CN104130943B - Neuron and the orderly co-culture device of neurogliocyte, preparation method and neuron and the orderly co-culture method of neurogliocyte - Google Patents
Neuron and the orderly co-culture device of neurogliocyte, preparation method and neuron and the orderly co-culture method of neurogliocyte Download PDFInfo
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Abstract
The present invention relates to cell biology, disclose a kind of neuronal cell and the orderly co-culture device of neurogliocyte and its preparation method and application.This device includes substrate and polydimethylsiloxane seal, and described polydimethylsiloxane seal compound removedly is on the substrate；Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively；The distance of described first through hole and the second through hole is 500 μm～2000 μm.The present invention inoculates neuronal cell and neurogliocyte by the first through hole and the second through hole being perpendicular to substrate, and when two kinds of cells are inoculated in respective region, uniformity is easily controllable, and spendable cell concentration increases, and population effect is obvious, is beneficial to observational study.
Description
Technical field
The present invention relates to cell biology, be specifically related to a kind of device, its preparation method and neuron co-cultured in order for neuron and neurogliocyte and method that neurogliocyte co-cultures in order.
Background technology
Result of study in recent years shows, neuron and neurogliocyte not only existence mass transter, there is also information exchange.Between neuron and glial cell, how interaction mode affects its material, communication for information, and the change of they interaction modes becomes the another study hotspot of neuroscience with the multiple brain diseases such as correlation research such as alzheimer's disease, parkinsonism.
Being presently available for research neuron is that Banker is equal to the cultural method for the purpose of developing approach cell set up the nineties in 20th century with glial cell interaction co culture system in vitro system, there is scholar to improve on this basis, be successfully established the co-culture method of neuron and glial cell.But said method is with glial cell for trophoderm, in its surface seeding epineural unit cell, two kinds of unordered growths straggly of cell, it is difficult to be used for studying neuron and glial cell repercussion study.
The patent No. be 200710117815.8 and 200710119997.2 patent of invention establish in gold surface various kinds of cell co-culture device and method based on microflow control technique, substrate used by the method for these two kinds adhesions and manipulation various kinds of cell all adopts to be prepared in glass surface evaporation method one layer golden, this substrate needs to utilize high-vacuum apparatus to prepare at ultra-clean chamber, expensive, and layer gold character is unstable, cannot preserve for a long time, be unfavorable for promoting at biology laboratory.
The patent of invention that the patent No. is 201010105018.X discloses a kind of device establishing based on microflow control technique and studying various kinds of cell interaction at glass surface and its production and use, various kinds of cell is inoculated in different miniflow ducts until it after substrate adheres to by this method respectively, remove miniflow duct, it is achieved various kinds of cell interacts.Exist a lot of not enough when but this device co-cultures in order for neuron and neurogliocyte, first, inoculating cell in miniflow duct, cell is skewness in long and narrow duct；Second, cell is few owing to cultivating base unit weight in miniflow duct, and the cells survival time is very short, and the shorter time does not ensure that neuron and glial cell are simultaneously in the cultured time period；If ceaselessly to change culture medium, easily make cells float, final unstable dead；3rd, in miniflow duct, cell-volume is few, and the population effect for studying two kinds of born of the same parents' interactions is inconspicuous, is unfavorable for observational study.Further, since neuronal cell can not Secondary Culture, and said apparatus requires that the unsuitable interval of various cell inoculation times is long, and therefore, said apparatus is difficult to use in neuron and neurogliocyte repercussion study.
Summary of the invention
In view of this, it is an object of the invention to provide a kind of device co-cultured in order for neuron and neurogliocyte and preparation method thereof and neuron and method that neurogliocyte co-cultures in order, make device of the present invention easily controllable cell uniformity when inoculating neuronal cell with glial cell, and two kinds of cells inoculations interval time can be extended, realizing co-culturing in order of neuron and neurogliocyte, increase can use cell concentration.
For realizing above goal of the invention, the present invention provides following technical scheme:
A kind of device co-cultured in order for neuron and neurogliocyte, including substrate and polydimethylsiloxane seal, described polydimethylsiloxane seal compound removedly is on the substrate；
Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively；
The distance of described first through hole and the second through hole is 500 μm～2000 μm.
For the ease of understanding the inventive principle of device of the present invention, can referring to Fig. 1, it should be noted that, shape therein, size, material are not defined by accompanying drawing of the present invention, it only to facilitate understand apparatus of the present invention core technology on the basis that the claims in the present invention describe, those skilled in the art on the basis of accompanying drawing of the present invention, can be reasonably expected that other of variously-shaped, size that the present invention limits and material are without departing from the device of inventive principle of the present invention.
