CN104130943A - Neuronal and neuroglial cell ordered co-culture device and preparation method thereof, and neuronal and neuroglial cell ordered co-culture method - Google Patents
Neuronal and neuroglial cell ordered co-culture device and preparation method thereof, and neuronal and neuroglial cell ordered co-culture method Download PDFInfo
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- CN104130943A CN104130943A CN201410363029.6A CN201410363029A CN104130943A CN 104130943 A CN104130943 A CN 104130943A CN 201410363029 A CN201410363029 A CN 201410363029A CN 104130943 A CN104130943 A CN 104130943A
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- 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
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
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- C12N2502/00—Coculture with; Conditioned medium produced by
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- C12N2533/32—Polylysine, polyornithine
Abstract
The invention relates to the cytobiology field, and discloses a neuronal cell and neuroglial cell ordered co-culture device and a preparation method and an application thereof. The device includes a substrate and a polydimethylsiloxane stamper, wherein the polydimethylsiloxane stamper can be removably compounded on the substrate; the polydimethylsiloxane stamper includes at least one micropore unit, the micropore unit includes at least one first through hole perpendicular to the substrate and at least one second through hole perpendicular to the substrate, the first through hole and the second through hole are arranged successively, and the first through hole and the second through hole respectively with the surface of the substrate form grooves having one end with an opening; the distance between the first through hole and the second through hole is 500 [mu]m-2000 [mu]m. Neuronal cells and neuroglial cells are inoculated through the first through hole and the second through hole which are perpendicular to the substrate, uniformity of the two kinds of cells is easy to control during inoculation in regions, cell quantity which can be used is increased, a population effect is obvious, and observation and study are facilitated.
Description
Technical field
The present invention relates to cytobiology field, be specifically related to a kind of for neurone and the neurogliocyte method that device, its preparation method and the neurone of cultivation and neurogliocyte are cultivated in order altogether altogether in order.
Background technology
Result of study in recent years shows, neurone and neurogliocyte not only exist exchange of substance, also have message exchange.Between neurone and spongiocyte, how interaction mode affects its material, information interchange, and the change of their interaction modes and multiple encephalopathy become the another study hotspot of neuroscience as the correlation research such as alzheimer's disease, parkinsonism.
Can be used at present studying neurone and spongiocyte interact external co-culture system be Banker equal to set up the nineties in 20th century to cultivate the cultural method of neuronal cell as object, there is scholar to improve on this basis, successfully set up the co-culture method of neurone and spongiocyte.But aforesaid method is taking spongiocyte as trophoderm, at its surface seeding epineural unit cell, two kinds of unordered growths straggly of cell, are difficult to use in research neurone and spongiocyte repercussion study.
The patent No. is that 200710117815.8 and 200710119997.2 patent of invention has been set up in gold surface various kinds of cell co-culture device and method based on microflow control technique, these the two kinds method substrates used that adhere to and handle various kinds of cell all adopt the method preparation at glass surface evaporation layer of gold, this substrate need to utilize high-vacuum apparatus preparation at ultra-clean chamber, price is more expensive, and gold layer character is unstable, cannot preserve for a long time, be unfavorable for promoting at biology laboratory.
The patent No. is that the patent of invention of 201010105018.X discloses and a kind of set up at interactional device of glass surface research various kinds of cell and its production and use based on microflow control technique, this method is inoculated in various kinds of cell respectively in different miniflow ducts until it after substrate adheres to, remove miniflow duct, realize various kinds of cell and interact.But it is a lot of not enough that this device exists while cultivation altogether in order for neurone and neurogliocyte, the first, inoculating cell in miniflow duct, cell is skewness in long and narrow duct; The second, cell is because cultivation base unit weight is few in miniflow duct, and the cells survival time is very short, and the shorter time can not ensure that neurone and spongiocyte are simultaneously in the cultured time period; If ceaselessly change substratum, easily make cell floating, final unstable death; The 3rd, in miniflow duct, cell-volume is few, not obvious for studying two kinds of interactional population effects of born of the same parents, is unfavorable for observational study.In addition, due to the neuronal cell cultivation of can not going down to posterity, and said apparatus requires the unsuitable interval of various cell inoculation times long, and therefore, said apparatus is difficult to use in neurone and neurogliocyte repercussion study.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of for neurone and the neurogliocyte method that device of cultivation and preparation method thereof and neurone and neurogliocyte are cultivated in order altogether altogether in order, make device of the present invention in the time of inoculation neuronal cell and spongiocyte, be easy to control cell homogeneity, and can extend two kinds of cells inoculation interval time, realize cultivating altogether in order of neurone and neurogliocyte, increase can be used cell concentration.
