CN106148285A - A kind of Three-dimensional cell culture matrix for screening anti-tumor medicine system and preparation thereof - Google Patents
A kind of Three-dimensional cell culture matrix for screening anti-tumor medicine system and preparation thereof Download PDFInfo
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Abstract
The invention discloses a kind of novel matrix for tumour cell dimensional culture, this kind of matrix can be used for parcel tumour cell and carries out dimensional culture, thus carries out the screening of antineoplastic further.Its preparation is to be mixed in the proper ratio by sodium alginate soln, sodium hyaluronate solution and extracellular medium matter Matrigel, then wraps up cell.Sodium alginate can directly wrap up cell, but is unfavorable for propagation and the differentiation of tumour cell, and Matrigel is as a kind of comparatively ideal three-dimensional cell cultivation matrix but its expensive bad mechanical strength, is unfavorable for screening anti-tumor medicine and pharmacological research.Present invention improves the mechanical performance of matrix, effectively facilitate the formation of cultured cell in vitro institutional framework simultaneously, reduce the use even substituting Matrigel, greatly reduce the cost of tumour cell dimensional culture, it is easy to carry out heterograft and zoopery to three-dimensional cell, and preparation method is simple, practical.
Description
Technical field
The present invention relates to a kind of biomaterial for tumour cell dimensional culture, be specifically related to the three-dimensional cell cultivation matrix that sodium alginate, Sodium Hyaluronate and Matrigel form with debita spissitudo and ratio.
Background technology
Cancer is the malignant disease of serious threat human health, and the World Health Organization estimates that the whole world in 2008 has 7,600,000 cancer patients dead, in state-owned 1,960,000 people, account for the 25.89% of whole world number of cancer deaths.The whole nation the 3rd time cause of the death is looked back sample investigation data and is shown, with the development of China's hygiene industry and improving constantly of medical technique level, some disease such as acute infectious disease, mother and baby's disease etc. have obtained the control of significant effective, mortality of malignant tumors then in obvious ascendant trend, has become as one of main cause of death affecting China's residents ' health.The World Health Organization (WHO) scholarly forecast, the year two thousand twenty population in the world 8,000,000,000, cancer new cases are up to 20,000,000, and 12,000,000 people die from cancer, and cancer will become first killer of the new century mankind, and become one of maximum public health problem in the whole world.Therefore, effective means research and development antineoplastic is taked just to seem of crucial importance.
In pharmacological evaluation, two dimension cell experiment is the most frequently used.But, two dimension cell is cultivated can not analog cell grows in vivo realistically microenvironment, and then cause the metabolism etc. of cell and the internal cell expression at cytomorphology, propagation and differentiation capability, cell viability, gene and albumen, stress ability to external world and the medicine of two dimension cultivation all to there is bigger difference, this greatly limits genetic development.And in terms of new drug development, tend to that false positive occurs by the medicine that cell in vitro screens.With the development of cell culture technology, three-dimensional cell cultivation technology reaches its maturity, and has shown good effect in terms of drug screening and pharmacological research.This technology is compared with cell monolayer, and its vitro Drug the selection result more can be consistent with experimental result in animal body.Experimental studies have found that, during antineoplastic and pharmacological research, use dimensional culture technology can improve the uniformity with the experiment effect in animal body to drug screening, the tumor cell culture technology imitating tissue with blood circulation especially researched and developed after repeatedly improving, can further improve the uniformity of external experiment in vivo.Therefore, use this imitative tissue three-dimensional cell culture technology, carry out Vitro Tumor medicine susceptibility and quick and precisely screen, important scientific meaning and using value are had to the treatment of malignant tumour Personalized Drug Administration.
It is one of current three-dimensional cell cultivation model that matrix covers culture model, and so-called matrix covers to cultivate and refers to that covering one layer of collagen in the bottom of cell culture apparatus forms substrate glue, makes cell grow in substrate glue, and differentiation forms three-dimensional structure.Matrix cover culture model essence be analog cell growth external environment (ECM), make cell be grown in in internal similar microenvironment.Internal cell be grown in by ECM around three-dimensional cell in, other cellular infiltration is in serum and tissue fluid, serum and in containing the factor that can promote that fibroblast breed, and tissue homeostasis can be translated into typical Wound healing and bone regeneration from normal differentiation function and react.The method is suitable in common lab carrying out, with a wide range of applications.But, it is expensive at present more ripe and conventional three-dimensional cell cultivation matrix Matrigel, and mechanical performance is poor, experimental cost can be caused high and be unfavorable for the experiment heterograft of dimensional culture multicellular animal, this is unfavorable for the popularization and application of three-dimensional cell cultivation technology.
