CN102174381A - Culture dish containing micro spherical arrays and application thereof - Google Patents
Culture dish containing micro spherical arrays and application thereof Download PDFInfo
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- CN102174381A CN102174381A CN2011100376158A CN201110037615A CN102174381A CN 102174381 A CN102174381 A CN 102174381A CN 2011100376158 A CN2011100376158 A CN 2011100376158A CN 201110037615 A CN201110037615 A CN 201110037615A CN 102174381 A CN102174381 A CN 102174381A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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Abstract
The invention relates to the field of bio-nanometer, in particular to a culture dish containing micro spherical arrays. The culture dish comprises a culture dish body 1 and a culture dish cover plate 2, wherein the surface of a base 3 of the culture dish body 1 is provided with at least one micro spherical array 4, and the diameter of a microsphere 5 in the micro spherical array 4 is of micron or nanometer grade. The invention also comprises a method for enhancing the voltage compliance type calcium ion channel functional responsibility of cells, which specifically comprises two steps of pre-treating with the culture dish and enhancing the voltage compliance type calcium ion channel functional responsibility of cells. The culture dish is prepared by a step-by-step etching method under an inclination condition; the micro spherical arrays of the culture dish are of micron or nanometer grade; the area of the prepared micro spherical arrays meets the requirements of culture dish application; and the culture dish has low production cost and is simple to operate. The method for enhancing the voltage compliance type calcium ion channel functional responsibility of cells by using the culture dish provides a new method and a new thought for drug screening.
Description
Technical field
The present invention relates to the biological nano field, particularly contain the culture dish and the utilization thereof of microballoon face array.
Background technology
Based on the biological detection of cell is that recent two decades comes the medicinal effect that the drug screening field grows up or the critical function screening means of toxicological effect.In this screening system, cell is integrated in the screening platform as a kind of biosensor, (high throughput screening HTS) also can be used as heavy body screening (high content screening, detection means HCS) both to have can be used as high flux screening.Compare to the biological chemistry detection means, cell screening method has from view of function and obtains the advantage of waiting to screen medicinal effect of molecule or toxicological effect, yet its screening cost but is markedly inferior to experimentation on animals.
In existing cell drug screening system, cell cultures adopts the common plane culture dish usually.Although existing substratum and chemical additive can satisfy the cultivation requirement of most cells in the human body, yet adopt the culture technique of plane culture dish clearly to ignore the micro physical property that cells in vivo is survived.Under the body situation, cells survival is in three-dimensional cell matrix, and this support matrix has complex surfaces topology microstructure.The difference of life condition has caused the culturing cell functional status to compare to inevitable difference in the body in vitro culture substrate and the body.For drug screening, the major reason that this may be that the screening flux is low, The selection result and later stage experimentation on animals result are not inconsistent.On the other hand, by culture dish substrate physical property, as the topological framework feature, design change the function responsiveness of screening cell, will provide new technological approaches for improving cell drug screening usefulness.
In recent years, the innovation that develops into drug screening technology of organizational project and cell engineering provides new opportunity.As, being established as in in-vitro simulated cells in vivo living environment of dimensional culture technology provides effective through engineering approaches means.Here the most successful example may be the multiple drug resistance research that three-dimensional tumour microballoon is used to tumor cell in vitro.On the other hand, the relation of topology microstructure and culturing cell growth behavior has had the research history of three more than ten years at the bottom of the substratum.Early stage research concentrates on the research of inoblast and osteocyte in a large number, and its result of study has been used to the structure of organizational project implant.Cell cultures substrate topological framework is owing to influence the form and the behaviour of cell, thereby is the important channel of analogue body inner cell microenvironment.For neurocyte, myocyte and gland cell etc. depend on ion channel function active excitable cell, the topological framework environment because influence cell adhesion, sprawl and geometric shape will may produce tangible influence to the function responsiveness of ionic channel, this has important application prospects in the screening of ionic channel targeted drug.
(voltage dependent calcium channel VDCC) is the important ionic channel of cell to the voltage-dependent calcium channel.Because it participates in the numerous physiological processs and the vital role in neural system and cardiovascular system diseases of cell, VDCC has become the important target effect passage of drug screening in pharmacy industry.With VDCC by the cell drug of target effect passage screening system at first require the employing target cell have expression and the corresponding VDCC function responsiveness of VDCC.Strong and weak or the size of VDCC function responsiveness directly has influence on the screening usefulness of medicine, as the screening flux and about drug effect and toxic information intensive amount.For the VDCC target passage drug screening based on neurocyte, the VDCC function responsiveness of cell lowly is the important factor that restriction screening usefulness improves.Cause that the low factor of neurocyte VDCC function responsiveness may comprise: (1) the neurocyte VDCC expression level that uses low; (2) differentiation condition that adopts is unfavorable for giving full expression to of neurocyte VDCC and ripe; (3) other ion channel function or the level of membrane potential of cell are unusual.Thereby strengthen the purpose that VDCC function responsiveness makes up the screening cell although adopt engineered method to reach, the genetic manipulation process is worth further estimating for the influence of The selection result.