For realizing above goal of the invention, the invention provides following technical scheme:
Be total in order for neurone and neurogliocyte a device of cultivating, comprise substrate and polydimethylsiloxane seal, described polydimethylsiloxane seal is compounded in described substrate removedly;
Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface;
The distance of described the first through hole and the second through hole is 500 μ m～2000 μ m.
For the ease of understanding the inventive principle of device of the present invention, can be referring to Fig. 1, it should be noted that, accompanying drawing of the present invention does not limit shape, size, material wherein, it is just for the ease of understanding apparatus of the present invention core technology on the basis of describing in the claims in the present invention, those skilled in the art can be on the basis of accompanying drawing of the present invention, Rational Pre expect different shape, size and material that the present invention limits other do not depart from the device of inventive principle of the present invention.
The defect at the interactional device of glass surface research various kinds of cell of setting up based on microflow control technique for existing patent 201010105018.X, the present invention is with novel cell co-culture device with low cost, simple to operation, solve that its inoculating cell homogeneity is wayward, the inoculation of two kinds of cells is short interval time, can use the less problem of cell concentration, be particularly useful for neurone and neurogliocyte and cultivate altogether in order.
Device for neurone and the orderly cultivation of neurogliocyte provided by the invention comprises substrate, and described substrate preferably has flat surface, to combine closely with polydimethylsiloxane seal, prevents leakage.Described substrate can be for can be used in arbitrarily the cover glass, slide glass, Tissue Culture Dish etc. of culturing cell, and the present invention there is no particular restriction to this.Described substrate surface can have been hatched extracellular matrix, also can be in use, and incubated cell epimatrix while more cultivation for neurone and neurogliocyte.As preferably, described extracellular matrix is poly-lysine, more preferably left-handed poly-lysine.
In described substrate, be compounded with removedly polydimethylsiloxane seal, polydimethylsiloxane seal can remove from substrate.Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface; The distance of described the first through hole and the second through hole is 500 μ m～2000 μ m.
As preferably, described polydimethylsiloxane is (CH
3o)
3si (CH
2) CN (OCH
2oCH
2)
ncH
3or (CH
3cH
2o)
3si (CH
2) CN (OCH
2oCH
2)
ncH
3, wherein, n is 3,6,17 or 100.
As preferably, described polydimethylsiloxane seal is the hexahedron with micro through hole, and as square or rectangular parallelepiped, its length, width and height specification can specifically 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, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate.The first through hole and the second through hole are arranged in order, and respectively perpendicular to substrate, 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 the first through hole and the second through hole is rectangle.More preferably, the length of described the first through hole is 1.0cm～2.0cm, and wide is 0.5cm～2.0cm, and its height (or being called thickness, the degree of depth) is looked polydimethylsiloxane seal height (or thickness or degree of depth) and determined; Side by side more preferably, the length of described the second through hole is 1.0cm～2.0cm, and wide is 0.5cm～2.0cm, and its height (or being called thickness, the degree of depth) is looked polydimethylsiloxane seal height (or thickness or degree of depth) and determined.As preferably, the distance of described the first through hole and the second through hole is 500 μ m～2000 μ m, more preferably 1000 μ m～1500 μ m.
As preferably, described micropore unit also comprises at least one third through-hole perpendicular to described substrate being arranged in order with the first through hole and the second through hole.The cross section of third through-hole is preferably rectangle, preferred, the length of described third through-hole is 1.0cm～2.0cm, and wide is 0.5cm～2.0cm, and its height (or being called thickness, the degree of depth) is looked polydimethylsiloxane seal height (or thickness or degree of depth) and determined.The distance of described third through-hole and described the second through hole is 500 μ m～2000 μ m, more preferably 1000 μ m～1500 μ m.