In order to solve this problem, mixed in the proper ratio by sodium alginate soln, sodium hyaluronate solution and extracellular medium matter Matrigel, improve the mechanical performance of matrix, effectively facilitate the formation of cultured cell in vitro institutional framework simultaneously, reduce the use even substituting Matrigel, greatly reduce the cost of tumour cell dimensional culture, it is simple to heterograft and zoopery are carried out to three-dimensional cell, and preparation method is simple, practical.
Content of the invention
The invention aims to reduce even replacement use in three-dimensional cell cultivation for the Matrigel, preferable effect is reached while reducing cost, strengthen the performance of three-dimensional cell cultivation matrix so that it is can directly be inoculated into animal In vivo culture simultaneously, reduce the step of experiment and loaded down with trivial details.
Technical scheme
The preparation of a kind of three-dimensional cell cultivation matrix for screening anti-tumor medicine system, uses moderately viscous alginate to be dissolved in HEPES buffer solution, high pressure steam sterilization then filtration treatment.2% alginate solution, sodium hyaluronate solution and matrigrl are mixed in the proper ratio (operation on ice), finally mix with cell suspending liquid, after mixing, place 4 DEG C of 10min, instilling mixed liquor in 0.1M CaCl2 solution, both are cross-linked to form the milky globule being enclosed with cell.
The preparation and application of above-mentioned a kind of matrix for tumour cell dimensional culture, specifically includes following steps:
(1) preparation of solution used in preparation process
The preparation of 0.1M CaCl2 solution weighs 5.5g CaCl2 solid and is dissolved in 500ml deionized water, stirring and dissolving, filters with 0.22 μm of filter, obtains aseptic CaCl2 solution.
The preparation of 0.15M NaCl-0.025M HEPES solution weighs 8.775g sodium chloride and 5.958g HEPES is dissolved in 1L deionized water simultaneously, stirring and dissolving, filters with 0.22 μm of filter, obtains the preparation of aseptic 0.15M NaCl-0.025M HEPES solution.
The preparation of 2% alginate solution weighs 1g Sodium Alginate powder and is slowly added to while stirring in the 0.15M NaCl-0.025MHEPES solution of 50 ° of heating water baths, and high pressure steam heating naturally cools to room temperature, filters with 0.22 μm of filter.
The preparation of 10mg/ml sodium hyaluronate solution weighs 200mg Hyal powder, adds 20ml phosphate buffer (PH7.4), overnight, filters with 0.22 μm of filter.
(2) cell is wrapped up with mixed gel
On ice by sodium hyaluronate solution and the 2% moderately viscous Sodium Alginate proportioning in certain proportion of matrigel and 10mg/ml, form mixed gel, then mix with the cell suspension of debita spissitudo and volume, then stand 10min on ice to eliminate bubble.The content finally making Matrigel is 8%-23% (v/v), the final concentration of 2-8mg/ml of Sodium Hyaluronate, the final concentration of 0.3%-1.5% of Sodium Alginate.Finally, the gel mixed liquor hanging drop being mixed with cell is instilled 0.1M CaCl2In solution, stand 10-20min, form milky alginate globule.Milky globule is moved to respectively washed once in phosphate buffer and serum free medium through the little spoon of sterilizing, each 3min.Finally, immigration culture dish add fresh complete medium to cultivate.
In above-mentioned preparation process, the content of matrigel, Sodium Hyaluronate and Sodium Alginate and ratio are wanted suitably, if matrigel and the too high levels of Sodium Hyaluronate, can be greatly increased cost, if content is too low, cell cannot grow into and the two dimension visibly different three-dimensional structure of cell.If the content of Sodium Alginate directly affects the mechanical performance of the milky alginate globule being enclosed with cell, too high globule is easily rupturable, it is impossible to the growth for cell provides support, and too low, the rigidity of globule is had a surplus and flexible deficiency, and globule is easily pulverized.Proportioning between matrigel, Sodium Hyaluronate and Sodium Alginate is formed into the key of above-mentioned a kind of applicable tumour cell dimensional culture matrix.
