CN107557426A - Based on the horizontal screening anti-tumor medicine kit of three-dimensional micro-assembly robot and its application method - Google Patents
Based on the horizontal screening anti-tumor medicine kit of three-dimensional micro-assembly robot and its application method Download PDFInfo
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Abstract
The present invention relates to a kind of tumour personalised drug screening reagent box horizontal based on three-dimensional micro-assembly robot and its application method.Including：Three-dimensional cell culture material；Histocyte dispersing agent；Protease inhibitors；Basal cell culture medium；Serum；Antibiotic；Balanced salt solution；Cell viability detection reagent；Cell filtering net.Application method includes, and patient's tumor tissues are disperseed, obtained tissue block, fragment of tissue, cell mass, and/or scattered individual cells；Then, patient's tumor tissues, cell sample after will be scattered be cultivated by the method for dimensional culture, cell is grown in three dimensions, are formed the three-dimensional structure with spherical and/or irregular three-dimensional shape；Finally, the tumour micro-assembly robot obtained with dimensional culture carries out antibiotics susceptibility test.The present invention is easy to operate, is detected suitable for the susceptibility of various solid tumors.
Description
Technical field
The present invention relates to a kind of tumour personalised drug screening reagent box horizontal based on three-dimensional micro-assembly robot and its user
Method.The composition of the horizontal tumour personalised drug screening reagent box of specifically a kind of three-dimensional micro-assembly robot, with the kit to patient
Tumour cell be collected and dimensional culture, carry out antibiotics susceptibility test.The kit has obtained tumour by three-dimensional culture method
Micro-assembly robot, the biological characteristics of tumour cell is kept in testing in vitro to greatest extent, reliable susceptibility detection can be obtained
Data, the formulation for clinical tumor personalized therapy program provide reference, improve the accuracy of medication.
Background technology
The antineoplastic species clinically commonly used at present are various, but curative effect is still undesirable.Because first, tumour is in itself
There is heterogeneity, even if same histological type, differentiation degree identical tumour also can be different to the sensitiveness of same medicine.
Second, patient has individual difference, and different individual cognition shows different susceptibilitys to different pharmaceutical.Clinic is mostly to pass through
Medication is tested, same chemotherapy regimen is applied to many patients with immobilizing, individual difference is not considered, so unsatisfactory curative effect.
Chemotherapeutics is generally also provided with stronger toxicity, and inaccurate chemotherapy can not only alleviate the state of an illness, allows patient to produce serious bad
Reaction, it is also possible to which induced tumor cell produces the multidrug resistance to multi-medicament, causes to lose best occasion for the treatment.Even and
Gene target medicine, also not energy 100% is corresponding by patient.Therefore how more effectively anticarcinogen is screened for each individual patient
Thing, prediction individual tumors to guiding clinical treatment, improve curative effect, are the passes of tumour personalized treatment to the sensitiveness of medicine
One of key problem.
Screening anticancer medicine, also referred to as anticancer sensitivity are tested, and have internal drug sensitive experiment and In vitro chemo-drug sensitive test two ways.
Internal drug sensitive experiment, antibiotics susceptibility test is mainly carried out by the heteroplastic transplantation knurl model (PDX) from patient.But
PDX animal model modeling success rates are low, the cycle is long, costly, it is difficult to the extensive popularization and application in clinical cancer therapy.
Drug sensitivity testing in vitro is the Critical policies for implementing individualized treatment.The in vitro culture of cell is most basic biology doctor
Learn research meanses.Traditional cell culture mode is 2D cultures.The cell of 2D cultures grows in monolayer adherence, its form and cell
It is all and different in vivo from the mode of environmental interaction, so cause the signal transduction of cell, protein expression that anomalous variation occurs,
Finally the cell of in vitro culture is set gradually to lose original biological characteristics, the experimental result obtained on this basis is difficult often
It is verified in testing in vivo.For example, Document system shows (Rasheena Edmondson, Jessica Jenkins
Broglie,Audrey F.Adcock,et al.ASSAY and Drug Development Technologies 2014,12
(7):207-219), suffered in drug development process, the medicine come out with the cell screening of 2D cultures, finally can be by clinical real
Only 5% tested, causes great cost and wastes.So due to the unreliability of 2D cultivation results, traditional is trained using 2D
The anticancer sensitivity that foster mode is assessed drugs against tumor effect is tested, and its result is difficult to instruct clinical application.
