：200mg～500mg colorectal cancer tumor tissues are taken, colorectal cancer tumor tissues is put in superclean bench
Enter the first centrifuge tube and mix, obtain the first product；
Step S2：Add the first reagent in the first product, the span of the addition of the first reagent be 10mL～
25mL, and mix, the second product is obtained, is covered tightly and under lid, room temperature, is placed in shaking table 40rpm～60rpm, 15min～20min；
Step S3：After the first reagent that superclean bench is exhausted in the first centrifuge tube, by the first centrifuge tube
Colorectal cancer tumor tissues insert the second centrifuge tube, obtain third product；
Step S4：Add the second reagent in third product, the span of the addition of the second reagent be 10mL～
25mL, and mix, the 4th product is obtained, is covered tightly and under lid, room temperature, is placed in shaking table 40rpm～60rpm, 8min～10min；
Step S5：Configure the PBS dilution of the 3rd reagent on the superclean bench, wherein, in dilution, the 3rd reagent with
The span of the volume ratio of PBS solution is 1: (6～12)；
Step S6：Take out the tumor tissues in the second centrifuge tube on superclean bench to be placed in the 3rd centrifuge tube, and
The PBS dilution of the 3rd reagent of 10mL～25mL is added in the 3rd centrifuge tube, obtains the 5th product, cover tightly lid after mixing,
Shaking table 40rpm～60rpm, 15min～20min is placed under room temperature；
Step S7：The colorectal cancer tumor tissues in the 3rd centrifuge tube are taken out on superclean bench and be placed to the 4th centrifuge tube
In, 10mL～25mL sterile PBS buffer is added in the 4th centrifuge tube, take out, insert the 5th centrifuge tube, and repeat after mixing
This step, until cause colorectal cancer tumor tissues to be placed to N centrifuge tube；
Step S8：After the PBS exhausted in N centrifuge tube on superclean bench, by N centrifuge tube
Colorectal cancer tumor tissues are inserted in tissue freezing pipe, obtain the 6th product；
6th product is shredded by step S9 application sterile scissors, obtains the 7th product；
Step S10：Be placed on 37 DEG C of incubators after the 4th reagent of 200 μ L～500 μ L is added in the 7th product, for the first time
Incubation more than 1h so that after the 7th product digestion, obtain the 8th product；
Step S11：Contain the RPMI-1640 training of 8%～15% (wt) hyclone and antibiotic to 300mL～600mL
The 5th reagent is added in foster base and is mixed, obtain hatch culture medium；
Step S12：The 8th product is moved into after culture dish on superclean bench, incubation culture is added into culture dish
Base, and colorectal cancer primitive cell culture is carried out under the conditions of 37 DEG C in incubator.
Wherein, the method for mixing includes reverse and/or application shaking table.
Wherein, the 8th product is moved into after culture dish on superclean bench, also includes to stand the step of 5min～10min
Suddenly, so as to ensure postdigestive tumor tissues equably confluent cultures ware bottom, strengthen stablizing for colorectal cancer primitive cell culture
Property.
Wherein, the 6th product is shredded by application sterile scissors, during obtaining the 7th product, the mark shredded by the 6th product
It is pasty state that will is the 6th product, and the agglomerate being wherein visible by naked eyes.
3～22 table 1 of embodiment
3～22 continued 1-2 of embodiment
3～22 continued 1-3 of embodiment
In application process, the first reagent of application has carried out one to the colorectal cancer primitive cell culture kit that the present invention is provided
Secondary sterilization, afterwards, all to add reagents and process the operations of tissue carried out under aseptic environment, equivalent to there is provided one
Two step sterilizations are planted, which not only simplify the handling process of specimens of colorectal, additionally it is possible to reduce the use of antibiotic, accordingly, it is capable to
No pollution is enough substantially carried out；Also, the digestion method that the kit is applied in application process be to the 7th product in add
37 DEG C of incubators are placed on after the 4th reagent of 200 μ L～500 μ L, for the first time incubation more than 1h so that the 7th product digestion, with machine
Tool separation is compared, and can be reduced colorectal cancer cells loss in this course, and can be reduced the pollution in operating process,
And improve digestive efficiency.Additionally, the first reagent being applied to during colorectal cancer primitive cell culture, second are tried by the kit
Agent, the 3rd reagent, the 4th reagent and the 5th reagent are individually encapsulated, and in application process, need not repeat to prepare each reagent,
Therefore, it is possible to cause operating process to be simplified, so as to improve the culture efficiency of colorectal cancer primary cell.Additionally, from reality of the present invention
Apply the experiment conclusion of example 3～22, it is known that, the colorectal cancer primitive cell culture kit for applying the present invention to provide, enable to training
Form power and reach 50%～60%.