Compared with prior art, there is advantages that
(1) present invention uses Sodium Hyaluronate and Sodium Alginate good biocompatibility, to cytotoxic side effect, friendly to human and environment, and price is relatively low, greatly reduces the cost of three-dimensional cell cultivation.
(2) present invention uses the three-dimensional cell cultivation matrix that matrigel, Sodium Hyaluronate and Sodium Alginate proportioning in the proper ratio is formed, and reaches good three-dimensional cell cultivation effect, is suitable for the dimensional culture of kinds of tumor cells, applied range.
(3) present invention instills, after using the three-dimensional cell cultivation matrix parcel cell that matrigel, Sodium Hyaluronate and Sodium Alginate proportioning in the proper ratio is formed, the milky globule good mechanical property being formed in calcium chloride solution, it is easy to carry out zoopery heterograft, and it is convenient to reclaim cell, it is easy to carry out subsequent analysis to the cell of dimensional culture.
(4) preparation process of the present invention is simple and easy to control, reproducible.
Brief description
Fig. 1 PC3 cells paraffin section H.E. dyeing after 14 days under three-dimensional cell cultivation matrix condition, microscope is observed.
Fig. 2 PC3 cells is under two dimension condition of culture, and microscope is observed.
Fig. 3 DU145 cells paraffin section H.E. dyeing after 14 days under three-dimensional cell cultivation matrix condition, microscope is observed.
Fig. 4 DU145 cells is under two dimension condition of culture, and microscope is observed.
The N-Cadherin immunofluorescence dyeing picture of the paraffin section that Fig. 5 is PC3 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Fig. 5-1 and Fig. 5-2 is paraffin section N-Cadherin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The N-Cadherin immunofluorescence dyeing picture of Fig. 6 cell climbing sheet that is PC3 cell under two dimension cultivation, and Fig. 6-1 and Fig. 6-2 be respectively PC3 cell under two dimension cultivation the N-Cadherin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of the paraffin section that Fig. 7 is PC3 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Fig. 7-1 and Fig. 7-2 is paraffin section Vimentin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of Fig. 8 cell climbing sheet that is PC3 cell under two dimension cultivation, and Fig. 8-1 and Fig. 8-2 be respectively PC3 cell under two dimension cultivation the Vimentin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
The N-Cadherin immunofluorescence dyeing picture of the paraffin section that Fig. 9 is DU145 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Fig. 9-1 and Fig. 9-2 is paraffin section N-Cadherin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The N-Cadherin immunofluorescence dyeing picture of Figure 10 cell climbing sheet that is DU145 cell under two dimension cultivation, and Figure 10-1 and Figure 10-2 be respectively PC3 cell under two dimension cultivation the N-Cadherin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of the paraffin section that Figure 11 is DU145 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Figure 11-1 and Figure 11-2 is paraffin section Vimentin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of Figure 12 cell climbing sheet that is DU145 cell under two dimension cultivation, and Figure 12-1 and Figure 12-2 be respectively PC3 cell under two dimension cultivation the Vimentin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
Detailed description of the invention
Above-mentioned purpose of the present invention is achieved by the following technical programs:
Embodiment 1
The three-dimensional cell cultivation matrix that matrigel, Sodium Hyaluronate and Sodium Alginate are mixed to get in appropriate proportions cultivates prostate cancer PC3 cells, then carries out H.E. dyeing, the form of observation of cell dimensional culture.Specifically comprise the following steps that
(1) sodium hyaluronate solution and 2% (W/V) the moderately viscous Sodium Alginate solution mixed proportion in the proper ratio of matrigel, 10mg/ml are mixed, the content finally making matrigel is 10%, the final concentration of 2.5mg/ml of Sodium Hyaluronate, final concentration of 0.5% (W/V) of Sodium Alginate.
(2) mixed liquor obtaining described in above-mentioned (1) is positioned over 10min on ice, to eliminate bubble, then to wrap up PC3 cell, make 1.0x106Cells/ milliliter mixed liquor.
