CN103014118B - Cell co-culture method for screening anti-cancer medicament - Google Patents
Cell co-culture method for screening anti-cancer medicament Download PDFInfo
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- CN103014118B CN103014118B CN201310008125.4A CN201310008125A CN103014118B CN 103014118 B CN103014118 B CN 103014118B CN 201310008125 A CN201310008125 A CN 201310008125A CN 103014118 B CN103014118 B CN 103014118B
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Abstract
The invention discloses a cell co-culture method for screening an anti-cancer medicament, comprising the following steps: splitting an enzyme labeling hole strip, inputting the split enzyme labeling hole strip into a six-hole plate, inoculating each independent enzyme labeling hole against the cancer cell and the normal cell, adding a medicament to be detected into each enzyme labeling hole after all the cells adhere to the wall, adding DMEM (dulbecco modified eagle medium) which is a culture solution into the six-hole plate, co-culturing to suck all the culture solution, and proliferating cells and detecting the cytotoxicity or apoptosis of the cells by adopting the MTT, the trypan blue staining method, the CCK-8, the morphological observation method or the hoechst staining method so as to screen out the anti-cancer medicament. The cell co-culture method disclosed by the invention is easy to operate and is low in cost and can adopt the steps of disassembling, cleaning and reusing according to experiment needs. Due to the adoption of the method, more than two cells can be co-cultured, the efficiency of experiments is improved greatly, the research and development expenses of the anti-cancer medicament is saved, and the microenvironment generated in the human body can be simulated better.
Description
Technical field
The present invention relates to Cell Co culturing Techenique, especially a kind of co-culture of cells method for screening anticancer medicine.
Background technology
The whole world has more than 700 ten thousand people to die from cancer every year, and cancer therapy drug can be killed cancer cells on the one hand, promotes patient's physiological loading to recover normal; Also may produce but then serious toxic side effect, therefore, in screening anticancer medicine, it be carried out to preclinical cell toxicant, cell proliferation or apoptosis detects, safety evaluation is significant.
Most scientific research personnel carries out screening anticancer medicine, cell toxicant, cell proliferation or apoptosis and detects research in 96 orifice plates, because 96 orifice plates cannot be dismantled, the metabolite of cancer cells cannot act on normal cell, therefore, a kind of cell of single culture cannot be considered influencing each other between the interior cancer cells of body and normal cell metabolite, and the co-culture of cells method in this invention has made up the deficiency of this respect.Cultivate cell though be useful on the Transwell of co-culture of cells on market, it is expensive, and cannot realize the common cultivation of two or more attached cells, has limited the development of co-culture of cells research.
Summary of the invention
The object of the invention is: a kind of co-culture of cells method for screening anticancer medicine is provided, the microenvironment that it generates in can better analogue body, improve greatly conventional efficient, save cancer drug development expense, there is feature with low cost, simple to operate, reliable for effect.
The present invention is achieved in that the co-culture of cells method for screening anticancer medicine, after enzyme mark hole bar is split, puts into 6 orifice plates, and volume is to 100ul, and density is 2 × 10
4the cancer cells of individual/ml and normal cell are inoculated into respectively in each independently enzyme mark hole, after all cell attachments, in each enzyme mark hole, add 10ul medicine to be measured, in 6 orifice plates, add DMEM nutrient solution 8ml again, enzyme mark hole is immersed in nutrient solution, cultivates altogether after 48h, exhaust nutrient solution, adopt mtt assay, trypan blue staining, CCK-8 method, morphological observation method or hoechst staining to carry out cell toxicant, cell proliferation or apoptosis detection, thereby realize the screening of cancer therapy drug.
Before enzyme mark hole bar is split, in the ethanol that is first 75% by enzyme mark hole in mass percent, soak 1.5～2.5h, then blower fan open mode blowing down 1h in Biohazard Safety Equipment, then irradiate 1～1.5h under ultraviolet ray.
Before enzyme mark hole bar is split, under aseptic condition, be coated with enzyme mark hole with gelatin or 100ug/ml poly-l-lysine.To promote cell attachment.
Described specifically refer to the coated enzyme mark of gelatin hole, gelatin is dissolved in ultrapure water, be mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, be under 121 ℃, the pressure condition that is 100～110KPA in temperature, sterilizing 30min, will take out through autoclaved gelatin, obtain the gelatin preparing; Under aseptic condition, to the gelatin that adds 15 ul to prepare in each enzyme mark hole, will at the bottom of enzyme mark hole, all cover; The CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used aseptic PBS or ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment.