Summary of the invention
In view of this, one of purpose of the present invention is to provide culture dish, the substrate of this culture dish contains sphere dense array structure, and this substrate contains sphere dense array structure can be supported the adhesion of institute's culturing cell and sprawl, and then strengthens the function responsiveness of screening cell VDCC.
For achieving the above object, technical scheme of the present invention is:
The culture dish that contains microballoon face array comprises culture dish body 1 and culture dish cover plate 2, and the substrate 3 of described culture dish body 1 is provided with at least one microballoon face array 4, and the diameter of microballoon 5 is micron or nano level in the described microballoon face array 4.
Further, described microballoon face array 4 is closely arranged for the microballoon 5 of spherical diameter 0.51-1.98 micron and is formed, and the spacing of two adjacent microballoons 5 in the described microballoon face array 4 is less than 0.5 micron;
Further, described microballoon 5 is little global 5-A and/or little hemisphere 5-B;
Further, the area of described microballoon face array 4 is at least greater than the area of single target cell;
Further, the area of described microballoon face array 4 is 0.25cm
2
Further, described culture dish also comprises liquid-inlet pipe 7 and drain pipe 8, and ware cover plate 2 is provided with connecting hole 6, and described liquid-inlet pipe 7 and drain pipe 8 stretch in the culture dish from connecting hole 6;
Further, the end that stretches in the culture dish of described drain pipe 8 contacts with substrate 3;
Further, the connecting hole on the culture dish cover plate 2 is provided with seal washer 9.
Two of purpose of the present invention is that the culture dish that provides a kind of utilization to contain microballoon face array strengthens the method that cell voltage is comply with formula calcium channel function responsiveness, and this method is simple to operate, and effect is obvious.
For achieving the above object, technical scheme of the present invention is:
Use described culture dish to comply with the method for formula calcium channel function responsiveness strengthening cell voltage, specifically may further comprise the steps:
The pre-treatment of A culture dish
With mounting lining liquid lining is mounted in the substrate 3 of described culture dish, got pretreated culture dish;
The B cell voltage is comply with the enhancing of formula calcium channel function responsiveness
To have the target cell that the voltage-dependent calcium channel expresses and in pretreated culture dish, cultivate at least 24 hours, and impose depolarize stimulate voltage-dependent calcium channel function responsiveness enhanced target cell.
Further, described culture dish is comply with the utilization of formula calcium channel function responsiveness strengthening cell voltage, specifically may further comprise the steps:
The pre-treatment of A culture dish
With mounting lining liquid lining is mounted in the substrate 3 of described culture dish, is mounted the lining time to be at least 2 hours, pretreated culture dish;
The B cell cultures
The neurocyte that will have the expression of voltage-dependent calcium channel is inoculated in through the pretreated culture dish of steps A;
The C cell voltage is comply with the enhancing of formula calcium channel fluorescence response
Cultured cells is behind the calcium ion fluorescent dyeing in the microballoon face array culture dish, gather the metabolic defect in cellular calcium ion fluorescent signal with common fluorescent microscope, laser confocal microscope, orifice plate reader or other calcium ion fluorescence records devices, imposing high potassium depolarize in signal acquisition process stimulates, and gets enhanced voltage-dependent calcium channel fluorescence response.
Beneficial effect of the present invention is: this culture dish adopts the step-etching method under the tilt condition to be prepared from, and its microballoon face diameter is micron or nano level, and the microballoon face array of preparation can reach 0.10 to 0.25 cm
2The big area scope, reach the requirement that culture dish is used, its production cost is low, and is simple to operate; This culture dish can significantly increase neurocyte VDCCs function responsiveness: in the time of the 0th day, 7 days, 14 days, more common culture dish can significantly improve the VDCC fluorescence response of target cell to the culture dish that contains microballoon face array in cell differentiation of nerve cord; The method of using this culture dish enhancing cell voltage to comply with formula calcium channel function responsiveness provides novel method and thinking for drug screening.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is the synoptic diagram that contains the culture dish body of microballoon face array;
Fig. 2 is the synoptic diagram that contains the culture dish cover plate of microballoon face array;
Fig. 3 is the culture dish substrate synoptic diagram that contains little global sphere array;
Fig. 4 is the culture dish substrate synoptic diagram that contains little hemisphere sphere array;
Fig. 5 is ENStem-A
TMHuman nerve stem cell is mounted the culture dish of lining substrate and the stereoscan photograph in little global sphere array substrate culture dish at the plane polystyrene.Be shown in the 2nd day (breaking up the 0th day) behind cell inoculation cell and mount the upgrowth situation of culture dish (a), the little global sphere array substrate culture dish of 1.98 μ m (b) and the little global sphere array substrate culture dish of 0.51 μ m (c) of lining substrate in the plane polystyrene, scale shows 25 μ m.