3D cell culture is provided close to internal 3 D stereo life because of the cell growth mode in analogue body for cell
Manage microenvironment so that cell can remain closest to internal form and physiology characteristic, be the extracorporeal biology of most worthy
Model.Chinese patent CN104111254A provides a kind of method that cell progress anticancer sensitivity test is cultivated in collagen, but
It is because its is complex for operation step, inconvenience is caused to clinical practice.
Therefore, how conveniently and efficiently with 3D tumor models outside the tumour cell construct from patient, and carry out quick
, the personalized anticancer sensitivity for having higher clinical reference value detection, be it is extensive carry out tumour personalized treatment there is an urgent need to
Solve the problems, such as.
The content of the invention
The present invention provides a kind of kit that personalised drug sensitivity assessment result is obtained by external 3D tumor models,
Purpose be for clinic provides standardization, easily, cost is rational, highly reliable, the tumour that can popularize on a large scale is individual
Property the quick detection means of chemical drug, improve the confidence level of In vitro chemo-drug sensitive test, shorten modeling time and the susceptibility inspection of Vitro Tumor model
Survey the cycle.
Technical scheme is as follows：
A kind of horizontal tumour personalised drug screening reagent box of three-dimensional micro-assembly robot, including：Three-dimensional cell culture material；Group
Knit cell dispersing agent；Protease inhibitors；Basal cell culture medium；Serum；Antibiotic；Balanced salt solution；Cell viability is examined
Test agent；Cell filtering net." the three-dimensional micro-assembly robot " refers to, 3 D stereo grows the three-dimensional to be formed to cell or tissue in vitro
Structure, can be any shape (such as spherical, three-dimensional netted, fibrous, tubulose, irregular shape) and/or chi
It is very little.
Three-dimensional cell culture material of the present invention, can be that any cell that can allow in vitro culture is empty in 3 D stereo
Between middle growth device, consumptive material and/or material, including but not limited to suspend culture plate, Hanging drop culture plate, microfluid dimensional culture
Plate and corollary apparatus, hydrogel, dimensional culture magnetic bead and corollary apparatus, solid tridimensional scaffold material, in Tissue Culture Plate
It is one or more.As preferable, the dimensional culture material is probably suspension culture plate, Hanging drop culture plate, dimensional culture water
Gel and/or solid tridimensional scaffold material and arbitrary Tissue Culture Plate, more preferably suspend culture plate, or is hydrogel
It is used cooperatively with Tissue Culture Plate.In some embodiments of the invention, the dimensional culture material, including at least suspension cell
Culture plate.In the other embodiment of the present invention, the dimensional culture material, including at least dimensional culture hydrogel and carefully
Born of the same parents' culture plate.
Histocyte dispersing agent of the present invention, can have to decompose extracellular matrix and/or destroy cell to connect
Any compound and combinations thereof.Including but not limited to：Trypsase, EDTA, EDTA- trypsase, clostridiopetidase A, neutral protein
Enzyme, pronase, papain, DNA enzymatic, hyaluronidase, pancreatin, TrypLE, Trypzean, Liberase,
One or more in Blendzyme, Accutase.The working concentration of the histocyte dispersing agent is 0.00001mg/mL
~10000mg/mL, preferably 0.001mg/mL~100mg/mL.The histocyte dispersing agent is soluble in balance salt
In solution or in cell culture medium, the ╳ of about 1 ╳~1000 storage liquid is made into.