For the existing patent 201010105018.X defect studying the device that various kinds of cell interacts at glass surface set up based on microflow control technique, the present invention is with novel cell co-culture device with low cost, simple to operation, solve that its inoculating cell uniformity is wayward, the inoculation of two kinds of cells is short for interval time, the less problem of cell concentration can be used, be particularly suited for neuron and co-culture in order with neurogliocyte.
The device cultivated in order for neuron and neurogliocyte provided by the invention includes substrate, and described substrate preferably has flat surface, in order to combine closely with polydimethylsiloxane seal, it is prevented that leakage.Described substrate can be arbitrarily can be used in cultivating the coverslip of cell, microscope slide, Tissue Culture Dish etc., and this be there is no particular restriction by the present invention.Described substrate surface can hatch extracellular matrix, it is also possible in use, incubated cell epimatrix again when namely cultivating for neuron and neurogliocyte.As preferably, described extracellular matrix is poly-D-lysine, more preferably left-handed poly-D-lysine.
Being compounded with polydimethylsiloxane seal in described substrate removedly, namely polydimethylsiloxane seal can remove from substrate.Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively；The distance of described first through hole and the second through hole is 500 μm～2000 μm.
As preferably, described polydimethylsiloxane is (CH3O)3Si(CH2)CN(OCH2OCH2)nCH3Or (CH3CH2O)3Si(CH2)CN(OCH2OCH2)nCH3, wherein, n is 3,6,17 or 100.
As preferably, described polydimethylsiloxane seal is the hexahedron with micro through hole, and such as square or cuboid, its length, width and height specification specifically can be formulated according to the needs of actual tests, and the present invention is not specifically limited.
Described polydimethylsiloxane seal comprises at least one micropore unit, and described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate.First through hole and the second through hole are arranged in order, and are respectively perpendicular to substrate, and namely through hole is vertical through hole.The cross section of the first through hole of the present invention and the second through hole can be any suitable shape, and as preferably, the cross section of described first through hole and the second through hole is rectangle.It is highly preferred that the length of described first through hole is 1.0cm～2.0cm, wide for 0.5cm～2.0cm, its height (or being called thickness, the degree of depth) is determined depending on polydimethylsiloxane seal height (or thickness or the degree of depth)；Side by side it is highly preferred that the length of described second through hole is 1.0cm～2.0cm, wide for 0.5cm～2.0cm, its height (or being called thickness, the degree of depth) is determined depending on polydimethylsiloxane seal height (or thickness or the degree of depth).As preferably, the distance of described first through hole and the second through hole is 500 μm～2000 μm, more preferably 1000 μm～1500 μm.
As preferably, described micropore unit also include being arranged in order with the first through hole and the second through hole at least one be perpendicular to the third through-hole of described substrate.The cross section of third through-hole is preferably rectangle, preferred, the length of described third through-hole is 1.0cm～2.0cm, wide for 0.5cm～2.0cm, and its height (or being called thickness, the degree of depth) is determined depending on polydimethylsiloxane seal height (or thickness or the degree of depth).The distance of described third through-hole and described second through hole is 500 μm～2000 μm, more preferably 1000 μm～1500 μm.
Described polydimethylsiloxane seal comprises at least one micropore unit, it is possible to need to comprise two, three even ten micropore unit according to the size of polydimethylsiloxane seal and use；Distance between described each micropore unit is preferably 2.0cm～3.0cm.Described micropore unit includes at least one first through hole and at least one second through hole, it is possible to use need to comprise two, three even ten through holes.
When polydimethylsiloxane seal is compound in substrate, the first through hole and the second through hole form the groove of one end open with substrate surface respectively, and namely one end of the first through hole and the second through hole is terminated by substrate.
The preparation method that present invention also offers a kind of device co-cultured in order for neuron and neurogliocyte, including:
Prepare polydimethylsiloxane seal, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and the distance of described first through hole and the second through hole is 500 μm～2000 μm；
Described polydimethylsiloxane seal is attached at substrate surface, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively.
The method comprises the steps of firstly, preparing polydimethylsiloxane seal, described polydimethylsiloxane seal is identical with technique scheme, and the present invention does not repeat them here.
As preferably, described polydimethylsiloxane seal is prepared in accordance with the following methods:
Micromachining technology is adopted to prepare at least one spill line style microstructure unit in template, described spill line style microstructure unit comprises be arranged in order, at least one first spill bar and at least one the second matrix bar, and the distance of described first matrix bar and the second matrix bar is 500 μm～2000 μm；
With polydimethylsiloxane, the spill line style microstructure unit in described template is carried out secondary and turn over film, obtain polydimethylsiloxane template；
Remove the bottom surface of the first matrix bar and the second matrix bar in described polydimethylsiloxane template, obtain polydimethylsiloxane seal.
