CN106544320B - It is a kind of to be used to study single neuron culture mould of barometric gradient damage and its preparation method and application - Google Patents
It is a kind of to be used to study single neuron culture mould of barometric gradient damage and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of single neuron culture mould for being used to study barometric gradient damage and its preparation method and application, the mould includes sheet glass, trapezoidal hydrogel layer and the hydrogel layer containing neuronal cell, the trapezoidal hydrogel layer is shaped to trapezoidal by dovetail groove, and the hydrogel layer containing neuronal cell is located at the head end of trapezoidal hydrogel layer（H）, trapezoidal hydrogel layer and the hydrogel layer containing neuronal cell are freezed solidly on sheet glass.The trapezoidal axon growth hydrogel of " head height tail is low " is prepared by photopolymerization twice in the present invention, and neuronal cell is fixed on to the head end of axon growth gel（H）, in the external force of the exsule long gel direction loading fixed size of vertical axis, gradient stress, the barometric gradient that the long aixs cylinder of imictron is experienced are formed using deformation degree difference after gel stress.
Description
Technical field
The invention belongs to cellular damage research field, more particularly to a kind of single neuron for being used to study barometric gradient damage
Cultivate mould and its preparation method and application.
Background technology
Cell in body experiences a variety of stress with location difference（Shearing force, pressure, stretching
Power etc.）.After cell stress exceeds tolerance range, damage just occurs, studies influence of this damage process to cell behavior
It is an important topic of biomedical sector.In order to study damage of the power to cell, various Mechanical loading systems are opened
Hair, wherein the flexcell systems in the U.S., can complete the loading to cell flow shearing force, pressure, pulling force etc..These loadings
System integrally carries out Mechanical loading both for cell greatly, i.e., the power that individual cells are subject in the same time be it is identical, can be with
Meet most research.But the Shortcomings in neuron stress research.Partial nerve member president's excess of export in nervous system
Long aixs cylinder（Centimeter Level）, the stress of such individual cells just becomes line from point, i.e. individual cells different parts are subject to difference
Power.Example the most typical is exactly retinal ganglial cells（RGC）, its cell space part is located in eyeball, is subject to intraocular pressure；
And its aixs cylinder is located at the sealed tube intracavitary with ventricles of the brain unicom, it is subject to the pressure of cerebrospinal fluid, and intraocular pressure is different, meeting from intracranial pressure
A barometric gradient is formed, when intraocular pressure and the cranium pressure great change of generation, it may occur that irreversible optic nerve injury, can seriously make
Into blindness.
The content of the invention
To solve the above problems, it is contemplated that establish a single neuron culture body for being used to study barometric gradient damage
System, there is provided a kind of to be used to study single neuron culture mould of barometric gradient damage and its preparation method and application.
The purpose of the present invention is what is be achieved through the following technical solutions：
It is a kind of be used for study barometric gradient damage single neuron culture mould, including sheet glass, trapezoidal hydrogel layer and
Hydrogel layer containing neuronal cell, the trapezoidal hydrogel layer be shaped to by dovetail groove it is trapezoidal, it is described to contain neuronal cell
Hydrogel layer be located at the trapezoidal head end H of trapezoidal hydrogel layer, trapezoidal hydrogel layer and the hydrogel layer containing neuronal cell coagulate
It is fixed on sheet glass.
Further, the sheet glass is via the processed lid glass of 3- (trimethoxy first silicon substrate) propyl methacrylates
Piece.
Further, the hydrogel layer（3）With the hydrogel layer containing neuronal cell（5）Prepared by hydrogel solution
Obtain, the hydrogel solution is the GelMA hydrogels of concentration 5% ~ 8%, and wherein contains 0.1% ~ 1% photosensitizer.
Further, the GelMA hydrogels are prepared by phosphate buffer solution.