Described polydimethylsiloxane seal comprises at least one micropore unit, can and use according to the size of polydimethylsiloxane seal and need to comprise two, three even ten micropore unit; Distance between described each micropore unit is preferably 2.0cm～3.0cm.Described micropore unit comprises at least one first through hole and at least one second through hole, can use and 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 respectively the groove of an end opening with substrate surface, i.e. substrate is by one end end-blocking of the first through hole and the second through hole.
It is a kind of for neurone and the neurogliocyte preparation method of the common device of cultivating in order that the present invention also provides, and comprising:
Prepare polydimethylsiloxane seal, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, the distance of described the first through hole and the second through hole is 500 μ m～2000 μ m;
Described polydimethylsiloxane seal is attached to substrate surface, and described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface.
First the present invention prepares polydimethylsiloxane seal, and 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:
Adopt micromachining technology in template, to prepare at least one spill line style microstructure unit, described spill line style microstructure unit comprise be arranged in order, at least one first spill bar and at least one the second matrix bar, the distance of described the first matrix bar and the second matrix bar is 500 μ m～2000 μ m;
With polydimethylsiloxane, the spill line style microstructure unit in described template is carried out to secondary and turn over film, obtain polydimethylsiloxane template;
The bottom surface of removing the first matrix bar and the second matrix bar in described polydimethylsiloxane template, obtains polydimethylsiloxane seal.
First adopt micromachining technology in template, to prepare at least one spill line style microstructure unit, described spill line style microstructure unit comprise be arranged in order, at least one first spill bar and at least one the second matrix bar, the distance of described the first matrix bar and the second matrix bar is 500 μ m～2000 μ m.In the present invention, the cross section of described the first matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm; The cross section of described the second matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm.The distance of described the first matrix bar and the second matrix bar is 500 μ m～2000 μ m, is preferably 1000 μ m～1500 μ m.The present invention is not particularly limited the height of described the first matrix bar and the second matrix bar, can select voluntarily as required.
Then with polydimethylsiloxane, the spill line style microstructure unit in described template is carried out to secondary and turn over film, the first step is turned over mould and is obtained convex bar, and second turns over mould obtains Baltimore groove; After secondary turns over mould, obtain polydimethylsiloxane template, polydimethylsiloxane template is identical with original template structure, comprise at least one spill line style microstructure unit, described spill line style microstructure unit comprise be arranged in order, at least one first spill bar and at least one the second matrix bar, the distance of described the first matrix bar and the second matrix bar is 500 μ m～2000 μ m.
Obtain after polydimethylsiloxane template, remove the bottom surface of the first matrix bar and the second matrix bar, make the first matrix bar and the second spill bar become through hole, can obtain polydimethylsiloxane seal.
As preferably, prepare spill line style microstructure unit in template time, described spill line style microstructure unit comprises the 3rd matrix article being arranged in order with the first matrix article and the second matrix article, turns over mould, removes on the polydimethylsiloxane seal obtaining behind bottom surface and comprise third through-hole through secondary.To this, those skilled in the art can be with reference to mentioned above, and the present invention does not repeat them here.
Obtain stating after polydimethylsiloxane seal, be attached at substrate surface, described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface, can obtain being total in order for neurone and neurogliocyte the device of cultivating.Wherein, described substrate surface has preferably been hatched extracellular matrix, and method is as follows:
Substrate surface is carried out to pre-treatment;
Dripping extracellular matrix to pretreated substrate surface hatches.
First use flying tiger acid solution (vitriol oil: hydrogen peroxide=3:1) to soak category of glass substrate, soak time is 20～60min; Then extremely neutral with distilled water washing, after being dried, complete the processing to substrate.
Then basad surface drips extracellular matrix, and the concentration of described extracellular matrix is preferably 0.1%, hatches 0.5h～1h, surplus solution is sucked, then the phosphate buffered saline buffer that is 7.4 by pH value washs 1～3 time, dries.