In template, at least one spill line style microstructure unit is prepared initially with micromachining technology, described spill line style microstructure unit comprises be arranged in order, at least one first spill bar and at least one the second matrix bar, and the distance of described first matrix bar and the second matrix bar is 500 μm～2000 μm.In the present invention, the cross section of described first matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm；The cross section of described second matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm.The distance of described first matrix bar and the second matrix bar is 500 μm～2000 μm, it is preferred to 1000 μm～1500 μm.The height of described first matrix bar and the second matrix bar is not particularly limited by the present invention, it is possible to select voluntarily as required.
Then with polydimethylsiloxane, the spill line style microstructure unit in described template being carried out secondary and turn over film, first step overmolded obtains convex bar, and the second overmolded obtains Baltimore groove；After secondary overmolded, obtain polydimethylsiloxane template, polydimethylsiloxane template is identical with original template structure, namely at least one spill line style microstructure unit is included, described spill line style microstructure unit comprises be arranged in order, at least one first spill bar and at least one the second matrix bar, and the distance of described first matrix bar and the second matrix bar is 500 μm～2000 μm.
After obtaining polydimethylsiloxane template, remove the first matrix bar and the bottom surface of the second matrix bar, make the first matrix bar and the second spill bar become through hole, polydimethylsiloxane seal can be obtained.
As preferably, when template prepares spill line style microstructure unit, described spill line style microstructure unit includes the 3rd matrix article being arranged in order with the first matrix article and the second matrix article, and the polydimethylsiloxane seal obtained behind secondary overmolded, removal bottom surface includes third through-hole.To this, those skilled in the art are referred to mentioned above, and the present invention does not repeat them here.
After obtaining stating polydimethylsiloxane seal, being attached at substrate surface, described first through hole and the second through hole form the groove of one end open with substrate surface respectively, can obtain the device co-cultured in order for neuron and neurogliocyte.Wherein, described substrate surface has preferably hatched extracellular matrix, and method is as follows:
Substrate surface is carried out pretreatment；
Drip extracellular matrix to pretreated substrate surface to hatch.
First soaking category of glass substrate with flying tiger acid solution (concentrated sulphuric acid: hydrogen peroxide=3:1), soak time is 20～60min；Then with distilled water washing to neutral, the process to substrate is namely completed after drying.
Then basad surface dropping extracellular matrix, the concentration of described extracellular matrix is preferably 0.1%, hatches 0.5h～1h, is sucked by surplus solution, then washs 1～3 time with the phosphate buffer that pH value is 7.4, dries.
Present invention also offers a kind of method that neuronal cell co-cultures in order with glial cell, including:
Incubated cell epimatrix in substrate；
Polydimethylsiloxane seal is connected in the substrate hatching extracellular matrix, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively；The distance of described first through hole and the second through hole is 500 μm-2000 μm；
Add in described second through hole in neurogliocyte aaerosol solution and the first through hole respectively and add Primary Neurons suspension, cultivate；
Until two kinds of cell adhesions after substrate, remove polydimethylsiloxane seal, surface adhesion has the substrate of neurogliocyte and neuronal cell be soaked in two kinds of cell co-cultivation bases, carry out co-culturing of neurogliocyte and neuronal cell.
The present invention is incubated cell epimatrix in substrate first, and as described above, the present invention does not repeat them here method.
Then being connected in the substrate hatching extracellular matrix by polydimethylsiloxane seal, as described above, the present invention does not repeat them here structure of described polydimethylsiloxane seal and preparation method thereof.
The present invention adds in described second through hole in neurogliocyte aaerosol solution and the first through hole respectively and adds Primary Neurons suspension, cultivates；Until two kinds of cell adhesions after substrate, remove polydimethylsiloxane seal, the ordered adhering neurogliocyte in same substrate and neuronal cell are co-cultured.
First neurogliocyte and neuronal cell are arranged in each independent groove and cultivate respectively by the present invention, now, the growth in the region each limited respectively of neurogliocyte and neuronal cell, stick to respectively after in substrate until it, remove polydimethylsiloxane seal, suprabasil neurogliocyte and neuronal cell are co-cultured, it is achieved the ordering growth of neurogliocyte and neuronal cell.In the present invention, the inoculation interval of neurogliocyte and neuronal cell can be 1min～10 day, when neurogliocyte and neuronal cell inoculation are longer for interval time, time such as more than 1h, after inoculation neurogliocyte, for preventing from polluting, preventing cell death, it is preferable that carry out first step cultivation, namely put it into and incubator carries out be cultured to neurogliocyte stick to substrate surface；Then inoculate neuronal cell and carry out second step cultivation, namely again put it into and incubator carries out be cultured to neuronal cell adhesion at substrate surface.As preferably, the present invention is firstly added neurogliocyte aaerosol solution, carries out the first step and is cultured to neurogliocyte and sticks to substrate surface；It is subsequently adding Primary Neurons suspension, carries out second step and be cultured to neuronal cell adhesion at substrate surface.