Protease inhibitors of the present invention, can be any compound with protease inhibition activity and combinations thereof.
Including but not limited to：Pancreatin neutralizer, Aprotinin, aprotinin, pancreatin inhibitor, one kind or more in protease inhibitors
Kind.
Basal medium of the present invention is any culture medium that can be grown with sertoli cell, is included but is not limited to：BME is thin
Born of the same parents' culture medium, IMDM cell culture mediums, the cell culture mediums of RMPI 1640, MEM cell culture mediums, DMEM cell culture mediums, F10
Cell culture medium, F12 cell culture mediums, DMEM/F12 cell culture mediums, McCoy's 5A cell culture mediums, 199 cell culture
Base, Fisher ' s cell culture mediums, CMRL cell culture mediums, GMEM cell culture mediums, Leibovitz's L-15 cell culture
Base, the cell culture mediums of MCDB 131, Ames' cell culture mediums, the one or more in serum free medium.The one of the present invention
In a little embodiments, the culture medium comprises at least a kind of basal medium.In the other embodiment of the present invention, the training
It is serum free medium to support base.
Serum of the present invention is any animal blood serum or serum substitute that can be used for cultivating cell.Including but it is unlimited
In：Cow's serum, horse serum, horse serum, rabbit anteserum, chicken serum, Swine serum, other animal blood serums, synthesize serum replacement, serum
One or more in substitute.
Balanced salt solution of the present invention, can be any solution for having and maintaining Premeabilisation of cells pressure effect.Including but not
It is limited to PBS, D-PBS, Hank ' s, the one or more in D-Hank ' s, EBBS.
Antibiotic of the present invention is any compound that can suppress microbial activity and/or medicine and combinations thereof.Bag
Include but be not limited to：Penicillin, streptomysin, gentamicin, D actinomycin D, neomycin, kanamycins, polymyxins, nystatin,
One or more in anphotericin, preferably penicillin and streptomysin.The working concentration of antibiotic of the present invention is
0.00001mg/mL~10000mg/mL, or 0.00001U/mL~10000U/mL, preferably preferably, be 0.001mg/mL~
100mg/mL.The antibiotic is soluble in balanced salt solution or in cell culture medium, is made into the ╳ of about 1 ╳~1000 storage
Liquid storage.
Cell viability detection reagent of the present invention may be any physics, chemistry, biological property using life or death cell
The reagent that is handled cell of otherness.Including but not limited to：AlamarBlue, trypan blue, MTT reagents, XTT reagents,
WST-1 reagents, CCK-8 reagents, the one or more in live/dead cell viability detection reagents.
Cell filtering net of the present invention may be the material of any fibrous reticular structure for having filtering function.The fiber
The pore size of network structure is 0.001 μm~1000 μm.It is preferred that sterile gauze, syringe filter (filter), plastic cell mistake
One or more in filter screen and/or stainless steel cell filtering net.Preferably 10 μm~1000 μm of the size of hole.
A kind of application method of the horizontal tumour personalised drug screening reagent box of three-dimensional micro-assembly robot, comprises the following steps：
Before experiment, first serum and antibiotic are added in basal medium and are configured to complete medium.Wherein, serum
Concentration is any range between 0%~99.9%.The concentration of antibiotic is between 0.00001mg/mL~10000mg/mL
Any range.
(1) the flesh tissue sample from patient is soaked in antibiotic solution and carried out disinfection, and use balanced salt solution
Cleaning；
(2) tissue block is shredded with operating theater instruments, with histocyte dispersing agent vitellophag interstitial；
(3) protease inhibitors is used, serum and/or the nutrient solution containing serum terminate the work of histocyte dispersing agent
With.
(4) use cell filtration net filtration, obtain fritter (a diameter of 10~500 μm) cell mass or independent dispersion it is thin
Born of the same parents；Cell is collected by centrifugation.