Embodiment
In order to make the purpose, technical solutions and advantages of the present invention clearer, below the preferred embodiments of the present invention are described in detail.
Embodiment 1 contains the preparation of the culture dish of microballoon face array
One material is prepared:
1.35 mm Tissue Culture Dish (Corning, the U.S.); Cell cultures six orifice plates (Nest Biotech Co., Ltd, China)
2. diameter is that the circular lid slide of 25 mm (is gone up the Nereid and taken turns industrial Glass Co., Ltd., China.Or Fisher Scientific, the U.S.)
3. polystyrene microsphere (Bangs Laboratories, Inc., the U.S..Or aladdin, China), diameter is respectively 0.51 micron and 1.98 ± 0.20 microns.
4.2.5% polystyrene solution.Get one of ordinary polystyrene Tissue Culture Dish, take by weighing polystyrene 2.5 grams after shredding, add the dissolving of 100mL toluene.The dark bottles of putting into polystyrene solution after the dissolving keeps in Dark Place standby.
5.10.16% polystyrene solution.Get one of ordinary polystyrene Tissue Culture Dish, take by weighing polystyrene 10.16 grams after shredding, add the dissolving of 100mL toluene.The dark bottles of putting into polystyrene solution after the dissolving keeps in Dark Place standby.
6.1.7% polystyrene microsphere work suspension
10% polystyrene microsphere suspension (supply of material concentration), 40 μ L
Distilled water 200 μ L
Behind the mixing concentration is 1.7% microballoon work suspension.
7. sloping platform is made by oneself.
8. polydimethylsiloxane [poly (dimethylsiloxane), PDMS] (Sylgard 184, Dow Corning, the U.S.)
Two, contain the design and the preparation of the culture dish of microballoon face array:
1. contain the culture dish design of microballoon face array
The culture dish body 1 that contains microballoon face array of the present invention sees Fig. 1 for details; Culture dish cover plate 2 sees Fig. 2 for details.Should be pointed out that the design carries out design description according to common 35mm culture dish.As change culture dish and orifice plate size or have the culture dish orifice plate (as 6 orifice plates, 12 orifice plates etc.) of a plurality of repeat unit structures and possess the rights protection scope that the culture dish of rights protection feature of the present invention and orifice plate design still belong to this patent.
1) contain the culture dish of microballoon face array, comprise culture dish body 1 and culture dish cover plate 2, the substrate 3 of described culture dish body 1 is provided with at least one microballoon face array 4, and diameter of micro ball is micron or nano level in the described microballoon face array 4, sees Fig. 1 for details.
2) described microballoon face array 4 is closely arranged for the microballoon 5 of spherical diameter 0.51-1.98 micron and is formed, and the spacing of two adjacent microballoons 5 in the described microballoon face array 4 is less than 0.5 micron;
3) the described culture dish that contains microballoon face array, described microballoon face 5 are little global 5-A and/or little hemisphere 5-B; Wherein, little whole world is meant miniature spheroid, and little hemisphere is meant miniature hemisphere.
4) the described culture dish that contains microballoon face array, the area of described microballoon face array 4 are at least greater than the area of single target cell;
5) the described culture dish that contains microballoon face array, the area of described microballoon face array 4 is 0.25cm
2
6) the described culture dish that contains microballoon face array, described culture dish also comprises liquid-inlet pipe 7 and drain pipe 8, described liquid-inlet pipe 7 and drain pipe 8 stretch in the culture dish from the connecting hole 6 on the culture dish plate lid 2.Do not opening under the condition that leaves standstill of culture dish cover plate, adopting suction function can drain whole liquid of culture dish body 1 down by drain pipe 8.Under the culture condition of non-detection, culture dish also can not use liquid-inlet pipe and drain pipe, directly changes liquid by opening the culture dish cover plate;
7) end that the described culture dish that contains microballoon face array, described drain pipe 8 stretch in the culture dish contacts with substrate 3;
8) the described culture dish that contains microballoon face array, the connecting hole on the culture dish cover plate 2 is provided with seal washer 9.Seal washer 9 is being connected of drain pipe 8 and liquid-inlet pipe 7 and soft silicone tube fixedly.Liquid drainage tube and sample introduction pipe make can finish liquid changing, cleaning and application of sample under the condition of leaving standstill in the fluoroscopic examination process, avoided mobile culture dish to cause that the detected visual field changes the influence to fluoroscopic examination.