Specifically, when the micropore unit on polydimethylsiloxane seal only includes the first through hole and the second through hole, cultural method is as follows:
In the second through hole of polydimethylsiloxane seal, add neurogliocyte aaerosol solution carry out the first step and be cultured to neurogliocyte and stick to substrate surface.Wherein, the cell density of described neurogliocyte aaerosol solution is 105～107Individual/mL；The temperature that the first step is cultivated is 37 DEG C, and carbon dioxide volumetric concentration is 5%, and incubation time is preferably 0.5h～1h.
After the first step is cultivated, in the first through hole of polydimethylsiloxane seal, add Primary Neurons suspension carry out second step and be cultured to neuronal cell adhesion at substrate surface.Wherein, the cell density of described Primary Neurons aaerosol solution is 104～106Individual/mL；The temperature that second step is cultivated is 37 DEG C, and carbon dioxide volumetric concentration is 5%, and incubation time is preferably 0.5h～1.5h.
As preferably, cultivating and second step incubation time can have bigger interval in the first step, such as 1h～7 day even 10 days etc., this be there is no particular restriction by the present invention.
After second step is cultivated, remove polydimethylsiloxane seal, the substrate that surface adhesion has neurogliocyte and neuronal cell is soaked in two kinds of cell co-cultivation bases, carry out co-culturing of neurogliocyte and neuronal cell, neurogliocyte and neuronal cell ordering growth can be made.In co-culturing process, first neurogliocyte and neuronal cell each limiting growth in region, complete two kinds of cell ordered adherings on the same base；Then, neurogliocyte neurad unit's cell direction is moved, and neuronal cell grows prominent to all directions, its prominent interface contacting formation neurogliocyte and neuronal cell contact with glial cell, it is possible to for studying the interaction of the two.In co-culturing, culture medium is volume ratio is the mixed culture medium of two kinds of cell culture mediums of 1:1, wherein, two kinds of cell culture mediums are respectively as follows: the Neurobasal culture medium containing 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 culture medium containing 10% serum.The described temperature co-cultured is 37 DEG C, and carbon dioxide volumetric concentration is 5%, and the described time co-cultured is not had particular/special requirement by the present invention, controls as required.
When the micropore unit on polydimethylsiloxane seal also includes being arranged in order with the first through hole and the second through hole at least one be perpendicular to the third through-hole of described substrate time, its cultural method is as follows:
In the second through hole of polydimethylsiloxane seal, add neurogliocyte aaerosol solution carry out the first step and be cultured to neurogliocyte and stick to substrate surface；
After the first step is cultivated, in first through hole and third through-hole of polydimethylsiloxane seal, add Primary Neurons suspension carry out second step and be cultured to neuronal cell adhesion at substrate surface；
After second step is cultivated, remove polydimethylsiloxane seal, surface adhesion has the substrate of neurogliocyte and neuronal cell co-culture, neurogliocyte and neuronal cell ordering growth can be made.
Can also cultivate in accordance with the following methods:
When the micropore unit on polydimethylsiloxane seal also includes being arranged in order with the first through hole and the second through hole at least one be perpendicular to the third through-hole of described substrate time, its cultural method is as follows:
In first through hole and third through-hole of polydimethylsiloxane seal, add neurogliocyte aaerosol solution carry out the first step and be cultured to neurogliocyte and stick to substrate surface；
After the first step is cultivated, in the second through hole of polydimethylsiloxane seal, add Primary Neurons suspension carry out second step and be cultured to neuronal cell adhesion at substrate surface；
After second step is cultivated, remove polydimethylsiloxane seal, surface adhesion has the substrate of neurogliocyte and neuronal cell co-culture, neurogliocyte and neuronal cell ordering growth can be made.
The present invention inoculates neuronal cell and neurogliocyte by the first through hole and the second through hole being perpendicular to substrate, and when two kinds of cells are inoculated in respective region, uniformity is easily controllable, and spendable cell concentration increases, and population effect is obvious, is beneficial to observational study.The more important thing is, the inoculation time interval of neuronal cell and neurogliocyte can extend to a couple of days, thus being more beneficial for the contact interface of neuronal cell and the neurogliocyte obtaining larger area at grade, it is more beneficial for two kinds of intercellular interactions of research.
Accompanying drawing explanation
Fig. 1 show device schematic diagram of the present invention；
Fig. 2 is the interface of neuronal cell provided by the invention and glial growth.