(5) cell is inoculated with and/or be wrapped in dimensional culture material surface and/or inside, add cell culture medium, carried out
Three-dimensional cell culture；
(6) cell is handled with antineoplastic drug solution to be measured；
(7) with cell viability detection reagent processing cell；
(8) with ELIASA, cell counter, laser confocal microscope and/or flow cytometer to treated cell
Carry out the collection of signal and/or data；
(9) using the signal and/or data analysis cell viability collected, suppression of the antineoplastic to tumour cell is obtained
Effect conclusion processed.
Preferably, the concentration of serum is any range between 0%~99.9%；The concentration of antibiotic is 0.00001mg/
Any range between mL~10000mg/mL.
It is furthermore preferred that the concentration of serum is any concentration between 5%~20%；The concentration of antibiotic is 0.001mg/mL
~100mg/mL.
Preferably, in the step 4) in Three-dimensional cell culture, the dimensional culture material is 3D micropore suspension culture plates,
It is 3000~8000/micropore to make final concentration of cells；Or dimensional culture water-setting gel, make final concentration of cells for 50,000~250,000/
mL。
Preferably, the duration of the step 5) Three-dimensional cell culture is about 1~7 day.
A kind of application method of the horizontal tumour personalised drug screening reagent box of three-dimensional micro-assembly robot of the present invention, first
Need to be disperseed patient's tumor tissues, obtain tissue block, fragment of tissue, cell mass and/or scattered individual cells.
The three-dimensional micro-assembly robot level refers to that the sample after disperseing is cultivated by the method for dimensional culture, with three-dimensional
The product of culture carries out anticancer sensitivity test.
The Three-dimensional cell culture is inoculation and/or is wrapped in dimensional culture material surface and/or inside, allows cell to exist
Grown in three-dimensional space, form the three-dimensional structure (dimensional culture with spherical and/or irregular any three-dimensional shape
Product), i.e. tumour micro-assembly robot.In some embodiments of the invention, cell is seeded in above suspension culture plate and cultivated.At this
In the other embodiment of invention, first cell is mixed with hydrogel, by cell encapsulation among hydrogel, cultivated.
Preferably, in some embodiments of the invention, the time of Three-dimensional cell culture is 1~7 day.
The anticancer sensitivity test, refers to after handling dimensional culture product with antineoplastic to be measured, test cell
Vigor.The method of drug-treated, added drug in cell culture fluid, be incubated jointly with cell.It is big to handle the duration
About 0.1 minute~30 days.Preferably, in some embodiments of the invention, the processing time of antineoplastic be 4~
48 hours.The medicine of each certain concentration is, it is necessary to set more than 3 parallel laboratory tests.
Cell viability analysis, it may be possible to will be gathered using analysis software, calculation formula and/or the method that manually calculates
To signal and/or data be converted into cell viability indication, finally draw cell viability percentage, the tested medicine pair of reflection
The inhibitory action of cell growth.
Compared with prior art, the present invention has the advantages that：
(1) thing of the present invention carries out drug sensitive experiment using the three-dimensional nodule micro-assembly robot of in vitro culture as raw model, can be maximum
The biological characteristics of tumour cell is kept in testing in vitro to limit, so reliable susceptibility detection data can be obtained, is realized
External hi-fi anticancer sensitivity is assessed.
(2) short time consumption of the present invention is short, can obtain testing result within time of a couple of days by one month.
(3) present invention is easy to operate, is detected suitable for the susceptibility of various solid tumors.
(4) cost of the present invention is cheap, can detect a variety of antineoplastics simultaneously, anti-to difference swollen so as to obtain given patient
The sensitiveness sequence of tumor medicine.
Brief description of the drawings
Fig. 1 is the breast cancer 3D tumour micro-assembly robots obtained in embodiment 1 with the breast cancer cell of kit culture patient
Fig. 2 is the cancer of the esophagus 3D tumour micro-assembly robots obtained in embodiment 2 with the esophageal cancer cell of kit culture patient
Embodiment
The following examples, detailed explanation will be done to the present invention, but be not limiting upon the scope of the invention.