The culture dish body that contains microballoon face array sees Fig. 1 for details;
The culture dish cover plate that contains microballoon face array sees Fig. 2 for details;
The culture dish substrate that contains little global sphere array sees Fig. 3 for details;
The culture dish substrate that contains little hemisphere sphere array sees Fig. 4 for details;
Above-mentioned microballoon face diameter can be micron order or submicron order.
Above-mentioned culture dish also can directly be designed to the structure that there is microballoon face array 4 on substrate 3 surfaces.
2, the preparation process that contains the culture dish of microballoon face array
Should be pointed out that as preferential exemplifying embodiment, is that the example description has the culture dish of microballoon face array substrate and the preparation process of orifice plate with 35mm culture dish and six orifice plates here.With other size or other by way of the little global sphere of realizing or little hemisphere sphere dense array substrate culture dish or orifice plate also within the protection of this patent authority.Now be described below with regard to this part implementation step.
For obtaining to contain little global sphere dense array substrate culture dish or orifice plate, step
With
Can successively exchange, the slim polystyrene that promptly can bond is earlier mounted culture dish or the orifice plate bottom of the cover glass of lining in perforate, constitute the plane polystyrene and mount the culture dish of lining substrate, carry out the preparation of little global sphere array again at basal surface, must contain the culture dish of little global sphere dense array substrate.
Little global sphere array substrate culture dish and little hemisphere sphere array substrate culture dish are the enhancing cell voltage and comply with the culture dish of formula calcium channel function responsiveness.From ordinary cells size (10-20 micron) and spherical diameter yardstick of the present invention (0.51-1.98 micron), as long as in the cell cultivation process cell can not be deep into the size in gap between the sphere and spherical diameter all can, and little global sphere array and little hemisphere sphere array should be the cell culturing surfaces of identical function.
The culture dish for preparing is carried out disinfection after the germicidal treatment, can be used for cell cultures and detection.
The enhancing cell voltage that embodiment 2 contains the culture dish of microballoon face array is comply with the method for formula calcium channel function responsiveness
The above culture dish that contains little global sphere array is carried out the evaluation of cell cultures and voltage-dependent calcium channel function responsiveness, confirm the implementation result of present method.Should be understood that, for the culture dish of little hemisphere sphere array, the topological microenvironment of neurocyte perception and little global sphere array are just the same, thereby because the yardstick of cell and growth behavior have determined cell can not be attached at the difference of this partial geometry form of bottom perception of microballoon and little hemisphere.The inventor has reason fully to infer that hemisphere sphere dense array has identical cytobiology effect with little global sphere dense array topological framework.
One, material is prepared:
1.ENStem-A
TMHuman nerve stem cell (Millipore company, the U.S.)
2. poly-ornithine (poly-L-ornithine hydrobromide) (Sigma product, the U.S.)
3. concentration is that the poly-ornithine of 40 μ g/mL is mounted lining liquid
Get poly-ornithine supply of material ampere bottle (containing poly-ornithine freeze-dried preparation 50mg), add the 5mL sterile purified water, the vortex mixing is made the storing solution that concentration is 10mg/mL.Above-mentioned storing solution is packed as 16 μ L/eppendorf pipe.Get the above-mentioned eppendorf pipe that contains 16 μ L poly-ornithine storing solutions, add the sterile purified water of 4mL, it is that the poly-ornithine of 40 μ g/mL is mounted lining liquid that the vortex mixing is made concentration.
4. mouse basement membrane layer Fibronectin [from EHS rat meat knurl (Engelbreth-Holm-Swarm murine sarcoma)] (Sigma product, the U.S.)
5. concentration is that the mouse basement membrane layer Fibronectin of 5 μ g/mL is mounted lining liquid
With concentration is 1mg/mL(supply of material concentration) mouse basement membrane layer Fibronectin storing solution be packed as 10 μ L/eppendorf pipe.Get the above-mentioned eppendorf pipe that contains 10 μ L mouse basement membrane layer Fibronectin storing solutions, add the sterile purified water of 2mL, it is that the mouse basement membrane layer Fibronectin of 5 μ g/mL is mounted lining liquid that the vortex mixing is made concentration.
6. concentration is the L-glutaminate (Sigma-Aldrich product, the U.S.) of 200mM
7.B27 additive (Invitrogen product, the U.S.)
8. concentration is recombinant human leukaemia inhibitory factor (recombinant human leukemia inhibitory factor, LIF) (Chemicon product, the U.S.) of 10 μ g/mL
9.Neurobasal basic medium (Invitrogen product, the U.S.)