A kind of method that the present invention also provides neuronal cell and spongiocyte to cultivate altogether in order, comprising:
Incubated cell epimatrix in substrate;
Polydimethylsiloxane seal is connected in the substrate of hatching extracellular matrix, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface; The distance of described the first through hole and the second through hole is 500 μ m-2000 μ m;
Add Primary Neurons suspension to adding in neurogliocyte aaerosol solution and the first through hole in described the second through hole respectively, cultivate;
After substrate, remove polydimethylsiloxane seal until two kinds of cell adhesions, have the substrate of neurogliocyte and neuronal cell to be soaked in two kinds of cell co-cultivation bases surface adhesion, carry out the common cultivation 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 polydimethylsiloxane seal is connected in the substrate of hatching extracellular matrix, as described above, the present invention does not repeat them here structure of described polydimethylsiloxane seal and preparation method thereof.
The present invention adds Primary Neurons suspension to adding in neurogliocyte aaerosol solution and the first through hole in described the second through hole respectively, cultivates; After substrate, remove polydimethylsiloxane seal until two kinds of cell adhesions, ordered adhering is carried out to common cultivation in neurogliocyte and the neuronal cell of same substrate.
First the present invention is arranged on neurogliocyte and neuronal cell separately independently and cultivates respectively in groove, now, neurogliocyte and neuronal cell are grown respectively in the region limiting separately, after it sticks to respectively in substrate, remove polydimethylsiloxane seal, suprabasil neurogliocyte and neuronal cell are carried out to common cultivation, realize 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 the inoculation of neurogliocyte and neuronal cell longer interval time, for example, while exceeding 1h, after inoculation neurogliocyte, for preventing polluting, prevent necrocytosis, preferably carry out the first step cultivation, put it into and in incubator, be cultured to neurogliocyte and stick to substrate surface; And then inoculation neuronal cell carries out second step cultivation, again put it into and in incubator, be cultured to neuronal cell and stick to substrate surface.As preferably, first the present invention adds neurogliocyte aaerosol solution, carries out the first step and is cultured to neurogliocyte and sticks to substrate surface; Then add Primary Neurons suspension, carry out second step and be cultured to neuronal cell and stick to substrate surface.
Particularly, in the time that the micropore unit on polydimethylsiloxane seal only comprises the first through hole and the second through hole, cultural method is as follows:
Be cultured to neurogliocyte and stick to substrate surface to adding in the second through hole of polydimethylsiloxane seal neurogliocyte aaerosol solution to carry out the first step.Wherein, the cell density of described neurogliocyte aaerosol solution is 10
5～10
7individual/mL; The temperature that the first step is cultivated is 37 DEG C, and carbonic acid gas volumetric concentration is 5%, and incubation time is preferably 0.5h～1h.
After the first step is cultivated, be cultured to neuronal cell and stick to substrate surface to adding in the first through hole of polydimethylsiloxane seal Primary Neurons suspension to carry out second step.Wherein, the cell density of described Primary Neurons aaerosol solution is 10
4～10
6individual/mL; The temperature that second step is cultivated is 37 DEG C, and carbonic acid gas volumetric concentration is 5%, and incubation time is preferably 0.5h～1.5h.
As preferably, cultivate and second step incubation time can have the larger timed interval in the first step, as 1h～7 day even 10 days etc., the present invention there is no particular restriction to this.
After second step is cultivated, remove polydimethylsiloxane seal, there is the substrate of neurogliocyte and neuronal cell to be soaked in two kinds of cell co-cultivation bases surface adhesion, carry out the common cultivation of neurogliocyte and neuronal cell, can make neurogliocyte and neuronal cell ordering growth.In common culturing process, first neurogliocyte and neuronal cell growing in localized area separately, complete two kinds of cell ordered adherings in same substrate; Then, neurogliocyte neuralward unit cell direction moves, and neuronal cell grows outstanding to all directions, and its outstanding contact with spongiocyte forms the interface that neurogliocyte contacts with neuronal cell, can be for studying the interaction of the two.In common cultivation, substratum is that 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: the Neurobasal substratum that contains 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 substratum that contains 10% serum.Described temperature of cultivating is altogether 37 DEG C, and carbonic acid gas volumetric concentration is 5%, and the present invention does not have particular requirement to the described time of cultivating altogether, controls as required.