Kit of the present invention will be more fully described by the case study on implementation of present device herein.It should be appreciated that this
Invention can be implemented in different forms, and embodiment is understood not to limit case study on implementation described in this paper.But pass through
These case study on implementation are provided, make it that it is thorough and complete that the present invention discloses, and will fully be passed on to those skilled in the art
The applicable scope of kit of the present invention.Tumor tissues used in following examples derive from hospital's sample.
Embodiment one：
A kind of horizontal personalised drug screening reagent box of three-dimensional micro-assembly robot, including following component：
(1) Three-dimensional cell culture material：Micropore 3D suspension culture plates.
(2) histocyte dispersing agent：0.05% pancreas enzyme -EDTA, -20 DEG C of refrigerations.
(3) cell culture medium：The cell culture mediums of RMPI 1640.
(4) hyclone.Serum is added in basal cell culture medium with 10% volume ratio using preceding.Serum need-
20 DEG C of refrigerations.
(5) balanced salt solution：PBS.
(6) 100 ╳ antibiotic：10000U/ml penicillin chain and 10mg/ml streptomysin mixed liquors.- 20 DEG C of refrigerations.Use
Before be added in culture medium, be diluted to 1 ╳ working concentrations.
(7) cell viability detection reagent：alamarBlue.
(8) cell filtering net：Aseptic plastic cell filtering net, 100 μm of aperture.
A kind of horizontal personalised drug screening reagent box of three-dimensional micro-assembly robot in the screening of breast cancer personalised drug
Application method, comprise the following steps：
Before experiment, first serum, antibiotic are added in basal medium, are made into complete medium, it is standby.
(1) under aseptic condition, the fresh breast tumor tissue performed the operation or tissue biopsy obtains is filled with PBS
Distinguish and wash, cut off fiber part, necrotic zone and sludged blood, again with PBS 1~2 time.
(2) after weighing, with 10 × it is dual anti-flood tissue, soak 5min.Use PBS.
(3) tissue block is placed in 9cm culture dishes, is cut into 0.5~1mm3Fritter.It is transferred in centrifuge tube.It is clear with PBS
Wash.
(4) 0.05% pancreatin is added, 4 DEG C stand 6~18 hours.Pancreatin dosage：10mL/g is organized.
(5) unnecessary pancreatin liquid is carefully removed, leaves behind the pancreatin being stained with tissue surface.37 DEG C of digestion 20-30min.
(6) complete medium in equal volume containing 10% serum is added.It is repeatedly soft to blow and beat for several times.Precipitate 2min, centrifugation
Remove liquid.
(7) filtered with 100 μm of cell strainers, collect filtered fluid.
(8) 50~100g centrifuge 3min, discard supernatant.
(9) 2mL complete mediums are added, fully blow and beat cell dispersion, are counted.750,000~2,100,000 cells are taken out, are supplemented
Complete culture solution makes cell concentration adjust to 1.5 ten thousand~700,000/mL to 3~10mL.Final concentration of cells suspends according to micropore 3D and trained
Support the number of micropore in plate and determine.
(10) cell suspension is added in 3D micropore suspension culture plates, makes the final concentration of cells be：3000~8000/micro-
Hole.
(11) 37 DEG C are placed in, 5%CO2After being cultivated 24 hours in incubator, liquid is softly changed, can not be assembled with removal thin
Born of the same parents.
(12) culture about 24 hours is continued.Period microexamination, treat that (such as Fig. 1 is tried cell with this for glomeration in micropore
The breast cancer 3D tumours micro-assembly robot that the breast cancer cell of agent box culture patient obtains) next step experiment can be carried out.
(13) injected with 200 μ L liquid transfer gun head in micropore, gently suction out individual cells ball and be put into 96 orifice plates, per Kong Fangyi
Individual ball.An if only micropore in each hole of micropore 3D suspension culture plates used.Then ignore this step.