10. Prostatropin (basic fibroblast growth factor, bFGF) (Sigma-Aldrich product, the U.S.)
11. bovine serum albumin (bovine serum albumine, BSA) (precious bio tech ltd, China are matched in Yancheng)
12.bFGF storing solution
Take by weighing bovine serum albumin 1g, add 100mL Neurobasal basic medium, to make the Neurobasal substratum that contains 1%BSA after the 0.2 micron pore size filter membrane sterile filtration.
Get bFGF supply of material ampere bottle (including 25 μ g bFGF freeze-dried preparation), add the Neurobasal substratum that 0.5 mL contains 1%BSA, making concentration is the bFGF storing solution of 50 μ g/mL.
13. neural stem cell amplification cultivation liquid
Concentration is L-glutaminate 0.5 mL of 200mM
B27 additive 1.0 mL
Concentration is the LIF 50 μ L of 10 μ g/mL
Concentration is the bFGF storing solution 20 μ L of 50 μ g/mL
Be settled to 50 mL with the Neurobasal basic medium
The final concentration that above-mentioned amplification cultivation liquid contains additive is: L-glutaminate 2 mM; BFGF 20 ng/mL; LIF 10 ng/mL; B-27 additive 2%.
14. cell differentiation of nerve cord nutrient solution
Concentration is L-glutaminate 0.5 mL of 200mM
B27 additive 1.0 mL
Concentration is the LIF 50 μ L of 10 μ g/mL
Be settled to 50 mL with the Neurobasal basic medium
The final concentration that above-mentioned amplification cultivation liquid contains additive is: L-glutaminate 2 mM; LIF 10 ng/mL; B-27 additive 2%.
15.0.2M dimethyl arsenic acid buffer liquid (pH7.2)
First liquid: sodium dimethylarsonate 4.28g, adding distil water is to 100ml
Second liquid: 0.2N hydrochloric acid
Get first liquid 50mL, second liquid 4.2mL, be settled to 200mL, promptly get pH and be 7.2 0.2M dimethyl arsenic acid buffer liquid with distilled water.
16.0.1M dimethyl arsenic acid buffer liquid (pH7.2)
Get the dimethyl arsenic acid buffer liquid 50mL of above-mentioned 0.2M, be settled to 100mL with distilled water and get final product.
17.8% glutaraldehyde storing solution (Electron Microscopy Sciences product, the U.S.)
18. contain the 0.1M sodium cacodylate buffer liquid (pH 7.2) of 2% glutaraldehyde
Adopt following prescription:
8% glutaraldehyde storing solution 5 mL
0.2M dimethyl arsenic acid buffer liquid 10 mL
19.4% perosmic anhydride storing solution
Get perosmic anhydride 1g, be settled to 25mL with distilled water.
20. contain the 0.1M sodium cacodylate buffer liquid (pH 7.2) of 1% perosmic anhydride
Adopt following prescription:
4% perosmic anhydride storing solution 5 mL
0.2M dimethyl arsenic acid buffer liquid 10 mL
21. gradient dehydration Different concentrations of alcohol
Adopt following prescription:
22. there is not phenol red Neurobasal basic medium (Invitrogen product, the U.S.)
23. dimethyl sulfoxide (DMSO) (dimethyl sulphoxide, DMSO) (the difficult to understand safe chemical industry in Jinan company limited, China)
24.Pluronic F-127 (Molecular Probes product, the U.S.)
25. foetal calf serum (Tianjin Hao ocean biological products science and technology limited Company, China)
26.Calcium Green-1 AM(Molecular Probes product, the U.S.)
27.Calcium Green-1 AM storing solution
Get Calcium Green-1 AM supply of material ampere bottle (containing 50 μ g Calcium Green-1 AM), add the dissolving of 39 μ L DMSO vortexs and make the Calcium Green-1 AM storing solution that concentration is 1mM.
28.Pluronic F-127 storing solution
Take by weighing Pluronic F-127 0.2g in the eppendorf of 1.5 mL centrifuge tube, add the DMSO of 1mL, it is 20% Pluronic F-127 storing solution that the vortex dissolving makes concentration.
29. high potassium Neurobasal storing solution
Take by weighing 0.738g Repone K, be dissolved in the no phenol red Neurobasal basic medium of 20mL, make the high potassium Neurobasal storing solution that K+ concentration is about 500mM.
30.Calcium Green-1 AM staining fluid
Get one of the eppendorf centrifuge tube of 1.5 mL, add Calcium Green-1 AM storing solution 5 μ L, Pluronic F-127 storing solution 1 μ L, B27 additive 20 μ L successively and do not have phenol red Neurobasal basic medium 974 μ L, the vortex mixing makes 1mL Calcium Green-1 AM staining fluid.Calcium Green-1 AM staining fluid contains 5 μ M Calcium Green-1 AM, 2% B-27 additive and 0.02% Pluronic F-127.