In the time that the micropore unit on polydimethylsiloxane seal also comprises at least one third through-hole perpendicular to described substrate being arranged in order with the first through hole and the second through hole, its cultural method is as follows:
Be cultured to neurogliocyte and stick to substrate surface to adding in the second through hole of polydimethylsiloxane seal neurogliocyte aaerosol solution to carry out the first step;
After the first step is cultivated, be cultured to neuronal cell and stick to substrate surface to adding in the first through hole of polydimethylsiloxane seal and third through-hole Primary Neurons suspension to carry out second step;
After second step is cultivated, remove polydimethylsiloxane seal, have the substrate of neurogliocyte and neuronal cell to carry out common cultivation surface adhesion, can make neurogliocyte and neuronal cell ordering growth.
Also can cultivate in accordance with the following methods:
In the time that the micropore unit on polydimethylsiloxane seal also comprises at least one third through-hole perpendicular to described substrate being arranged in order with the first through hole and the second through hole, its cultural method is as follows:
Be cultured to neurogliocyte and stick to substrate surface to adding in the first through hole of polydimethylsiloxane seal and third through-hole neurogliocyte aaerosol solution to carry out the first step;
After the first step is cultivated, be cultured to neuronal cell and stick to substrate surface to adding in the second through hole of polydimethylsiloxane seal Primary Neurons suspension to carry out second step;
After second step is cultivated, remove polydimethylsiloxane seal, have the substrate of neurogliocyte and neuronal cell to carry out common cultivation surface adhesion, can make neurogliocyte and neuronal cell ordering growth.
The present invention is by the first through hole perpendicular to substrate and the second through hole inoculation neuronal cell and neurogliocyte, and two kinds of cells homogeneity in the time that region is inoculated is separately easy to control, 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, thereby be more conducive to obtain at grade the contact interface of larger area neuronal cell and neurogliocyte, be more conducive to study two kinds of intercellular interactions.
Brief description of the drawings
Figure 1 shows that device schematic diagram of the present invention;
Fig. 2 is the interface of neuronal cell provided by the invention and spongiocyte growth.
Embodiment
The invention discloses a kind of device of cultivating altogether in order for neurone and neurogliocyte and its production and use, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Device of the present invention, preparation method and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
Embodiment 1
1) get 3*3 centimetre of cover glass, first surface uses flying tiger acid solution (vitriol oil: hydrogen peroxide=3:1) to soak 40 minutes, then clean to neutral with distilled water, dry, drip the poly-lysine of 0.8 milliliter 0.1% on its surface, hatch 1.0 hours, surplus solution is sucked, with phosphate buffered saline buffer (pH 7.4) washing 3 times, dry, stand-by;
2) use micromachining technology, on a poly (methyl methacrylate) plate, prepare one group of matrix line style microstructure unit, this matrix line style microstructure unit is made up of two grooves that array 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, on this polydimethylsiloxane template lower surface, have micro groove unit, this micro groove unit is made up of two grooves that array 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;
Remove respectively the bottom surface of two grooves, 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 micropore holes is 2000 microns; Described two through holes form 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 and step 1 after autoclave sterilization) described substrate of glass carries out contact and is connected, and makes PATENT left side via and right side through hole form respectively the groove of an end opening with substrate surface;
5) prepare the aaerosol solution of spongiocyte, cell density is 10
6individual/ml, is then added dropwise to spongiocyte in right groove;
6) prepare neuronic primary cell suspension, cell density is 10
5individual/ml; Neuronal cell is added dropwise to step 5) device left groove in, note with pipettor, cell being dispelled, neuronal cell is uniformly distributed, left groove adds neuronal cell substratum, and notes it is not mixed with spongiocyte substratum, and device is put into cell culture incubator, at 37 DEG C, carbonic acid gas volumetric concentration 5%, cultivates 1 hour, and neuronal cell is sticked in substrate;
7) adhere to completely after substrate until spongiocyte and neuronal cell, take polydimethylsiloxane seal off, two kinds of cell culture fluids that add volume ratio 1:1 to mix, at 37 DEG C, in the cell culture incubator of carbonic acid gas volumetric concentration 5%, carry out two kinds of co-culture of cells, wherein, two kinds of cell culture fluids are respectively: the Neurobasal substratum that contains 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 substratum that contains 10% serum, two kinds of cells, first growing in localized area separately, are realized two kinds of cell ordered adherings in same substrate;
Then, spongiocyte starts neuralward unit cell direction and moves, neuronal cell grows prominent place to all directions, its prominent place contacts two kinds of cells contacting length of formation and is not less than the interface of 1.0 centimetres with colloid, can be for observing neurone and the interactional research of spongiocyte, referring to Fig. 2, Fig. 2 is the interface of neuronal cell provided by the invention and spongiocyte growth.As shown in Figure 2, method provided by the invention obtains neuronal cell and spongiocyte ordering growth are in good condition.