Described specifically refer to the coated enzyme mark of poly-lysine hole, get molecular weight and be 15～300,000 poly-l-lysine, be dissolved in PBS or ultrapure water, be made into the storage liquid of 1mg/ml, storage liquid is filtered and is placed in-20 ℃ of refrigerators and stores, when use by concentration dilution to 100ug/ml; Then under aseptic condition to the poly-l-lysine that adds 15 microlitre 100ug/ml in each enzyme mark hole, to at the bottom of enzyme mark hole, all cover, again 37 ℃ of CO2gas incubator are put into 5 hours in the enzyme mark hole that has been coated with poly-l-lysine or spend the night after take out, by aseptic PBS or ultrapure water washed twice, for next step inoculating cell experiment.
Described aseptic PBS is, take 1.6g sodium-chlor, 0.04g Repone K, 0.04g potassium primary phosphate, 0.44g Sodium phosphate dibasic (molecular formula band 7 in conjunction with water) or 0.588g Sodium phosphate dibasic (molecular formula band 12 in conjunction with water), add ultrapure water, dissolve and be settled to 200ml, autoclaving saves backup at 4 ℃.
Described common cultivation refers to trypsinase and digests respectively and collect cancer cells and normal cell, counts, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively 10ul medicine to be measured, and there is cell culture fluid as without medicine control group take what do not add medicine, take acellular nutrient solution as blank group, put into the CO2gas incubator that 37 ℃ of gas concentration lwevels are 5% and cultivate altogether
The product that pancreatin can adopt hyclone company to produce, also can prepare voluntarily, and compound method is: claim 0.25g trypsinase powder to add in 100mlPBS, 4 ℃ are spent the night, the membrane filtration of 0.22um, and-20 ℃ save backup.
Owing to having adopted technique scheme, compared with prior art, the co-culture of cells method that the present invention adopts is simple to operate, the enzyme mark hole bar using, ethanol, gelatin, 6 orifice plates or 24 orifice plates are cheap, can need to dismantle according to experiment, clean, recycling, and can realize the common cultivation of two or more cells, wherein the 6 every holes of orifice plate can hold 10 enzyme mark holes simultaneously, can inoculate 10 kinds of cells simultaneously, improve greatly conventional efficient, save cancer drug development expense, in addition, the microenvironment that this method generates in can better analogue body, at observation of cell and cell, interaction between cell and culture environment and inquire into cancer therapy drug mechanism of action and the medical field such as the target spot that may act on will be widely used.The inventive method is simple, and result of use is good.
Accompanying drawing explanation
Fig. 1 is the impact (400 ×) of different concns Stink Bug hemolymph effect 48h on SGC-7901 cellular form;
A1. be control group; A2 ~ A4. is respectively 20mg/L, 30mg/L, 40mg/L Stink Bug hemolymph treatment group;
Fig. 2 is the impact (400 ×) of different concns Stink Bug hemolymph effect 48h on BGC-823 cellular form;
B1. be control group; B2 ~ B4 is respectively 20mg/L, 30mg/L, 40mg/L Stink Bug hemolymph treatment group;
Fig. 3 is the restraining effect of Stink Bug hemolymph effect 24h to two strain cell proliferations;
Fig. 4 is the restraining effect of Stink Bug hemolymph effect 48h to two strain cell proliferations;
Fig. 5 is the restraining effect of Stink Bug hemolymph effect 72h to two strain cell proliferations;
Fig. 6 is SGC-7901 cell Hoechst33258 fluorescent dye (400 ×);
A. without medicine control group; B.20mg/ml drug dose group; C.30mg/ml drug dose treatment group; D40 mg/ml drug dose group;
Fig. 7 is rabbit aorta unstriated muscle CCC-SMC-1 cell Hoechst33258 fluorescent dye;