31.Calcium Green-1 AM de-esterifying Incubating Solution
Get one of the eppendorf centrifuge tube of 1.5 mL, add Pluronic F-127 storing solution 1 μ L, B27 additive 20 μ L, foetal calf serum 30 μ L successively and do not have phenol red Neurobasal basic medium 949 μ L, the vortex mixing makes 1mL Calcium Green-1 AM de-esterifying Incubating Solution.Calcium Green-1 AM de-esterifying Incubating Solution contains 2% B-27 additive, 3% foetal calf serum and 0.02% Pluronic F-127.
Two, the cultivation of neurocyte in culture dish
ENStem-A
TMHuman nerve stem cell is available from U.S. Millipore company.According to cell supplier's suggestion, to mount lining liquid and concentration be that the mouse basement membrane layer Fibronectin of 5 μ g/mL is mounted lining liquid and mounted lining respectively at least 2 hours after concentration is the poly-ornithine of 40 μ g/ml earlier to be used for culture dish that ordinary cells culture dish that cell amplification cultivates and the little global sphere array substrate culture dish that is used for the cytodifferentiation experiment and plane polystyrene mount the lining substrate.Neural stem cell increases with neural stem cell amplification cultivation liquid in the common culture dish of 35 mm, changes liquid next day of cell.When the cytogamy degree reaches 90-100%, make cell suspension with mechanical process.With about 1.2 * 10
6Cell inoculation is in new culture dish.For Analytical Chemical Experiment, cell be inoculated in through poly-ornithine and mouse basement membrane layer Fibronectin mount respectively containing of lining little global sphere array substrate culture dish and the plane polystyrene mount the culture dish of lining substrate.Behind cell inoculation 2 days (differentiation back the 0th day), nutrient solution is replaced by the cell differentiation of nerve cord nutrient solution.The 0th, 6,7,11 and 13 change liquid entirely after differentiation, partly change liquid after differentiation on the the 2nd, 4 and 9 day.In cell cultivation process, culture dish and little global sphere array substrate culture dish that the plane polystyrene is mounted the lining substrate all can adopt common culture dish lid, and must not adopt the special culture dish cover plate 2 that contains liquid-inlet pipe and drain pipe and seal washer.Cellular form and growth behavior are observed with Nikon ECLIPSE TE300 inverted microscope and are carried out microimaging with Nikon D100 digital camera.
Fig. 5 shows ENStem-A
TMHuman nerve stem cell is mounted the upgrowth situation of culture dish (a), the little global sphere array substrate culture dish of 1.98 μ m (b) and the little global sphere array substrate culture dish of 0.51 μ m (c) of lining substrate the 2nd day (breaking up the 0th day) at the plane polystyrene in the inoculation back.The array region that shows sphere array type culture dish of the present invention among the figure is the same with planar substrates, adhesion that can sustenticular cell and sprawling.
Three, contain of the influence of the culture dish of little global sphere array to neural stem cell VDCCs function responsiveness
The evaluation of VDCCs function responsiveness adopts the dynamic response of flowing in the metabolic defect in cellular calcium ion under the high potassium solution of 50 mM stimulates to estimate.It is worthy of note that as a kind of preferential embodiment and for estimating the implementation result that the present invention contains little global sphere array substrate culture dish more accurately, following embodiment adopts laser confocal microscope to carry out.In view of the common fluorescence reading instrument detection principle identical, have reason to believe and adopt this quasi-instrument can obtain identical implementation result with laser confocal microscope.
The the 0th, 7 and 14 day of cell differentiation of nerve cord, the plane polystyrene that takes out culturing cell is mounted the culture dish and the culture dish that contains little global sphere array of lining substrate, common culture dish lid is replaced by the special culture dish cover plate 2 that contains liquid-inlet pipe and drain pipe and seal washer carries out following operation and detection.The culture dish that the plane polystyrene is mounted the lining substrate cleans 2 times through there being phenol red Neurobasal basic medium with containing in the culture dish of little global sphere array cultured cells, dyes 1 hour in 37 ℃ of carbonic acid gas incubators with Calcium Green-1 AM staining fluid.Cell does not clean the back and hatches at 37 ℃ of carbonic acid gas incubators in 1mL Calcium Green-1 AM de-esterifying Incubating Solution again and carried out the dyestuff de-esterifying in 45 minutes there to be phenol red Neurobasal basic medium.With the scanning of 488 nm argon lasers, gather the laser co-focusing image with the long logical filter disc of 515 nm.Image acquisition rates is per 3 seconds 1 width of cloth.In image acquisition process, in cell Incubating Solution (being Calcium Green-1 AM de-esterifying Incubating Solution), add the high potassium Neurobasal of 100 μ L storing solution and make Incubating Solution K from the liquid-inlet pipe of culture dish cover plate 2
+Final concentration is 50 mM.VDCCs function responsiveness show as that under high potassium unpolarizing intracellular calcium concentration increases and the cell that causes thus in Calcium Green-1 fluorescence intensity increase.With Calcium Green-1 fluorescence intensity the time is mapped, trying to achieve high potassium stimulation back Calcium Green-1 fluorescence intensity is the relative response amplitude of VDCCs with respect to the relative increasing degree that stimulates preceding fluorescence intensity.