Although preferred embodiments of the present invention have been described, but those skilled in the art once know basic creation
Property concept, then can make other change and modification to these embodiments.So, claims are intended to be construed to include excellent
Select embodiment and fall into being had altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without deviating from the present invention to the present invention
God and scope.So, if these modifications of the present invention and modification belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising these changes and modification.
Claims (10)
1. a kind of colorectal cancer primitive cell culture kit, it is characterised in that including the first reagent, the second reagent, the 3rd reagent,
4th reagent and the 5th reagent,
Active ingredient in first reagent includes chelated iodine, and the mass concentration of its available iodine is 5000mg/L；
Active ingredient and substance withdrawl syndrome in second reagent includes：EDTA, 0.5mmol/L；NaCl, 137mmol/L；
KCl, 2.7mmol/L；Na2HPO4·12H2O, 10mmol/L；K2HPO4, 1.76mmol/L
Active ingredient in 3rd reagent includes NaClO, and the span of its weight/mass percentage composition is 8%～12%；
Active ingredient and mass concentration in 4th reagent includes：Clostridiopetidase A I, 10mg/mL；Clostridiopetidase A II, 10mg/mL；
Clostridiopetidase A IV, 5mg/L；
Active ingredient and content in 5th reagent includes：EGF, 5 μ g；Y27632,1.6mg；Hydrocortisone, 250 μ g；
Cholera toxin, 4.2 μ g；PBS solution：5mL；
First reagent, the second reagent, the 3rd reagent, the 4th reagent, the 5th reagent are individually encapsulated, wherein, described
One reagent, the second reagent, the 3rd reagent, the 4th reagent, the volume parts ratio of the 5th reagent are 200mL: 200mL: 20mL: 5mL:
5mL.
2. colorectal cancer primitive cell culture kit according to claim 1, it is characterised in that the guarantor of first reagent
The condition of depositing includes normal temperature, lucifuge；
Preferably, the preservation condition of second reagent includes normal temperature；
Preferably, the preservation condition of the 3rd reagent is preserved 0.5 month～1.5 months at a temperature of including 0 DEG C～4 DEG C；
Preferably, the preservation condition of the 4th reagent is preserved 0.5 month～1.5 months at a temperature of including 0 DEG C～4 DEG C；
Preferably, the preservation condition of the 5th reagent is preserved 0.5 month～1.5 months at a temperature of including 0 DEG C～4 DEG C.
3. colorectal cancer primitive cell culture kit according to claim 1, it is characterised in that also include that the first encapsulation is held
Device, the second packaging container, the 3rd packaging container, the 4th packaging container, the 5th packaging container and chuck,
First packaging container is used for encapsulating first reagent, and second packaging container is used for encapsulating the described second examination
Agent, the 3rd packaging container are used for encapsulating the 3rd reagent, and the 4th packaging container is used for encapsulating the 4th reagent,
5th packaging container is used for encapsulating the 5th reagent；
The chuck includes chuck body, in the chuck body at least provided with first through hole, the second through hole, third through-hole,
Four through holes and fifth hole；
First packaging container can be arranged in the first through hole, and second packaging container can be arranged in described
In two through holes, the 3rd packaging container can be arranged in the third through-hole, and the 4th packaging container can be arranged in
In the fourth hole, the 5th packaging container can be arranged in the fifth hole.