The preparation method of the single neuron culture mould for being used to study barometric gradient damage, comprises the following steps：
1）The preparation of photomask
Prepare axon growth photomask（1-1）With cell capture point photomask（1-2）；
2）The preparation of hydrogel solution
GelMA is dissolved into phosphate buffer solution, configures the GelMA hydrogel solutions of 5% ~ 8% final concentration, and is added
0.1% ~ 1% photosensitizer, obtains hydrogel solution；
3）The preparation of trapezoidal GelMA hydrogels
By step 2）The hydrogel solution being prepared is circulated into the dovetail groove being coated with by poly-D-lysine, above according to
Secondary cover glass piece, axon growth photomask, then photopolymerization is carried out with ultraviolet light, obtain the trapezoidal hydrogel layer of axon growth；
4）The preparation of GelMA hydrogel single cell suspensions
According to 1 × 104A/ml ~ 0.7x106The neuron of in vitro culture is resuspended to concentration as 3% ~ 8% by a/ml concentration
In GelMA hydrogel solutions, the GelMA suspensions containing neuron are obtained；
5）The unicellular capture of neuron
By step 4）The prepared GelMA suspensions containing neuron are filled into rectangle poly-D-lysine substrate groove, will be walked
Rapid 3）The hydrogel layer of obtained axon growth is placed in rectangular channel, trapezoidal hydrogel layer is dipped in the GelMA containing neuron and is hanged
In liquid, axon growth photomask is changed to cell capture point photomask, then photopolymerization is carried out with ultraviolet light, completes neuron list
Cell capture, that is, obtain the single neuron culture mould for being used to study barometric gradient damage.
Further, the photomask is film material, the axon growth photomask（1-1）Printing opacity at it is rectangular,
The cell capture point photomask（1-2）Printing opacity at it is rounded.
Further, axon growth photomask（1-1）Rectangle printing opacity at length and width be respectively 1cm, 20 μm, cell capture
Point photomask（1-2）Circular printing opacity at a diameter of 150 μm.
The mould is used for the method for studying the single neuron culture of barometric gradient damage, comprises the following steps：
1）The single celled culture of neuron
Mould as described above is placed in horizontal culture environment, neuron conditioned medium is added, is caught positioned at cell
The neuron axon obtained a little can be grown along the direction of axon growth gel, and aixs cylinder is grown after cultivating 3-4 weeks；
2）Barometric gradient loads
It is poor to prepare different height（4mm~20mm）Trapezoidal hydrogel layer developing approach it is unicellular, by with neuron
Trapezoidal hydrogel is placed in horizontal culture environment, on the top load vertical direction active force, using gel itself deformation not
With caused differential stress, there is provided the barometric gradient of long aixs cylinder neuron axon part, is studied under barometric gradient effect,
The change of long aixs cylinder neuron axoplasmic flow transport and dependent interaction mechanism.
Further, the neuron conditioned medium is containing 1%B27, 10% serum neuronal culture.
Further, the method is suitable for the neuron including retinal ganglial cells, Deiter's cells
Cell.
The present invention having the beneficial effect that compared with prior art：
1st, the present invention uses gelatin-methacrylate（GelMA）Material, by adding photosensitizer, makes it have light and gathers
Conjunction ability, by first time photopolymerization, is prepared the trapezoidal axon growth hydrogel of " head height tail is low ", is gathered by second of light
Close, neuronal cell is fixed on to the head end of axon growth gel（H）, it is thin that long aixs cylinder neuron is obtained after in vitro culture
Born of the same parents, in the external force of the exsule long gel direction loading fixed size of vertical axis, are formed using deformation degree difference after gel stress
Gradient stress, the barometric gradient that the long aixs cylinder of imictron is experienced；
2nd, this method can be used for the preparation of vitro disease model, is distributed by designing different-stiffness barometric gradient, regulation and control regard
The function of nerve cell simultaneously matches with internal disease model, and effective tool is provided for drug screening.
Brief description of the drawings
Fig. 1 is axon growth photomask（1-1）Structure diagram；
Fig. 2 is cell capture point photomask（1-2）Structure diagram；
Fig. 3 prepares schematic diagram for trapezoidal hydrogel；
Fig. 4 is unicellular capture schematic diagram；
Fig. 5 is the single neuron culture mold structure diagram for studying barometric gradient damage being prepared；
Fig. 6 is effect schematic diagram of the barometric gradient to long aixs cylinder neuron；
Fig. 7 is effect detailed schematic diagram of the barometric gradient to long aixs cylinder neuron；
Fig. 8 is effect schematic diagram of the different pressures gradient to long aixs cylinder neuron.