Table one is ENStem-A
TMHuman nerve stem cell is mounted the culture dish and the suprabasil VDCCs function of the culture dish responsiveness that contains the little global sphere array of 1.98 μ m of lining substrate at the plane polystyrene.The relative increased value of fluorescence intensity under the VDCCs reactivity stimulates with the high potassium solution of 50mM in the table is an index.Cell have reactive basis for estimation be under high potassium solution stimulates cell fluorescence intensity increase by 15% or more than.By this standard, no matter break up preceding or differentiation back the 7th day and the 14th day, mount the culture dish of lining substrate or the suprabasil cell response ratio of the little global sphere array culture dish of 1.98 μ m all near 100% at the plane polystyrene.Shown in the table mean value of the relative increased value of cell fluorescence intensity.As seen from the table, mount the relative increased value of cell fluorescence intensity (0.81 ± 0.53) and the suprabasil analog value of culture dish (0.83 ± 0.79) there was no significant difference that contains the little global sphere array of 1.98 μ m in the culture dish substrate of lining substrate (P〉0.2) at differentiation frontal plane polystyrene.At the 7th day that breaks up, the VDCCs function responsiveness (0.95 ± 0.46) that the plane polystyrene is mounted cell in the culture dish substrate of lining substrate nothing before the differentiation obviously increases (P〉0.20), and cell VDCCs function responsiveness (1.20 ± 0.48) obviously increases (P＜0.01) in the culture dish substrate of the little global sphere array of 1.98 μ m before differentiation but contain.Differentiation the 14th day, the plane polystyrene is mounted the twice above (P＜0.01) before the VDCCs function responsiveness (2.68 ± 1.21) of cell in the culture dish substrate of lining substrate increases to differentiation, and contain cell VDCCs function responsiveness (5.12 ± 2.42) in the culture dish substrate of the little global sphere array of 1.98 μ m increase to before the differentiation more than 5 times (P＜0.01).At the 7th day and the 14th day of differentiation, contain in the culture dish substrate of the little global sphere array of 1.98 μ m cell VDCCs function responsiveness and mount the suprabasil analog value of culture dish that serves as a contrast substrate than the plane polystyrene and obviously increase (P＜0.01).
Table one ENStem-A
TMHuman nerve stem cell is mounted the comparison of the suprabasil VDCCs function of the culture dish responsiveness of lining substrate (the high potassium of 50mM stimulate under Calcium Green-1 fluorescence intensity increased value) containing little global sphere array substrate of 1.98 μ m and plane polystyrene
Table two shows ENStem-A
TMHuman nerve stem cell is mounted culture dish substrate that serves as a contrast substrate and the suprabasil VDCCs function of the culture dish responsiveness that contains the little global sphere array of 0.51 μ m at the plane polystyrene.By aforesaid standards, before the differentiation or differentiation back the 7th day and the 14th day, the suprabasil cell response ratio of culture dish of mounting the culture dish substrate of lining substrate or containing the little global sphere array of 0.51 μ m at the plane polystyrene is equally near 100%.As seen from the table, before differentiation, contain the suprabasil analog value of culture dish (0.76 ± 0.60) that the relative increased value of cell fluorescence intensity (1.03 ± 0.78) is mounted the lining substrate than the plane polystyrene in the culture dish substrate of the little global sphere array of 0.51 μ m and obviously increase (P＜0.01).At the 7th day that breaks up, the VDCCs function responsiveness (0.82 ± 0.31) that the plane polystyrene is mounted cell in the culture dish substrate of lining substrate nothing before the differentiation obviously increases (P〉0.20), and cell VDCCs function responsiveness (1.30 ± 0.29) obviously increases (P＜0.01) in the culture dish substrate of the little global sphere array of 0.51 μ m before differentiation but contain.Meanwhile, contain the suprabasil analog value of culture dish that cell VDCCs function responsiveness is mounted the lining substrate than the plane polystyrene in the culture dish substrate of the little global sphere array of 0.51 μ m and obviously increase (P＜0.01).Differentiation the 14th day, the suprabasil analog value of culture dish that the plane polystyrene is mounted the VDCCs function responsiveness (1.40 ± 0.95) of cell in the culture dish substrate of lining substrate and contained the little global sphere array of 0.51 μ m before the differentiation and differentiation further increased (P＜0.01) on the 7th day, do not have remarkable significant difference (P〉0.20) but contain the suprabasil analog value of culture dish that cell VDCCs function responsiveness in the culture dish substrate of the little global sphere array of 0.51 μ m mounts the lining substrate than the plane polystyrene.