A. without medicine control group; B.20mg/ml drug dose group; C.30mg/ml drug dose group; D.40 mg/ml drug dose group.
Embodiment
Embodiments of the invention 1: morphological observation method detects the gastric carcinoma cell lines BGC-823 of Aspongopus extract to common cultivation and the restraining effect of SGC-7901 in-vitro multiplication, SGC-7901 BGC-823 and SGC-7901 are to form by building after the low differentiation adenocarcinoma of stomach of human body and the nodus lymphoideus transferring rate of human stomach gland cancer respectively, are representing that gastric epithelial cell cancerates and shifted for two stages.In the ethanol that is 75% in mass percent by enzyme mark hole bar, soak 2h, put in Biohazard Safety Equipment and dry up, the lower 1h that irradiates of ultraviolet ray, gelatin is dissolved in ultrapure water, is mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, be under 121 ℃, the pressure condition that is 100～110KPA in temperature, sterilizing 30min, will take out through autoclaved gelatin, obtain the gelatin preparing; Under aseptic condition, all cover at the bottom of by enzyme mark hole to the gelatin that adds in each enzyme mark hole 15 ul to prepare; The CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment; This enzyme mark hole bar is splitted into 3,3 hole, and 6 orifice plates are put in 11, hole, get SGC-7901 cell and BGC-823 Cells, count, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively liquid of haemolymph (medicine to be measured) 10ul of different concns, in reaction system, hemolymph final concentration is 20mg/L, 30mg/L, 40mg/L, 5 repeating holes are set, 1 blank, in 6 orifice plates, add 8ml nutrient solution again, enzyme mark hole is immersed in nutrient solution, under the gas concentration lwevel that 37 ℃, volumetric concentration are 5% in CO2gas incubator, cultivates altogether after 48h the state (seeing accompanying drawing 1,2) of observation of cell under inverted microscope.
Embodiments of the invention 2:MTT method detects the gastric carcinoma cell lines BGC-823 of Aspongopus extract to common cultivation and the restraining effect of SGC-7901 in-vitro multiplication, SGC-7901 BGC-823 and SGC-7901 are to form by building after the low differentiation adenocarcinoma of stomach of human body and the nodus lymphoideus transferring rate of human stomach gland cancer respectively, are representing that gastric epithelial cell cancerates and shifted for two stages.In the ethanol that is 75% in mass percent by enzyme mark hole bar, soak 2h, put in Biohazard Safety Equipment and dry up, the lower 1h that irradiates of ultraviolet ray, gelatin is dissolved in ultrapure water, is mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, be under 121 ℃, the pressure condition that is 100～110KPA in temperature, sterilizing 30min, will take out through autoclaved gelatin, obtain the gelatin preparing, under aseptic condition, all cover at the bottom of by enzyme mark hole to the gelatin that adds in each enzyme mark hole 15 ul to prepare, the CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment, this enzyme mark hole bar is splitted into 3,3 hole, and 6 orifice plates are put in 11, hole, get SGC-7901 cell and BGC-823 Cells, count, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively liquid of haemolymph (medicine to be measured) 10ul of different concns, in reaction system, hemolymph final concentration is 10mg/L, 20mg/L, 30mg/L, 40mg/L, 5 repeating holes are set, 1 blank, take acellular nutrient solution as blank group, in 6 orifice plates, add 8ml nutrient solution again, enzyme mark hole is immersed in nutrient solution, in CO2gas incubator 37 ℃, volumetric concentration is to exhaust nutrient solution after cultivating altogether 48h under 5% gas concentration lwevel, every hole adds the MTT solution 10 μ l of 5g/L, continue to hatch 4h at 37 ℃, every hole adds 100 μ l Formazan lysates, continue to cultivate 4h, abundant dissolving to be crystallized, measure OD value at wavelength 570nm place.The calculation formula of inhibiting rate is: inhibiting rate (%)=1-(medicine group OD value-blank OD value)/(control group OD value-blank group OD value)] × 100%(sees attached list 1, Fig. 3,4,5).
Embodiments of the invention 3:hoechst staining detects the cancer of the stomach SGC-7901 of Aspongopus extract to common cultivation and the cells apoptosis of rabbit aorta unstriated muscle CCC-SMC-1, in the ethanol that is 75% in mass percent by enzyme mark hole bar, soak 2h, put in Biohazard Safety Equipment and dry up, the lower 1h that irradiates of ultraviolet ray, gelatin is dissolved in ultrapure water, be mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, it is 121 ℃ in temperature, pressure is under the condition of 100～110KPA, sterilizing 30min, to take out through autoclaved gelatin, the gelatin that acquisition prepares, under aseptic condition, all cover at the bottom of by enzyme mark hole to the gelatin that adds in each enzyme mark hole 15ul to prepare, the CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment, this enzyme mark hole bar is splitted into 3,3 hole, and 6 orifice plates are put in 11, hole, get SGC-7901 cell and rabbit aorta unstriated muscle CCC-SMC-1 cell, count, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively liquid of haemolymph (medicine to be measured) 10ul of different concns, in reaction system, hemolymph final concentration is 20mg/L, 30mg/L, 40mg/L, 5 repeating holes are set, 1 blank, take acellular nutrient solution as blank group, in 6 orifice plates, add 8ml nutrient solution again, enzyme mark hole is immersed in nutrient solution, in CO2gas incubator 37 ℃, volumetric concentration is to exhaust nutrient solution after cultivating altogether 48h under 5% gas concentration lwevel, in each enzyme mark hole, add 0.5ml stationary liquid, fix 15 minutes, remove stationary liquid, wash twice with PBS, each 3 minutes, exhaust liquid, add 0.5mlHoechst33258 staining fluid, dye 5 minutes, remove staining fluid, wash twice, each 3 minutes with PBS, exhaust liquid, drip anti-quenching of fluorescence mounting liquid, fluorescence microscopy Microscopic observation, excitation wavelength 350nm, emission wavelength 460nm (seeing accompanying drawing 6,7).