Embodiment 2
1) Tissue Culture Dish in 25 millimeters, cut-off footpath, drips the left-handed poly-lysine of 1.0 milliliter 0.1% in its bottom surface, hatch 1.0 hours, and surplus solution is sucked, and with phosphate buffered saline buffer (pH7.4) washing 3 times, dries, stand-by;
2) use micromachining technology, prepare at least one group of matrix line style microstructure unit on a poly (methyl methacrylate) plate, this matrix line style microstructure unit is made up of three grooves that array 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 at least one group of matrix line style microstructure unit that obtains carries out secondary and turns over film, obtain polydimethylsiloxane template, on this polydimethylsiloxane seal lower surface, there is micro groove unit, this micro groove unit is made up of three grooves that array 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;
Remove respectively the bottom surface of described three grooves, 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 form 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 and step 1 after autoclave sterilization) described substrate of glass contacts and is closely connected, and makes PATENT left side via, intermediate throughholes and right side through hole form respectively the groove of an end opening with substrate surface;
5) prepare the aaerosol solution of spongiocyte, cell density is 10
6individual/ml, is then added dropwise to spongiocyte in intermediate throughholes, notes with pipettor, cell being dispelled, spongiocyte is uniformly distributed, at 37 DEG C, in the incubator of carbonic acid gas volumetric concentration 5%, cultivates, spongiocyte is sticked in substrate, use every day spongiocyte substratum to change liquid once;
6) prepare neuronic primary cell suspension, cell density is 10
5individual/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 with pipettor, cell being dispelled, neuronal cell is uniformly distributed, grooves on two sides adds neuronal cell substratum, and notes it is not mixed with spongiocyte substratum, at 37 DEG C, in the cell culture incubator of carbonic acid gas volumetric concentration 5%, cultivate 1 hour, neuronal cell is sticked in substrate;
7) take polydimethylsiloxane seal off, two kinds of cell culture mediums that add volume ratio 1:1 to mix in the culture dish of neurone and spongiocyte will be stained with, at 37 DEG C, in the cell culture incubator of carbonic acid gas volumetric concentration 5%, carry out two kinds of co-culture of cells, wherein, two kinds of cell culture fluids are respectively: the Neurobasal substratum that contains 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 substratum that contains 10% serum, first cell growing in localized area separately, completes two kinds of cell ordered adherings in same substrate;
Then, spongiocyte starts to move to both sides neuronal cell direction, and both sides neuronal cell grows prominent place to all directions, and its prominent place contacts two two kinds of cells contacting length of formation and is not less than the interface of 1.8 centimetres with the colloid of intermediate strap.