Detailed description of the invention
The invention discloses a kind of device co-cultured in order for neuron and neurogliocyte and its production and use, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention.Device of the present invention, preparation method and application are described already by preferred embodiment, method described herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention.
Embodiment 1
1) 3*3 centimetre of coverslip is taken, first surface soaks 40 minutes with flying tiger acid solution (concentrated sulphuric acid: hydrogen peroxide=3:1), then clean to neutral with distilled water, dry, drip the poly-D-lysine of 0.8 milliliter 0.1% on its surface, hatch 1.0 hours, surplus solution is sucked, washing 3 times with phosphate buffer (pH7.4), dry, stand-by；
2) micromachining technology is used, a poly (methyl methacrylate) plate is prepared one group of matrix line style microstructure unit, this matrix line style microstructure unit is made up of two grooves arrayed from left to right, wherein, the cross section of described left groove is rectangle, length is 1.0 centimetres, and width is 0.5 centimetre；The cross section of described right groove is rectangle, and length is 1.0 centimetres, and width is 1.0 centimetres；Left groove and right groove spacing are 2000 microns；
3) with polydimethylsiloxane to step 2) poly (methyl methacrylate) plate with one group of matrix line style microstructure unit that obtains carries out secondary and turns over film, obtain polydimethylsiloxane template, having micro groove unit on this polydimethylsiloxane template lower surface, this micro groove unit is made up of two grooves arrayed from left to right；Wherein, the cross section of described left groove is rectangle, and length is 1.0 centimetres, and width is 0.5 centimetre；The cross section of described right groove is rectangle, and length is 1.0 centimetres, and width is 1.0 centimetres；Left groove and right groove spacing are 2000 microns；
Removing the bottom surface of two grooves respectively, forming two cross sections is rectangular through hole, obtains polydimethylsiloxane seal, and wherein, described PATENT left side via length is 1.0 centimetres, and width is 0.5 centimetre；Described right side through hole length is 1.0 centimetres, and width is 1.0 centimetres；Spacing between described two Micro-v oid is 2000 microns；Said two through hole constitutes micropore unit, and the length of described micropore unit is 2.0 centimetres, and width is 2.8 centimetres；
Then polydimethylsiloxane seal autoclave sterilization；
4) by step 3) aseptic polydimethylsiloxane seal after autoclave sterilization and step 1) described substrate of glass carries out contact and is connected, and makes PATENT left side via and right side through hole form the groove of one end open with substrate surface respectively；
5) preparing the aaerosol solution of glial cell, cell density is 106Individual/ml, is then added dropwise to glial cell in right groove；
6) preparing neuronic primary cell suspension, cell density is 105Individual/ml；Neuronal cell is added dropwise to step 5) in the left groove of device, note being dispelled by cell with pipettor, neuronal cell is made to be uniformly distributed, left groove adds neuronal cell culture medium, and notes making it not mix with glial cell culture medium, and device puts into cell culture incubator, at 37 DEG C, carbon dioxide volumetric concentration 5%, cultivates 1 hour, makes neuronal cell adhesion in substrate；
7) after glial cell and neuronal cell adhere to substrate completely, take polydimethylsiloxane seal off, add two kinds of cell culture fluids of volume ratio 1:1 mixing, at 37 DEG C, the cell culture incubator of carbon dioxide volumetric concentration 5% carries out two kinds of co-culture of cells, wherein, two kinds of cell culture fluids are respectively as follows: the Neurobasal culture medium containing 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 culture medium containing 10% serum, two kinds of cell first growths in each restriction region, it is achieved two kinds of cell ordered adherings are in same substrate；
Then, glial cell starts neurad unit cell direction and moves, neuronal cell grows prominent place to all directions, its prominent place contacts with colloid and forms the interface that two kinds of cells contacting length are not less than 1.0 centimetres, may be used for observing the research that neuron interacts with glial cell, referring to the interface that Fig. 2, Fig. 2 are neuronal cell provided by the invention and glial growth.As shown in Figure 2, the neuronal cell that method provided by the invention obtains is in good condition with glial cell ordering growth.