(14) medicine that addition is dissolved with complete culture solution, per the μ L of hole 100.Control group adds the complete culture of not drug containing
Liquid.3~5 repetitions of every group of setting.Culture 48 hours.
(15) nutrient solution is gently sopped up, with PBS twice.
(16) the μ L of alamarBlue 100 are added, 37 DEG C of incubation 4h, blank pair are used as using the alamarBlue of no cell
According to.
(17) light absorption value surveyed at 570nm and 600nm.Light absorption value is corrected with OD 570nm-OD 600nm.
(18) cytoactive is calculated according to the following formula
Cytoactive (%)=(ODsample–OD0)/(ODcontrol–OD0)×100。ODsample、ODcontrolAnd OD0Generation respectively
The light absorption value of table sample sheet, control group and alamarBlue.
(19) with T-test statistical methods analysis experimental result, the antitumous effect of medicine is ranked up, you can sieve
The sensitive medicine of patient is selected, personalized therapy program is customized for patient.
Table 1 is to detect the susceptibility knot of obtained certain breast cancer patients to three kinds of breast cancer medicines with this kit in embodiment
Fruit
Table 1.
Contrast experiment
By the cell concentration according to the form below data in the step (10) in embodiment 1, remaining is same as Example 1, experiment knot
Fruit such as, table 2, it can be seen that 3000~8000/micropore of final concentration of cells of embodiment 1 is to be best suitable for cell balling-up, again can be with
Ensure the preferred plan of all cytoactives in ball.
| Cell concentration | Incubation time | As a result |
| 1000~2000/micropore | 48h | Cell ball can not be formed |
| 2000~3000/micropore | 48h | Cell ball can not be formed |
| 8000~12000/micropore | 48h | The necrosis of cell ball central area |
| 10000~20000/micropore | 48h | The necrosis of cell ball central area |
| 20000~50000/micropore | 48h | The necrosis of cell ball central area |
Embodiment two：
A kind of horizontal personalised drug screening reagent box of three-dimensional micro-assembly robot, including following component：
(1) Three-dimensional cell culture material：96 porocyte culture plates and 3D cell culture hydrogels.
(2) histocyte dispersing agent：0.05% pancreas enzyme -EDTA, 1000U/mL clostridiopetidase As.- 20 DEG C of refrigerations.
(3) cell culture medium：The cell culture mediums of RMPI 1640.
(4) hyclone.Serum is added in basal cell culture medium with 10% volume ratio using preceding.Serum need-
20 DEG C of refrigerations.
(5) balanced salt solution：PBS.
(6) 100 ╳ antibiotic：10000U/ml penicillin chain and 10mg/ml streptomysin mixed liquors.- 20 DEG C of refrigerations.Use
Before be added in culture medium, be diluted to 1 ╳ working concentrations.
(7) cell viability detection reagent：alamarBlue.
(8) cell filtering net：Aseptic plastic cell filtering net, 100 μm of aperture.
A kind of use in cancer of the esophagus personalised drug of the horizontal personalised drug screening reagent box of three-dimensional micro-assembly robot
Method, comprise the following steps：
Before experiment, first serum, antibiotic are added in basal medium, are made into complete medium, it is standby.
(1) it is under aseptic condition, the fresh human esophageal carcinoma PBS performed the operation or tissue biopsy obtains is fully clear
Wash, cut off fiber part, necrotic zone and sludged blood, again with PBS 1~2 time.
(2) after weighing, with 10 × it is dual anti-flood tissue, soak 5min.Use PBS.
(3) tissue block is placed in 9cm culture dishes, is cut into 0.5~1mm3Fritter.It is transferred in centrifuge tube.It is clear with PBS
Wash.
(4) 0.05% pancreatin is added, 4 DEG C stand 6~18 hours.Pancreatin dosage：10mL/g is organized.
(5) unnecessary pancreatin liquid is carefully removed, leaves behind the pancreatin being stained with tissue surface.37 DEG C of digestion 20-30min.