Embodiment 3
1) 25 millimeters, cut-off footpath Tissue Culture Dish, drips the poly-lysine of 1.0 milliliter 0.1% in its bottom surface, hatch 1.0 hours, and surplus solution is sucked, and with phosphate buffered saline buffer (pH7.4) washing 3 times, dries, stand-by;
2) use micromachining technology, prepare at least one group of matrix line style microstructure unit on poly (methyl methacrylate) plate, this matrix line style microstructure unit is made up of three grooves that array 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 at least one group of matrix line style microstructure unit that obtains carries out secondary and turns over film, obtain polydimethylsiloxane template, on this polydimethylsiloxane seal lower surface, have micro groove unit, this micro groove unit is made up of two grooves that array 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;
Remove 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 form micropore unit, and the length of described micropore unit is 1.8 centimetres, 2.2 centimetres of width;
Then polydimethylsiloxane seal autoclave sterilization;
4) by step 3) aseptic polydimethylsiloxane seal and step 1 after autoclave sterilization) described substrate of glass carries out contact and is connected, and makes PATENT left side via, intermediate throughholes and right side through hole form respectively the groove of an end opening with substrate surface;
5) prepare the aaerosol solution of spongiocyte, cell density is 10
6individual/ml, is then added dropwise to spongiocyte in the through hole of the left and right sides, notes with pipettor, cell being dispelled, spongiocyte is uniformly distributed, at 37 DEG C, in the incubator of carbonic acid gas volumetric concentration 5%, cultivates, spongiocyte is sticked in substrate, use every day spongiocyte substratum to change liquid once; ;
6) prepare neuronic primary cell suspension, cell density is 10
5individual/ml, the first step is cultivated interval and after 5 days, neuronal cell is added dropwise in middle micro groove, note with pipettor, cell being dispelled, neuronal cell is uniformly distributed, grooves on two sides adds neuronal cell substratum, and notes it is not mixed with spongiocyte substratum, at 37 DEG C, in the cell culture incubator of carbonic acid gas volumetric concentration 5%, cultivate 1 hour, neuronal cell is sticked in substrate;
7) take polydimethylsiloxane seal off, two kinds of cell culture mediums that add volume ratio 1:1 to mix in the culture dish of neurone and spongiocyte will be stained with, at 37 DEG C, in the cell culture incubator of carbonic acid gas volumetric concentration 5%, carry out two kinds of co-culture of cells, wherein, two kinds of cell culture fluids are respectively: the Neurobasal substratum that contains 2% (volume ratio) neurotrophic factor B27 and the DMEM/F12 substratum that contains 10% serum, first cell growing in localized area separately, completes two kinds of cell ordered adherings in same substrate;
Then, spongiocyte starts the first cell direction of neuralward and moves, and neuronal cell grows prominent place to all directions, and its prominent place contacts two two kinds of cells contacting length of formation and is not less than the interface of 1.8 centimetres with colloid.
Comparative example 1
The device of cultivating altogether in order for the preparation of cell according to the 1 disclosed method in step 1～6 of embodiment in Chinese patent CN201010105018.X, then preparing respectively cell density is 10
6the aaerosol solution of the spongiocyte of individual/ml and cell density are 10
5the neuronal cell suspension of individual/ml, adds respectively Article 2 miniflow duct and Article 5 miniflow duct, and device is placed in to cell culture incubator, under 37 DEG C, the condition of carbonic acid gas volumetric concentration 5%, cultivates 1h;
Take polydimethylsiloxane seal off, there is the glass surface of neurone and spongiocyte to put into two kinds of cell culture fluids that volume ratio 1:1 mixes growth, experimental result shows, at outlet and the ingress spongiocyte dense accumulation in Article 2 duct, in middle part, Article 2 duct, spongiocyte is more sparse, and cell distribution is even not; At outlet and the ingress neuronal cell dense accumulation in Article 5 duct, in middle part, Article 5 duct, spongiocyte is more sparse, and cell distribution is even not.
Comparative example 2
The device of cultivating altogether in order for the preparation of cell according to the 1 disclosed method in step 1～6 of embodiment in Chinese patent CN201010105018.X, then preparing respectively cell density is 10
6the aaerosol solution of the spongiocyte of individual/ml adds and device is placed in to cell culture incubator behind Article 2 miniflow duct and carries out the first step cultivation, cultivates every day and change liquid 4-5 time under 37 DEG C, the condition of carbonic acid gas volumetric concentration 5%; Cultivate spongiocyte after 2 days, neuronal cell is joined to Article 5 miniflow duct, be placed in 1h under the condition of 37 DEG C of cell culture incubators, carbonic acid gas volumetric concentration 5%;
Take polydimethylsiloxane seal off, there is the glass surface of neurone and spongiocyte to put into two kinds of cell culture fluids that volume ratio 1:1 mixes growth, result shows, spongiocyte growth conditions is bad, under microscope, observe spongiocyte transparent not, Cytoplasmic inclusions is more, is tending towards 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 make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (15)
1. be total in order for neurone and neurogliocyte a device of cultivating, it is characterized in that, comprise substrate and polydimethylsiloxane seal, described polydimethylsiloxane seal is compounded in described substrate removedly;
Described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface;
The distance of described the first through hole and the second through hole is 500 μ m～2000 μ m.