Embodiment 2
1) Tissue Culture Dish in 25 millimeters of cut-off footpath, drips the left-handed poly-D-lysine of 1.0 milliliter 0.1%, hatches 1.0 hours, sucked by surplus solution, washing 3 times with phosphate buffer (pH7.4), dry in its bottom surface, stand-by；
2) using micromachining technology, prepare least one set matrix line style microstructure unit on a poly (methyl methacrylate) plate, this matrix line style microstructure unit is made up of three grooves arrayed from left to right；Wherein, the cross section of described left groove is rectangle, and length is 1.8 centimetres, and width is 0.5 centimetre；The cross section of described intermediate groove is rectangle, and length is 1.8 centimetres, and width is 1.1 centimetres；The cross section of described right groove is rectangle, and length is 1.8 centimetres, and width is 0.5 centimetre；The distance of left groove and intermediate groove, intermediate groove and right groove is 1000 microns；
3) with polydimethylsiloxane to step 2) poly (methyl methacrylate) plate with least one set matrix line style microstructure unit that obtains carries out secondary and turns over film, obtain polydimethylsiloxane template, this polydimethylsiloxane seal lower surface there is micro groove unit, this micro groove unit is made up of three grooves arrayed from left to right: wherein, the cross section of described left groove is rectangle, length is 1.8 centimetres, and width is 0.5 centimetre；The cross section of described intermediate groove is rectangle, and length is 1.8 centimetres, and width is 1.1 centimetres；The cross section of described right groove is rectangle, and length is 1.8 centimetres, and width is 0.5 centimetre；The distance of left groove and intermediate groove, intermediate groove and right groove is 1000 microns；
Removing the bottom surface of described three grooves respectively, forming three cross sections is rectangular through hole, obtains polydimethylsiloxane seal；Wherein, the length of described PATENT left side via is 1.8 centimetres, and width is 0.5 centimetre；The length of described intermediate throughholes is 1.8 centimetres, and width is 1.1 centimetres；The length of described right side through hole is 1.8 centimetres, and width is 0.5 centimetre；Distance between the first through hole and the second through hole, the second through hole and third through-hole is 1000 microns；Described three through holes constitute micropore unit, and the length of described micropore unit is 2.0 centimetres, and width is 2.3 centimetres；
Then polydimethylsiloxane seal autoclave sterilization；
4) by step 3) aseptic polydimethylsiloxane seal after autoclave sterilization and step 1) described substrate of glass carries out contacting and compact siro spinning technology, makes PATENT left side via, intermediate throughholes and right side through hole form the groove of one end open with substrate surface respectively；
5) preparing the aaerosol solution of glial cell, cell density is 106Individual/ml, is then added dropwise to glial cell in intermediate throughholes, notes being dispelled by cell with pipettor, make glial cell be uniformly distributed, at 37 DEG C, the incubator of carbon dioxide volumetric concentration 5% is cultivated, make glial cell stick in substrate, use every day glial cell culture medium to change liquid once；
6) preparing neuronic primary cell suspension, cell density is 105Individual/ml, the first step is cultivated interval and after 2 days, neuronal cell is added dropwise in the through hole of the left and right sides, note being dispelled by cell with pipettor, neuronal cell is made to be uniformly distributed, grooves on two sides adds neuronal cell culture medium, and notes making it not mix with glial cell culture medium, at 37 DEG C, the cell culture incubator of carbon dioxide volumetric concentration 5% is cultivated 1 hour, makes neuronal cell adhesion in substrate；
7) polydimethylsiloxane seal is taken off, the volume ratio 1:1 two kinds of cell culture mediums mixed are added by being stained with in the culture dish of neuron and glial cell, at 37 DEG C, the cell culture incubator of carbon dioxide volumetric concentration 5% carries out two kinds of co-culture of cells, wherein, two kinds of cell culture fluids are respectively as follows: the Neurobasal culture medium containing 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 culture medium containing 10% serum, first cell each limiting growth in region, completes two kinds of cell ordered adherings in same substrate；
Then, glial cell starts to move to neuronal cell direction, both sides, and both sides neuronal cell grows prominent place to all directions, and its prominent place contacts with the colloid of intermediate strap and forms the interface that two two kinds of cells contacting length are not less than 1.8 centimetres.