(3) the mixed liquor hanging drop containing cell obtaining described in (2) is entered 0.1M CaCl2In solution, stand 10-20min, form milky Sodium Alginate globule.
(4) each once with the culture medium washing Sodium Alginate globule of phosphate buffer and serum-free, each 3min.
(5) Sodium Alginate globule dislocation 100mn culture dish will add fresh culture cultivate.Example of spatial compartmentalizationis.After 14 days, Sodium Alginate globule phosphate buffer is washed 3 times, each 3min, then fixing overnight with 10% neutral formalin.
(6) the Sodium Alginate globule after fixing is washed 3 times by phosphate buffer, each 5min.
(7) it is dehydrated.Globule after washing is put into 30%, and 50%, 70%, 80%, 90% ethanol solutions at different levels are dehydrated each 40min, are respectively put into 95% and 100% ethanol two grades, every grade of 20min.
(8) transparent: 100% alcohol and dimethylbenzene equivalent mixed liquor soak globule 2 times, each 10min.Xylene soak 2 times, each 30min.
(9) saturating wax: globule is immersed 15min in dimethylbenzene and paraffin equivalent mixed liquor, places into the saturating each 20-30min of wax in paraffin I and paraffin II.Saturating wax is carried out in insulating box, temperature 55 DEG C.
(10) embed, section, then carry out hematoxylin eosin staining.Result is as shown in Figure 1.The cell cultivated with two dimension compares (Fig. 2), and in the matrix of three-dimensional cell cultivation of the present invention, the cellular morphology of growth is significantly different, defines three-dimensional architecture.
Embodiment 2
The three-dimensional cell cultivation matrix that matrigel, Sodium Hyaluronate and Sodium Alginate are mixed to get in appropriate proportions cultivates prostate cancer DU145 cells, then carries out H.E. dyeing, the form of observation of cell dimensional culture.Specifically comprise the following steps that
(1) sodium hyaluronate solution and 2% (W/V) the moderately viscous Sodium Alginate solution mixed proportion in the proper ratio of matrigel, 10mg/ml are mixed, the content finally making matrigel is 10%, the final concentration of 2.5mg/ml of Sodium Hyaluronate, final concentration of 0.5% (W/V) of Sodium Alginate.
(2) mixed liquor obtaining described in upper is positioned over 10min on ice, to eliminate bubble, then to wrap up PC3 cell, make 1.0x106Cells/ milliliter mixed liquor.
(3) the mixed liquor hanging drop containing cell obtained above is entered 0.1M CaCl2In solution, stand 10-20min, form milky Sodium Alginate globule.
(4) each once with the culture medium washing Sodium Alginate globule of phosphate buffer and serum-free, each 3min.
(5) Sodium Alginate globule dislocation 100mn culture dish will add fresh culture cultivate.Example of spatial compartmentalizationis.
After (6) 14 days, Sodium Alginate globule phosphate buffer is washed 3 times, each 3min, then fixing overnight with 10% neutral formalin.
(7) the Sodium Alginate globule after fixing is washed 3 times by phosphate buffer, each 5min.
(8) it is dehydrated.Globule after washing is put into 30%, and 50%, 70%, 80%, 90% ethanol solutions at different levels are dehydrated each 40min, are respectively put into 95% and 100% ethanol two grades, every grade of 20min.
(9) transparent.100% alcohol and dimethylbenzene equivalent mixed liquor soak globule 2 times, each 10min.Xylene soak 2 times, each 30min.
(10) saturating wax.Globule is immersed 15min in dimethylbenzene and paraffin equivalent mixed liquor, places into the saturating each 20-30min of wax in paraffin I and paraffin II.Saturating wax is carried out in insulating box, temperature 55 DEG C.
(11) embed, section, then carry out hematoxylin eosin staining.Result is as shown in Figure 3.The cell cultivated with two dimension compares (Fig. 4), and in the matrix of three-dimensional cell cultivation of the present invention, the cellular morphology of growth is significantly different, defines three-dimensional architecture.