2. device according to claim 1, is characterized in that, described micropore unit also comprises at least one third through-hole perpendicular to described substrate being arranged in order with the first through hole and the second through hole.
3. device according to claim 2, is characterized in that, the distance of described third through-hole and described the second through hole is 500 μ m～2000 μ m.
4. device according to claim 1, is characterized in that, the cross section of described the first through hole and the second through hole is rectangle.
5. device according to claim 4, is characterized in that, the length of described the first through hole cross section is 1.0cm～2.0cm, and wide is 0.5cm～2.0cm;
The length of described the second through hole cross section is 1.0cm～2.0cm, and wide is 0.5cm～2.0cm.
6. device according to claim 1, is characterized in that, described substrate surface has been hatched extracellular matrix.
7. for neurone and a neurogliocyte preparation method for the common device of cultivating in order, comprising:
Prepare polydimethylsiloxane seal, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, the distance of described the first through hole and the second through hole is 500 μ m～2000 μ m;
Described polydimethylsiloxane seal is attached to substrate surface, and described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface.
8. preparation method according to claim 7, is characterized in that, described polydimethylsiloxane seal is prepared in accordance with the following methods:
Adopt micromachining technology in template, to prepare at least one spill line style microstructure unit, described spill line style microstructure unit comprise be arranged in order, at least one first spill bar and at least one the second matrix bar, the distance of described the first matrix bar and the second matrix bar is 500 μ m～2000 μ m;
With polydimethylsiloxane, the spill line style microstructure unit in described template is carried out to secondary and turn over film, obtain polydimethylsiloxane template;
The bottom surface of removing the first matrix bar and the second matrix bar in described polydimethylsiloxane template, obtains polydimethylsiloxane seal.
9. preparation method according to claim 8, is characterized in that, the cross section of described the first matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm; The cross section of described the second matrix bar is rectangle, and length is 1.0～2.0cm, and wide is 0.5～2.0cm.
10. preparation method according to claim 7, is characterized in that, described micropore unit also comprises at least one third through-hole perpendicular to described substrate being arranged in order with the first through hole and the second through hole.
The method that 11. 1 kinds of neuronal cells and spongiocyte are cultivated in order altogether, is characterized in that, comprising:
Incubated cell epimatrix in substrate;
Polydimethylsiloxane seal is connected in the substrate of hatching extracellular matrix, described polydimethylsiloxane seal comprises at least one micropore unit, described micropore unit comprise be arranged in order, at least one first through hole perpendicular to described substrate and at least one the second through hole perpendicular to described substrate, described the first through hole and the second through hole form respectively the groove of an end opening with substrate surface; The distance of described the first through hole and the second through hole is 500 μ m-2000 μ m;
Add Primary Neurons suspension to adding in neurogliocyte aaerosol solution and the first through hole in described the second through hole respectively, cultivate;
After cultivation, remove polydimethylsiloxane seal, have the substrate of neurogliocyte and neuronal cell to carry out common cultivation surface adhesion.
12. methods according to claim 11, is characterized in that, described micropore unit also comprises at least one third through-hole perpendicular to described substrate being arranged in order with the first through hole and the second through hole;
In described the second through hole, add neurogliocyte aaerosol solution and add Primary Neurons suspension in described the first through hole and third through-hole, cultivate.
13. according to the method described in claim 11 or 12, it is characterized in that, first adds neurogliocyte aaerosol solution, carries out the first step and is cultured to neurogliocyte and sticks to substrate surface;
Then add Primary Neurons suspension, carry out second step and be cultured to neuronal cell and stick to substrate surface.
14. methods according to claim 13, is characterized in that, the cell density of described neurogliocyte aaerosol solution is 10
5～10
7individual/mL; Described Primary Neurons suspension cell density is 10
4～10
6individual/mL.
15. methods according to claim 13, is characterized in that, the time that the described the 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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