Embodiment 3
1) 25 millimeters of cut-off footpath Tissue Culture Dish, drips the poly-D-lysine of 1.0 milliliter 0.1%, hatches 1.0 hours, sucked by surplus solution, washing 3 times with phosphate buffer (pH7.4), dry in its bottom surface, stand-by；
2) using micromachining technology, prepare least one set matrix line style microstructure unit on poly (methyl methacrylate) plate, this matrix line style microstructure unit is made up of three grooves arrayed from left to right；Wherein, the cross section of described left groove is rectangle, and length is 1.8 centimetres, and width is 0.8 centimetre；The cross section of described intermediate groove is rectangle, and length is 1.8 centimetres, and width is 0.5 centimetre；The cross section of described right groove is rectangle, and length is 1.8 centimetres, and width is 0.8 centimetre；The distance of left groove and intermediate groove, intermediate groove and right groove is 500 microns；
3) with polydimethylsiloxane to step 2) poly (methyl methacrylate) plate with least one set matrix line style microstructure unit that obtains carries out secondary and turns over film, obtain polydimethylsiloxane template, having micro groove unit on this polydimethylsiloxane seal lower surface, this micro groove unit is made up of two grooves arrayed from left to right；Wherein, the cross section of described left groove is rectangle, and length is 1.8 centimetres, and width is 0.8 centimetre；The cross section of described intermediate groove is rectangle, and length is 1.8 centimetres, and width is 0.5 centimetre；The cross section of described right groove is rectangle, and length is 1.8 centimetres, and width is 0.8 centimetre；The distance of left groove and intermediate groove, intermediate groove and right groove is 500 microns；
Removing the bottom surface of described three grooves, forming three cross sections is rectangular through hole, obtains polydimethylsiloxane seal；The length of wherein said PATENT left side via is 1.8 centimetres, and width is 0.8 centimetre；The length of described intermediate throughholes is 1.8 centimetres, and width is 0.5 centimetre；The length of described right side through hole is 1.8 centimetres, and width is 0.8 centimetre；The distance of PATENT left side via and intermediate throughholes, intermediate throughholes and right side through hole is 500 microns；Described three through holes constitute micropore unit, and the length of described micropore unit is 1.8 centimetres, width 2.2 centimetres；
Then polydimethylsiloxane seal autoclave sterilization；
4) by step 3) aseptic polydimethylsiloxane seal after autoclave sterilization and step 1) described substrate of glass carries out contact and is connected, and makes PATENT left side via, intermediate throughholes and right side through hole form the groove of one end open with substrate surface respectively；
5) preparing the aaerosol solution of glial cell, cell density is 106Individual/ml, is then added dropwise to glial cell in the through hole of the left and right sides, notes being dispelled by cell with pipettor, make glial cell be uniformly distributed, at 37 DEG C, the incubator of carbon dioxide volumetric concentration 5% is cultivated, make glial cell stick in substrate, use every day glial cell culture medium to change liquid once；；
6) preparing neuronic primary cell suspension, cell density is 105Individual/ml, the first step is cultivated interval and after 5 days, neuronal cell is added dropwise in middle micro groove, note being dispelled by cell with pipettor, neuronal cell is made to be uniformly distributed, grooves on two sides adds neuronal cell culture medium, and notes making it not mix with glial cell culture medium, at 37 DEG C, the cell culture incubator of carbon dioxide volumetric concentration 5% is cultivated 1 hour, makes neuronal cell adhesion in substrate；
7) polydimethylsiloxane seal is taken off, the volume ratio 1:1 two kinds of cell culture mediums mixed are added by being stained with in the culture dish of neuron and glial cell, at 37 DEG C, the cell culture incubator of carbon dioxide volumetric concentration 5% carries out two kinds of co-culture of cells, wherein, two kinds of cell culture fluids are respectively as follows: the Neurobasal culture medium containing 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 culture medium containing 10% serum, first cell each limiting growth in region, completes two kinds of cell ordered adherings in same substrate；
Then, glial cell starts neurad unit cell direction and moves, and neuronal cell grows prominent place to all directions, and its prominent place contacts with colloid and forms the interface that two two kinds of cells contacting length are not less than 1.8 centimetres.
Comparative example 1
The device that method preparation disclosed in embodiment 1 step 1～6 in Chinese patent CN201010105018.X co-cultures in order for cell, then preparing cell density respectively is 106The aaerosol solution of the glial cell of individual/ml and cell density are 105The neuronal cell suspension of individual/ml, is separately added into Article 2 miniflow duct and Article 5 miniflow duct, is placed in cell culture incubator by device, 37 DEG C, carbon dioxide volumetric concentration 5% when cultivate 1h；
Take polydimethylsiloxane seal off, growth have the glass surface of neuron and glial cell put in two kinds of cell culture fluids of volume ratio 1:1 mixing, test result indicate that, outlet in Article 2 duct and porch glial cell dense accumulation, in middle part, Article 2 duct, glial cell ratio is sparse, and cell distribution is uniform not；Outlet in Article 5 duct and porch neuronal cell dense accumulation, in middle part, Article 5 duct, glial cell ratio is sparse, and cell distribution is uniform not.
Comparative example 2
The device that method preparation disclosed in embodiment 1 step 1～6 in Chinese patent CN201010105018.X co-cultures in order for cell, then preparing cell density respectively is 106Device is placed in cell culture incubator and carries out first step cultivation after adding Article 2 miniflow duct by the aaerosol solution of the glial cell of individual/ml, 37 DEG C, carbon dioxide volumetric concentration 5% when cultivate and change liquid 4-5 time every day；Cultivate glial cell after 2 days, neuronal cell joined Article 5 miniflow duct, be placed in cell culture incubator 37 DEG C, carbon dioxide volumetric concentration 5% when 1h；
Taking polydimethylsiloxane seal off, have the glass surface of neuron and glial cell to put in two kinds of cell culture fluids of volume ratio 1:1 mixing growth, result shows, glial growth is out of order, basis of microscopic observation is transparent not to glial cell, and Cytoplasmic inclusions is more, tends to apoptosis.