(6) complete medium in equal volume containing 10% serum is added.It is repeatedly soft to blow and beat for several times.Precipitate 2min, centrifugation
Remove liquid.
(7) addition is with nutrient solution or the PBS final concentration of 200U/mL prepared clostridiopetidase A 3mL, and soft piping and druming is for several times.
It is placed in 25 bottles of T, 37 DEG C of digestion 2h.Gently blow and beat.
(8) filtered with 100 μm of cell strainers, collect filtered fluid.
(9) 50~100g centrifuge 3min, discard supernatant.
(10) 2mL complete mediums are added, fully blow and beat cell dispersion, are counted.By cell suspension adjust to 100,000~
500000/mL, 20 μ L are taken out with hydrogel according to 1:1 ratio mixes, and is inoculated in 96 orifice plates.10~60 minutes, treat that gel is consolidated
Change.
(11) 200 μ L complete mediums are added.37 DEG C are placed in, 5%CO2Cultivated 24~72 hours in incubator.
(12) treat to adhere to growth in gel by cell that (such as Fig. 2 is obtained with the esophageal cancer cell of this kit culture patient
Cancer of the esophagus 3D tumours micro-assembly robot) next step experiment can be carried out.
(13) culture matrix, the medicine that addition is dissolved with complete medium, per the μ L of hole 100 are discarded.Control group is added and is free of
The complete medium of medicine.3~5 repetitions of every group of setting.Culture 48 hours.
(14) nutrient solution is gently sopped up, with PBS twice.
(15) the μ L of alamarBlue 100 are added, 37 DEG C of incubation 4h, blank pair are used as using the alamarBlue of no cell
According to.
(16) light absorption value surveyed at 570nm and 600nm.Light absorption value is corrected with OD 570nm-OD 600nm.
(17) cytoactive is calculated according to the following formula
Cytoactive (%)=(ODsample–OD0)/(ODcontrol–OD0)×100。ODsample、ODcontrolAnd OD0Generation respectively
The light absorption value of table sample sheet, control group and alamarBlue.
(18) with T-test statistical methods analysis experimental result, the antitumous effect of medicine is ranked up, you can sieve
The sensitive medicine of patient is selected, personalized therapy program is customized for patient.
Table 3 is to detect the medicine of obtained certain Patients With Carcinoma of Esophagus to three kinds of Chemotherapy in Esophageal Cancer schemes with this kit in embodiment
Quick result
| Medicine | Cell viability (%) | Susceptibility sorts |
| Cis-platinum+5-FU | 26.87±3.33 | 1 |
| Taxol+cis-platinum | 48.66±2.32 | 3 |
| Taxol+carboplatin | 55.71±1.90 | 2 |
Table 3.
Claims (10)
1. based on the horizontal screening anti-tumor medicine kit of three-dimensional micro-assembly robot, it is characterised in that including Three-dimensional cell culture material
Material；Histocyte dispersing agent；Protease inhibitors；Basal cell culture medium；Serum；Antibiotic；Balanced salt solution；Cell is lived
Power detection reagent；Cell filtering net.
2. kit according to claim 1, it is characterised in that the Three-dimensional cell culture material includes suspension culture plate,
Hanging drop culture plate, microfluid dimensional culture plate and corollary apparatus, hydrogel, dimensional culture magnetic bead and corollary apparatus, solid three
Dimensional scaffold material, the one or more in Tissue Culture Plate.
3. kit according to claim 2, it is characterised in that the Three-dimensional cell culture material is the suspension culture of 3D micropores
Plate, or hydrogel and Tissue Culture Plate using polysaccharide and/or polypeptide as main component are used cooperatively.