Embodiments of the invention 4:MTT method detects the cancer of the stomach SGC-7901 of Aspongopus extract to common cultivation and the cytotoxicity of rabbit aorta unstriated muscle CCC-SMC-1, in the ethanol that is 75% in mass percent by enzyme mark hole bar, soak 2h, put in Biohazard Safety Equipment and dry up, the lower 1h that irradiates of ultraviolet ray, gelatin is dissolved in ultrapure water, be mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, be under 121 ℃, the pressure condition that is 100～110KPA in temperature, sterilizing 30min, to take out through autoclaved gelatin, obtain the gelatin preparing, under aseptic condition, all cover at the bottom of by enzyme mark hole to the gelatin that adds in each enzyme mark hole 15 ul to prepare, the CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment, this enzyme mark hole bar is splitted into 3,3 hole, and 6 orifice plates are put in 11, hole, get SGC-7901 cell and rabbit aorta unstriated muscle CCC-SMC-1 cell, count, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively liquid of haemolymph (medicine to be measured) 10ul of different concns, in reaction system, hemolymph final concentration is 20mg/L, 30mg/L, 40mg/L, 5 repeating holes are set, 1 blank, take acellular nutrient solution as blank group, in 6 orifice plates, add 8ml nutrient solution again, enzyme mark hole is immersed in nutrient solution, in CO2gas incubator 37 ℃, volumetric concentration is to exhaust nutrient solution after cultivating altogether 48h under 5% gas concentration lwevel, every hole adds the MTT solution 10 μ l of 5g/L, continue to hatch 4h at 37 ℃, every hole adds 100 μ l Formazan lysates, continue to cultivate 4h, abundant dissolving to be crystallized, measure OD value at wavelength 570nm place.The calculation formula of inhibiting rate is: inhibiting rate (%)=1-(medicine group OD value-blank OD value)/(control group OD value-blank group OD value)] × 100%
Embodiments of the invention 5:CCK-8 method detects the cancer of the stomach SGC-7901 of Aspongopus extract to common cultivation and the cytotoxicity of rabbit aorta unstriated muscle CCC-SMC-1, in the ethanol that is 75% in mass percent by enzyme mark hole bar, soak 2h, put in Biohazard Safety Equipment and dry up, the lower 1h that irradiates of ultraviolet ray, gelatin is dissolved in ultrapure water, be mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, be under 121 ℃, the pressure condition that is 100～110KPA in temperature, sterilizing 30min, to take out through autoclaved gelatin, obtain the gelatin preparing, under aseptic condition, all cover at the bottom of by enzyme mark hole to the gelatin that adds in each enzyme mark hole 15ul to prepare, the CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment, this enzyme mark hole bar is splitted into 3,3 hole, and 6 orifice plates are put in 11, hole, get SGC-7901 cell and rabbit aorta unstriated muscle CCC-SMC-1 cell, count, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively liquid of haemolymph (medicine to be measured) 10ul of different concns, in reaction system, hemolymph final concentration is 20mg/L, 30mg/L, 40mg/L, 5 repeating holes are set, 1 blank, take acellular nutrient solution as blank group, in 6 orifice plates, add 8ml nutrient solution again, enzyme mark hole is immersed in nutrient solution, in CO2gas incubator 37 ℃, volumetric concentration is to exhaust nutrient solution after cultivating altogether 48h under 5% gas concentration lwevel, add the new nutrient solution of 100ul to every enzyme mark hole, add CCK solution 10 μ l simultaneously, in incubator, hatch 4h, measure OD value at wavelength 450nm place.The calculation formula of inhibiting rate is: inhibiting rate (%)=1-(medicine group OD value-blank OD value)/(control group OD value-blank group OD value)] × 100%.