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (12)
1. the device co-cultured in order for neuron and neurogliocyte, it is characterized in that, including substrate and polydimethylsiloxane seal, on the substrate, described substrate surface has hatched extracellular matrix to described polydimethylsiloxane seal compound removedly；
Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively；
The distance of described first through hole and the second through hole is 500 μm～2000 μm, and the cross section of described first through hole and the second through hole is rectangle, and the length of described first through hole cross section is 1.0cm～2.0cm, wide for 0.5cm～2.0cm；The length of described second through hole cross section is 1.0cm～2.0cm, wide for 0.5cm～2.0cm.
2. device according to claim 1, it is characterised in that described micropore unit also include being arranged in order with the first through hole and the second through hole at least one be perpendicular to the third through-hole of described substrate.
3. device according to claim 2, it is characterised in that the distance of described third through-hole and described second through hole is 500 μm～2000 μm.
4. a preparation method for the device co-cultured in order for neuron and neurogliocyte, including:
Prepare polydimethylsiloxane seal, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to substrate and at least one is perpendicular to the second through hole of substrate, the distance of described first through hole and the second through hole is 500 μm～2000 μm, the cross section of described first through hole and the second through hole is rectangle, the length of described first through hole cross section is 1.0cm～2.0cm, wide for 0.5cm～2.0cm；The length of described second through hole cross section is 1.0cm～2.0cm, wide for 0.5cm～2.0cm；
Described polydimethylsiloxane seal is attached at substrate surface, and described substrate surface has hatched extracellular matrix, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively.
5. preparation method according to claim 4, it is characterised in that described polydimethylsiloxane seal is prepared in accordance with the following methods:
Micromachining technology is adopted to prepare at least one spill line style microstructure unit in template, described spill line style microstructure unit comprises be arranged in order, at least one first spill bar and at least one the second matrix bar, and the distance of described first matrix bar and the second matrix bar is 500 μm～2000 μm；
With polydimethylsiloxane, the spill line style microstructure unit in described template is carried out secondary and turn over film, obtain polydimethylsiloxane template；
Remove the bottom surface of the first matrix bar and the second matrix bar in described polydimethylsiloxane template, obtain polydimethylsiloxane seal.
6. preparation method according to claim 5, it is characterised in that the cross section of described first matrix bar is rectangle, and length is 1.0～2.0cm, wide is 0.5～2.0cm；The cross section of described second matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm.
7. preparation method according to claim 4, it is characterised in that described micropore unit also include being arranged in order with the first through hole and the second through hole at least one be perpendicular to the third through-hole of described substrate.
8. the method that a neuronal cell co-cultures in order with glial cell, it is characterised in that including:
Incubated cell epimatrix in substrate；
Polydimethylsiloxane seal is connected in the substrate hatching extracellular matrix, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprises the first through hole that be arranged in order, that at least one is perpendicular to described substrate and at least one is perpendicular to the second through hole of described substrate, and described first through hole and the second through hole form the groove of one end open with substrate surface respectively；The distance of described first through hole and the second through hole is 500 μm-2000 μm, and the cross section of described first through hole and the second through hole is rectangle, and the length of described first through hole cross section is 1.0cm～2.0cm, wide for 0.5cm～2.0cm；The length of described second through hole cross section is 1.0cm～2.0cm, wide for 0.5cm～2.0cm；
Add in described second through hole in neurogliocyte aaerosol solution and the first through hole respectively and add Primary Neurons suspension, cultivate；
After cultivation, remove polydimethylsiloxane seal, surface adhesion has the substrate of neurogliocyte and neuronal cell co-culture.
9. method according to claim 8, it is characterised in that described micropore unit also include being arranged in order with the first through hole and the second through hole at least one be perpendicular to the third through-hole of described substrate；
In described second through hole, add neurogliocyte aaerosol solution and in described first through hole and third through-hole, add Primary Neurons suspension, cultivating.
10. method according to claim 8 or claim 9, it is characterised in that be firstly added neurogliocyte aaerosol solution, carries out the first step and is cultured to neurogliocyte and sticks to substrate surface；
It is subsequently adding Primary Neurons suspension, carries out second step and be cultured to neuronal cell adhesion at substrate surface.
11. method according to claim 10, it is characterised in that the cell density of described neurogliocyte aaerosol solution is 105～107Individual/mL；Described Primary Neurons suspension cell density is 104～106Individual/mL.
12. method according to claim 10, it is characterised in that the time that the described first step is cultivated is 0.5h～1.5h；The time that described second step is cultivated is 0.5h～1.5h.
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