Table two ENStem-A
TMHuman nerve stem cell is mounted the comparison of the suprabasil VDCCs function of the culture dish responsiveness (Calcium Green-1 fluorescence intensity increased value under the high potassium stimulation of 50mM) of lining substrate at culture dish substrate that contains the little global sphere array of 0.51 μ m and plane polystyrene
At neurocyte is that the raising of voltage-dependent calcium channel function responsiveness will greatly improve relevant fluorescent screening efficient in the cell drug screening on basis.Neural stem cell has good representativeness, and the present invention is an example with the neurocyte only, but is not limited only to neurocyte, and its atomization has been represented the different developmental phases of cell.The present invention has designed the culture dish that contains micro-sphere array on this basis by the substep plasma etching, microspheres prepared face array area has reached the culture dish application requiring, and feasible application with microballoon face array structure enhancing voltage-dependent calcium channel function responsiveness becomes possibility.Exemplifying embodiment shows that the sphere dense array type culture dish substrate of micron order (1.98 μ m) and submicron order (0.51 μ m) has the voltage-dependent of enhancing calcium channel function responsiveness effect, has reached its intended purposes.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (10)
1. the culture dish that contains microballoon face array, comprise culture dish body (1) and culture dish cover plate (2), it is characterized in that: the substrate (3) of described culture dish body (1) is provided with at least one microballoon face array (4), and the diameter of microballoon (5) is micron or nano level in the described microballoon face array (4).
2. the culture dish that contains microballoon face array according to claim 1, it is characterized in that: described microballoon face array (4) is that the microballoon (5) of 0.51-1.98 micron is closely arranged and formed by diameter, and the spacing of two adjacent microballoons (5) in the described microballoon face array (4) is less than 0.5 micron.
3. the culture dish that contains microballoon face array according to claim 1 is characterized in that: described microballoon (5) is little whole world (5-A) and/or little hemisphere (5-B).
4. the culture dish that contains microballoon face array according to claim 1 is characterized in that: the area of described microballoon face array (4) is at least greater than the area of single target cell.
5. the culture dish that contains microballoon face array according to claim 4 is characterized in that: the area of described microballoon face array (4) is 0.25cm
2
6. the culture dish that contains microballoon face array according to claim 1, it is characterized in that: described culture dish also comprises liquid-inlet pipe (7) and drain pipe (8), be provided with connecting hole (6) on the culture dish plate lid (2), described liquid-inlet pipe (7) and drain pipe (8) stretch in the culture dish from connecting hole (6).
7. the culture dish that contains microballoon face array according to claim 6 is characterized in that: the end that described drain pipe (8) stretches in the culture dish contacts with substrate (3).
8. the culture dish that contains microballoon face array according to claim 6 is characterized in that: the connecting hole on the culture dish cover plate (2) is provided with seal washer (9).
9. each described culture dish of utilization claim 1-8 is comply with the method for formula calcium channel function responsiveness strengthening cell voltage, it is characterized in that, specifically may further comprise the steps:
The pre-treatment of A culture dish
With mounting lining liquid lining is mounted in the substrate (3) of described culture dish, got pretreated culture dish;
The B cell voltage is comply with the enhancing of formula calcium channel function responsiveness
To have the target cell that the voltage-dependent calcium channel expresses and in pretreated culture dish, cultivate at least 24 hours, and impose depolarize stimulate voltage-dependent calcium channel function responsiveness enhanced target cell.
10. comply with the method for formula calcium channel function responsiveness according to the described culture dish of the described utilization of claim 9 strengthening cell voltage, it is characterized in that, specifically may further comprise the steps:
The pre-treatment of A culture dish
With mounting lining liquid lining is mounted in the substrate (3) of described culture dish, is mounted the lining time to be at least 2 hours, pretreated culture dish;
The B cell cultures
The neurocyte that will have the expression of voltage-dependent calcium channel is inoculated in through the pretreated culture dish of steps A;
The C cell voltage is comply with the enhancing of formula calcium channel fluorescence response
Cultured cells is behind the calcium ion fluorescent dyeing in the microballoon face array culture dish, gather the metabolic defect in cellular calcium ion fluorescent signal with common fluorescent microscope, laser confocal microscope, orifice plate reader or other calcium ion fluorescence records devices, imposing high potassium depolarize in signal acquisition process stimulates, and gets enhanced voltage-dependent calcium channel fluorescence response.
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