Claims (7)
1. for a co-culture of cells method for screening anticancer medicine, it is characterized in that: after enzyme mark hole bar is split, put into 6 orifice plates, volume is to 100ul, density is 2 × 10
4the cancer cells of individual/ml and normal cell are inoculated into respectively in each independently enzyme mark hole, after all cell attachments, in each enzyme mark hole, add 10ul medicine to be measured, in 6 orifice plates, add DMEM nutrient solution 8ml again, enzyme mark hole is immersed in nutrient solution, cultivates altogether after 48h, exhaust nutrient solution, carry out cell proliferation, cell toxicant or apoptosis detects by mtt assay, trypan blue staining, CCK-8 method, morphological observation method or hoechst staining, thereby realize the screening of cancer therapy drug.
2. the co-culture of cells method for screening anticancer medicine according to claim 1, it is characterized in that: before enzyme mark hole bar is split, in the ethanol that is first 75% by enzyme mark hole in mass percent, soak 1.5～2.5h, then blower fan open mode blowing down 1h in Biohazard Safety Equipment, then irradiate 1～1.5h under ultraviolet ray.
3. the co-culture of cells method for screening anticancer medicine according to claim 1, is characterized in that: before enzyme mark hole bar is split, be coated with enzyme mark hole, to promote cell attachment under aseptic condition with gelatin or 100ug/ml poly-l-lysine.
4. the co-culture of cells method for screening anticancer medicine according to claim 3, it is characterized in that: described specifically refer to the coated enzyme mark of gelatin hole, gelatin is dissolved in ultrapure water, be mixed with the gelatin solution of 0.2g/100ml, after gelatin dissolves completely, be under 121 ℃, the pressure condition that is 100～110KPA in temperature, sterilizing 30min, to take out through autoclaved gelatin, obtain the gelatin preparing; Under aseptic condition, to the gelatin that adds 15 ul to prepare in each enzyme mark hole, will at the bottom of enzyme mark hole, all cover; The CO2gas incubator of the enzyme mark hole that has been coated with gelatin being put into 37 ℃ was again taken out after 1 hour, finally used aseptic PBS or ultrapure water washed twice, and each 100ul, for next step inoculating cell experiment.
5. the co-culture of cells method for screening anticancer medicine according to claim 3, it is characterized in that: described specifically refer to the coated enzyme mark of poly-lysine hole, get molecular weight and be 15～300,000 poly-l-lysine, be dissolved in PBS or ultrapure water, be made into the storage liquid of 1mg/ml, storage liquid is filtered and is placed in-20 ℃ of refrigerators and stores, when use by concentration dilution to 100ug/ml; Then under aseptic condition to the poly-l-lysine that adds 15 microlitre 100ug/ml in each enzyme mark hole, to at the bottom of enzyme mark hole, all cover, again 37 ℃ of CO2gas incubator are put into 5 hours in the enzyme mark hole that has been coated with poly-l-lysine or spend the night after take out, by aseptic PBS or ultrapure water washed twice, for next step inoculating cell experiment.
6. according to the co-culture of cells method for screening anticancer medicine described in claim 4 or 5, it is characterized in that: described aseptic PBS is to take 1.6gNaCl, 0.04gKCl, 0.04gKH
2pO
4, 0.44gNa
2hPO
47H
2o, adds ultrapure water, dissolves and is settled to 200ml, and autoclaving saves backup at 4 ℃.
7. the co-culture of cells method for screening anticancer medicine according to claim 1, is characterized in that: described common cultivation refers to trypsinase and digests respectively and collect cancer cells and normal cell, counts, respectively with 2 × 10 with under inverted phase contrast microscope
4the concentration of individual/ml is inoculated in each enzyme mark hole, every hole 100ul, after cell attachment, add respectively 10ul medicine to be measured, and there is cell culture fluid as without medicine control group take what do not add medicine, take acellular nutrient solution as blank group, put into the CO2gas incubator that 37 ℃ of gas concentration lwevels are 5% and cultivate altogether.
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Non-Patent Citations (6)
| Title |
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| 一种电镜下观察贴壁培养细胞原位形态的新方法;赵永岐等;《军事医学科学院院刊》;20040831;第28卷(第4期);397-398 * |
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| 杨军等.相互间非接触性共培养对神经干细胞和骨髓基质细胞的影响.《南方医科大学学报》.2010,第30卷(第4期),823-826. |
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| 蒋泽生等.人骨髓来源多能成体祖细胞与人肝细胞体外直接共培养向肝样细胞分化的实验研究.《广东医学》.2007,第28卷(第2期),202-204. |
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