Embodiment 3
Immunofluorescence dyeing Germicidal efficacy N-Cadherin, Vimentin expression situation
The N-Cadherin immunofluorescence dyeing picture of the paraffin section that Fig. 5 is PC3 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Fig. 5-1 and Fig. 5-2 is paraffin section N-Cadherin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The N-Cadherin immunofluorescence dyeing picture of Fig. 6 cell climbing sheet that is PC3 cell under two dimension cultivation, and Fig. 6-1 and Fig. 6-2 be respectively PC3 cell under two dimension cultivation the N-Cadherin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of the paraffin section that Fig. 7 is PC3 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Fig. 7-1 and Fig. 7-2 is paraffin section Vimentin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of Fig. 8 cell climbing sheet that is PC3 cell under two dimension cultivation, and Fig. 8-1 and Fig. 8-2 be respectively PC3 cell under two dimension cultivation the Vimentin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
The N-Cadherin immunofluorescence dyeing picture of the paraffin section that Fig. 9 is DU145 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Fig. 9-1 and Fig. 9-2 is paraffin section N-Cadherin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The N-Cadherin immunofluorescence dyeing picture of Figure 10 cell climbing sheet that is DU145 cell under two dimension cultivation, and Figure 10-1 and Figure 10-2 be respectively PC3 cell under two dimension cultivation the N-Cadherin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of N-Cadherin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of the paraffin section that Figure 11 is DU145 cell after dimensional culture matrix of the present invention is cultivated 14 days, and Figure 11-1 and Figure 11-2 is paraffin section Vimentin immunofluorescence dyeing after dimensional culture matrix of the present invention is cultivated 14 days for the PC3 cell respectively and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
The Vimentin immunofluorescence dyeing picture of Figure 12 cell climbing sheet that is DU145 cell under two dimension cultivation, and Figure 12-1 and Figure 12-2 be respectively PC3 cell under two dimension cultivation the Vimentin immunofluorescence dyeing of cell climbing sheet and the picture of seedless dye and only core dye without the picture of Vimentin immunofluorescence label.
Result is as it can be seen, green fluorescence represents destination protein, blue expression nucleus.Be can be observed by immunofluorescence dyeing picture, the prostate gland cancer cell PC3 cells being cultivated by Three-dimensional cell culture matrix of the present invention and the expression of the biomarker such as N-Cadherin, Vimentin of DU145 cells raise than the expression under two dimension condition of culture.
Specific experiment step is as follows:
Prostate gland cancer cell dimensional culture paraffin section immunofluorescence dyeing
(1) by the three-dimensional cell cultivation matrix that gained matrigel of the present invention, Sodium Hyaluronate and Sodium Alginate are mixed to get in appropriate proportions, dimensional culture, example of spatial compartmentalizationis are carried out to prostate cancer PC3 cells and DU145 cells.
It after (2) 2 weeks, is enclosed with the milky alginates globule 3 times of cell, each 5min with phosphate buffer washing.Then fixing overnight with 4% paraformaldehyde 4 DEG C.After Gu Ding, milky alginates globule phosphate buffer is washed, then FFPE, section.
(3) paraffin section de-waxing is to water: section is put into dimethylbenzene I 15min-dimethylbenzene II 15min-absolute ethyl alcohol I 5min-absolute ethyl alcohol II 5min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5min-distillation washing successively.
(4) antigen retrieval: histotomy is placed in the reparation box filling with EDTA antigen retrieval buffer solution (PH8.0) and carries out antigen retrieval in micro-wave oven.In moderate heat 8min to boiling truce 8min, low fire 7min, should prevent buffer solution excessive vaporization, be sure not dry plate during this.Naturally after cooling down, slide is placed in and PBS (PH7.4) rocks on decolorization swinging table washing 3 times, each 5min.
(5) add one anti-: section is drawn a circle (preventing antibody from flowing away) by groupization pen after slightly drying around tissue, and dropping 3%BSA room temperature closes 30min in enclosing, get rid of the anti-covering tissue that BSA dropping dilutes by a certain percentage.Section lies against 4 DEG C of overnight incubation in wet box.
(6) add two anti-: slide is placed in PBS (PH7.4) and rocks washing 3 times, each 5min on decolorization swinging table.After section slightly drying, in circle, dropping and resists two anti-covering tissues of corresponding kind, lucifuge incubated at room 60min.
(7) slide is placed in PBS (PH7.4) on decolorization swinging table, rocks washing 3 times, each 5min.