Embodiment
Present embodiments provide a kind of single neuron culture mould for being used to study barometric gradient damage, its preparation method
For：
1）The preparation of photomask
Prepare axon growth photomask 1-1 and cell capture point photomask 1-2, the photomask as shown in Figure 1 and Figure 2, axis
It is rectangular at the printing opacity of exsule long photomask 1-1, length and width be respectively 1cm, 20 μm；At the printing opacity of cell capture point photomask 1-2
It is rounded, a diameter of 150 μm；The photomask is film material；
The photomask is prepared by Shenzhen Guang Yi Co., Ltds；
2）The preparation of hydrogel solution
GelMA is dissolved into phosphate buffer solution（DPBS）In, the GelMA hydrogel solutions of 5% final concentration of configuration, and
0.5% photosensitizer is added, obtains hydrogel solution；
The photosensitizer is Irgacure 2959；
3）The preparation of trapezoidal GelMA hydrogels
As shown in figure 3, by step 2）The hydrogel solution being prepared is circulated into the ladder being coated with by poly-D-lysine 4-1
In shape groove, cover glass piece 2, axon growth photomask 1-1 successively, then use 2.95mW/cm above2The ultraviolet light of energy carries out light
Polymerization, obtains the trapezoidal hydrogel layer 3 of axon growth；
The sheet glass is 3- (trimethoxy first silicon substrate) propyl methacrylate（TMSPMA）Processed coverslip；
4）The preparation of GelMA hydrogel single cell suspensions
According to 1 × 104Extracorporeal neuron is resuspended to concentration in 5% GelMA hydrogel solutions, to obtain by a/ml concentration
GelMA suspensions containing neuron；
5）The unicellular capture of neuron
As shown in figure 4, by step 4）The prepared GelMA suspensions containing neuron are filled into rectangle poly-D-lysine substrate
In 4-2 grooves, by step 3）The hydrogel layer of obtained axon growth is placed in rectangular channel, trapezoidal hydrogel layer is dipped in containing nerve
In the GelMA suspensions of member, axon growth photomask is changed to cell capture point photomask, then use 2.95mW/cm2The purple of energy
Outer light carries out photopolymerization, completes the unicellular capture of neuron, forms the hydrogel layer 5 containing neuronal cell, that is, obtains described
For studying the single neuron culture mould of barometric gradient damage.
The mould is used for the method for single neuron culture for studying barometric gradient damage, is comprised the following steps：
1）The culture of RGC cells
As shown in figure 5, the above-mentioned mould being prepared is placed in horizontal culture environment, neuron conditioned medium is added
（Containing 1%B27, 10% serum neuronal culture）, the neuron axon positioned at cell capture point can be along axon growth gel
Direction is grown, and aixs cylinder is grown after cultivating 3-4 weeks, treats to carry out pressure-loaded in next step；
2）Barometric gradient loads
As shown in fig. 6, the trapezoidal hydrogel layer developing approach for preparing different height difference is unicellular, by with neuron
Trapezoidal hydrogel is placed in horizontal culture environment, on the top load vertical direction active force, using gel itself deformation not
With caused differential stress, there is provided the barometric gradient of long aixs cylinder neuron axon part, is studied under barometric gradient effect,
The change of long aixs cylinder neuron axoplasmic flow transport and dependent interaction mechanism.
Specifically, different pressure is formed using the compression factor difference of material.In the figure 7, the position of boundary line mark
Before and after putting, RGC cells bear different pressure.H4 in figure is labeled as the height of material after shaping.Because what cell space was subject to
Power can decline suddenly after by boundary line position, so being designed as h1 is more than h2.The difference of h2 and h3 is trapezoidal gel
Difference in height, for forming barometric gradient, and this difference is fixed 16mm.
For more preferable simulated pressure gradient, pass through the pressure for adjusting the size of each h in Fig. 7 to simulate under different situations
Gradient.
As shown in upper table and Fig. 8, we are retouched the pressure that pericaryon and aixs cylinder are experienced with high, medium and low
State, the ratio of corresponding gel compression is also to be high, medium and low.The h being introduced into Fig. 7 is right in the case of h4 fixations upon compression
Material before loading, stress are proportionate with h differences.