4. colorectal cancer primitive cell culture kit according to claim 3, it is characterised in that
First packaging container is coordinated with the first through hole gap；And/or,
Second packaging container is coordinated with second via clearance；And/or,
3rd packaging container is coordinated with the third through-hole gap；And/or,
4th packaging container is coordinated with the fourth hole gap；And/or,
5th packaging container is coordinated with the fifth hole gap.
5. colorectal cancer primitive cell culture kit according to claim 3, it is characterised in that first packaging container
And/or second the bottom surface of packaging container and/or the 3rd packaging container and/or the 4th packaging container and/or the 5th packaging container be in
Arc-shaped transition.
6. colorectal cancer primitive cell culture kit according to claim 3, it is characterised in that also include packing box, institute
Stating packing box includes box body, and the shape of cross section of the box body is adapted with the shape of the chuck；
Preferably, the packing box also includes lid, the upper lid can cover the box body；
Preferably, the upper lid is split-type structural with the box body；
Preferably, hinged between the upper lid and the box body；
Preferably, the box body is provided with the sub- end of a buckle structure, described on cover the female end for being provided with the buckle structure,
The sub- end is adapted with the female end.
7. colorectal cancer primitive cell culture kit according to claim 3, it is characterised in that the shape choosing of the chuck
From rectangle or circle.
8. in claim 1～7 arbitrary described colorectal cancer primitive cell culture kit application process, it is characterised in that
Comprise the following steps：
Take 200mg～500mg colorectal cancer tumor tissues, in the superclean bench by the colorectal cancer tumor tissues insert first from
Heart pipe is simultaneously mixed, and obtains the first product；
Add first reagent in first product, the span of the addition of first reagent be 10mL～
25mL, and mix, obtain the second product；
After the first reagent that superclean bench is exhausted in first centrifuge tube, by the large intestine in first centrifuge tube
Cancerous swelling tumor tissue inserts the second centrifuge tube, obtains third product；
Add second reagent in the third product, the span of the addition of second reagent be 10mL～
25mL, and mix, obtain the 4th product；
The PBS dilution of the 3rd reagent is configured on superclean bench, wherein, in dilution, the 3rd reagent and PBS
The span of the volume ratio of solution is 1: (6～12)；
The tumor tissues taken out on superclean bench in second centrifuge tube are placed in the 3rd centrifuge tube, and to described
The PBS dilution of the 3rd reagent described in 10mL～25mL is added in three centrifuge tubes, obtains the 5th product after mixing；
The colorectal cancer tumor tissues taken out on superclean bench in the 3rd centrifuge tube are placed in the 4th centrifuge tube, to institute
Addition 10mL～25mL sterile PBS buffer in the 4th centrifuge tube is stated, is taken out after mixing, the 5th centrifuge tube is inserted, and repeats this
Step, until cause colorectal cancer tumor tissues to be placed to N centrifuge tube；
After the PBS exhausted on superclean bench in the N centrifuge tube, will be big in the N centrifuge tube
Intestinal cancer tumor tissues are inserted in tissue freezing pipe, obtain the 6th product；
6th product is shredded by application sterile scissors, obtains the 7th product；
Add in the 7th product and 37 DEG C of incubators after the 4th reagent described in 200 μ L～500 μ L, are placed on, be incubated for the first time
More than 1h so that after the 7th product digestion, obtain the 8th product；
Contain addition institute in the RPMI-1640 culture medium of 8%～15% (wt) hyclone and antibiotic to 300mL～600mL
State the 5th reagent and mix, obtain hatch culture medium；
The 8th product is moved into after culture dish on superclean bench, the incubation culture is added into the culture dish
Base, and colorectal cancer primitive cell culture is carried out under the conditions of 37 DEG C in incubator.
9. the application process of colorectal cancer primitive cell culture kit according to claim 8, it is characterised in that described mixed
Even method includes reverse and/or application shaking table.
10. the application process of colorectal cancer primitive cell culture kit according to claim 8, it is characterised in that Yu Chao
The 8th product is moved into after culture dish on net workbench, also include the step of standing 5min～10min；
Preferably, the 6th product is shredded by application sterile scissors, and during obtaining the 7th product, the 6th product quilt
It is pasty state that the mark for shredding is the 6th product, and the agglomerate being wherein visible by naked eyes.
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