(8) DAPI redyes nucleus: slide is placed in PBS (PH7.4) and rocks washing 3 times, each 5min on decolorization swinging table.Section drips DAPI dye liquor, lucifuge incubated at room 10min after slightly drying in circle.
(9) mounting: slide is placed in PBS (PH7.4) and rocks washing 3 times, each 5min on decolorization swinging table.With anti-fluorescent quenching mountant mounting after section slightly drying.
(10) microscopy is taken pictures: cuts into slices and observes under Nikon inverted fluorescence microscope and gather image.Such as Fig. 5, Fig. 5-1, Fig. 5-2, Fig. 7, Fig. 7-1, Fig. 7-2, Fig. 9, Fig. 9-1, Fig. 9-2, Figure 11, Figure 11-1, shown in Figure 11-2.
Prostate gland cancer cell two dimension cultured cells creep plate immunofluorescence dyeing
(1) passage, counting, creep plate.
(2) cell is fixed: 4% paraformaldehyde fixes 30min, and PBS washes 3 times, each 5min.
(3) cell rupture of membranes: creep plate is drawn a circle (preventing antibody from flowing away) by the position that groupization pen is evenly distributed at cover glass intermediate cell after slightly drying, and adds 50-100 μ l rupture of membranes working solution, incubated at room 10min, and PBS washes 3 times, each 5min.
(4) adding one anti-: remove PBS, having diluted by a certain percentage is anti-covers tissue.Creep plate lies against 4 DEG C of overnight incubation in refrigerator.
(5) add two anti-: creep plate PBS (PH7.4) washs 3 times, each 5min.After removing PBS, in circle, dropping resists two anti-coverings of corresponding kind to organize to one, lucifuge incubated at room 50min.
(6) DAPI redyes nucleus: creep plate PBS (PH7.4) washs 3 times, each 5min.Drip DAPI dye liquor, lucifuge incubated at room 10min in circle after removing PBS.
(7) mounting: creep plate PBS (PH7.4) washs 3 times, each 5min.Creep plate faces down have cell one after slightly drying, by anti-fluorescent quenching mountant, slide is locked in mounting on slide.
(8) microscopy is taken pictures: cuts into slices and observes under Nikon inverted fluorescence microscope and gather image.Result such as Fig. 6, Fig. 6-1, Fig. 6-2, Fig. 8, Fig. 8-1, Fig. 8-2, Figure 10, Figure 10-1, Figure 10-2, Figure 12, Figure 12-1, shown in Figure 12-2.
Claims (5)
1. a three-dimensional cell cultivation matrix, it is characterised in that can be used for the dimensional culture of solid tumor cell.
2. a three-dimensional cell cultivation matrix, it is characterised in that can be used for carrying out screening anti-tumor medicine.
3. a three-dimensional cell cultivation matrix, it is characterised in that: use matrigel, the natural polymer such as Sodium Hyaluronate and moderately viscous Sodium Alginate
Sub-material is a kind of three-dimensional cell cultivation matrix of raw material.
4. a three-dimensional cell cultivation matrix, it is characterised in that: use matrigel, Sodium Hyaluronate and the one that medium-viscosity Sodium Alginate is raw material
Three-dimensional cell cultivation matrix, the content of matrigel is 5%-30% (v/v), the final concentration of 2-10mg/ml of Sodium Hyaluronate, medium-viscosity
The final concentration of 0.1%-2.0% (w/v) of Sodium Alginate.
5. a three-dimensional cell cultivation matrix, it is characterised in that its preparation and application is: at transparent by matrigel and 10mg/ml on ice
Matter acid sodium solution and 2% moderately viscous Sodium Alginate proportioning in certain proportion, form mixed gel, then thin with debita spissitudo and volume
Born of the same parents' suspension mixes, and then stands 10min on ice to eliminate bubble.The content finally making Matrigel is 5%-40% (v/v), hyaluronic acid
The final concentration of 2-10mg/ml of sodium, the final concentration of 0.1%-2.0% of Sodium Alginate.Finally, the gel mixed liquor hanging drop being mixed with cell is dripped
Enter 0.1M CaCl2In solution, stand 10-20min, form milky alginate globule.Milky globule is little through sterilize
Spoon moves to respectively washed once in phosphate buffer and serum free medium, each 3min.Finally, move in culture dish add fresh complete
Full medium culture.
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