4. kit according to claim 1, it is characterised in that
The histocyte dispersing agent is trypsase, EDTA- trypsase, clostridiopetidase A, neutral proteinase, pronase
Enzyme, papain, DNA enzymatic, hyaluronidase, pancreatin, TrypLE, Trypzean, Liberase, Blendzyme,
One or more in Accutase；The protease inhibitors is pancreatin neutralizer, Aprotinin, aprotinin, pancreatin suppression
Preparation, the one or more of protease inhibitors；
The basal cell culture medium includes：BME cell culture mediums, IMDM cell culture mediums, the cell culture mediums of RMPI 1640,
MEM cell culture mediums, DMEM cell culture mediums, F10 cell culture mediums, F12 cell culture mediums, DMEM/F12 cell culture mediums,
McCoy's 5A cell culture mediums, 199 cell culture mediums, Fisher ' s cell culture mediums, CMRL cell culture mediums, GMEM cells
Culture medium, Leibovitz's L-15 cell culture mediums, the cell culture mediums of MCDB 131, one kind in Ames' cell culture mediums
It is or a variety of；
The serum includes：Cow's serum, horse serum, horse serum, rabbit anteserum, chicken serum, Swine serum, other animal blood serums, synthesis
Serum replacement, the one or more in serum substitute；The antibiotic includes：Penicillin, streptomysin, gentamicin, put
Line rhzomorph, neomycin, kanamycins, polymyxins, nystatin, the one or more in anphotericin；The balance salt is molten
Liquid includes PBS, D-PBS, Hank ' s, the one or more in D-Hank ' s, EBBS；The cell filtering net includes：Sterile yarn
One or more in the reticular fibre of cloth, plastic cell screen pack, metal cell screen pack and/or other materials；
The cell viability detection reagent includes：AlamarBlue, trypan blue, MTT reagents, XTT reagents, WST-1 reagents, CCK-
8 reagents, the one or more in live/dead cell viability detection reagents.
5. the application method of any kits of claim 1-4, it is characterised in that first, patient's tumor tissues are carried out
It is scattered, obtained tissue block, fragment of tissue, cell mass, and/or scattered individual cells；Then, patient's tumour after will be scattered
Tissue, cell sample are cultivated by the method for dimensional culture, cell is grown in three dimensions, are formed with spherical
And/or the three-dimensional structure of irregular three-dimensional shape；Finally, the tumour micro-assembly robot obtained with dimensional culture carries out antibiotics susceptibility test.
6. application method according to claim 5, it is characterised in that specifically comprise the following steps：
1) the flesh tissue sample from patient is soaked in antibiotic solution and carried out disinfection, and cleaned with balanced salt solution；
2) histocyte dispersing agent vitellophag interstitial is used；
3) cell filtration net filtration is used, obtains a diameter of 10~500 μm of cell mass or the cell of independent dispersion；
4) cell is seeded in dimensional culture material surface and/or inside, adds cell culture medium, carry out Three-dimensional cell culture；
5) cell is handled with antineoplastic drug solution to be measured；
6) with cell viability detection reagent processing cell；
7) line number is entered to treated cell with ELIASA, cell counter, laser confocal microscope and/or flow cytometer
According to collection；
8) using the signal and/or data analysis cell viability collected, suppression effect of the antineoplastic to tumour cell is obtained
Fruit conclusion.
7. application method according to claim 6, it is characterised in that the concentration of serum be 0%~99.9% between appoint
Meaning scope；Any range of the concentration of antibiotic between 0.00001mg/mL~10000mg/mL.
8. application method according to claim 7, it is characterised in that the concentration of serum be 5%~20% between it is any
Concentration；The concentration of antibiotic is 0.001mg/mL~100mg/mL.
9. application method according to claim 6, it is characterised in that described in the step 4) in Three-dimensional cell culture
Dimensional culture material is 3D micropore suspension culture plates, and it is 3000~8000/micropore to make final concentration of cells；Or dimensional culture water-setting
Gel, it is 50,000~250,000/mL to make final concentration of cells.
10. application method according to claim 6, it is characterised in that the step 4) Three-dimensional cell culture it is lasting when
Between be about 1~7 day.
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