（1）Normal gradient situation：Respectively hn1, hn2, hn3, the numerical value of other situations is compared with normal condition；
（2）The situation of H pressure rises：Hh1 is more than hn1, other to be respectively worth equal, the change increase of hh1 after forced compression,
That is cell space stress increase, and aixs cylinder stress is constant；
（3）The situation that T pressure reduces：Hl1 is equal to hn1；Hl2, hl3 are less than hn2 and hn3, after forced compression, hl2/
The change of hl3 reduces, i.e., aixs cylinder stress reduces, and cell space stress is constant.
By adjusting the numerical value of h1-4, different pressure can be applied to cell and adjust barometric gradient, cut suitable for each
The culture of kind neuron.
Claims (10)
1. a kind of single neuron culture mould for being used to study barometric gradient damage, it is characterised in that the mould includes glass
Piece（2）, trapezoidal hydrogel layer（3）With the hydrogel layer containing neuronal cell（5）, the trapezoidal hydrogel layer determined by dovetail groove
Shape is trapezoidal, the hydrogel layer containing neuronal cell（5）Positioned at trapezoidal hydrogel layer（3）Head end（H）, trapezoidal hydrogel
Layer and the hydrogel layer containing neuronal cell freeze solidly on sheet glass（2）On；The horizontal stroke of wherein described trapezoidal hydrogel on the glass sheet
Section is rectangle, and longitudinal section is trapezoidal.
2. the single neuron culture mould according to claim 1 for being used to study barometric gradient damage, it is characterised in that institute
It is via 3- (trimethoxy first silicon substrate) the processed coverslip of propyl methacrylate to state sheet glass.
3. the single neuron culture mould according to claim 1 for being used to study barometric gradient damage, it is characterised in that institute
State trapezoidal hydrogel layer（3）With the hydrogel layer containing neuronal cell（5）It is prepared by hydrogel solution, the hydrogel
Solution is the GelMA hydrogels of concentration 5% ~ 8%, and wherein contains 0.1% ~ 1% photosensitizer.
4. the single neuron culture mould according to claim 3 for being used to study barometric gradient damage, it is characterised in that institute
GelMA hydrogels are stated to be prepared by phosphate buffer solution.
A kind of 5. system for the single neuron culture mould for being used to study barometric gradient damage such as claim 1-4 any one of them
Preparation Method, it is characterised in that the preparation method comprises the following steps：
1）The preparation of photomask
Prepare axon growth photomask（1-1）With cell capture point photomask（1-2）；
2）The preparation of hydrogel solution
GelMA is dissolved into phosphate buffer solution, configures the GelMA hydrogel solutions of 5% ~ 8% final concentration, and add 0.1%
~ 1% photosensitizer, obtains hydrogel solution；
3）The preparation of trapezoidal hydrogel layer
By step 2）The hydrogel solution being prepared is circulated into by poly-D-lysine（4-1）In the dovetail groove being coated with, above
Cover glass piece, axon growth photomask, then carry out photopolymerization with ultraviolet light successively, obtain the trapezoidal hydrogel of axon growth
Layer；
4）The preparation of GelMA hydrogel single cell suspensions
Extracorporeal neuron is resuspended to concentration in 5% ~ 8% GelMA hydrogel solutions, to obtain the GelMA suspensions containing neuron；
5）The unicellular capture of neuron
By step 4）The prepared GelMA suspensions containing neuron are filled into rectangle poly-D-lysine substrate（4-2）In groove, it will walk
Rapid 3）The hydrogel layer of obtained axon growth is placed in rectangular channel, trapezoidal hydrogel layer is dipped in the GelMA containing neuron and is hanged
In liquid, axon growth photomask is changed to cell capture point photomask, then photopolymerization is carried out with ultraviolet light, completes neuron list
Cell capture, that is, obtain the single neuron culture mould for being used to study barometric gradient damage.
6. preparation method according to claim 5, it is characterised in that the photomask is film material, the aixs cylinder life
Long photomask（1-1）Printing opacity at rectangular, the cell capture point photomask（1-2）Printing opacity at it is rounded.
7. preparation method according to claim 6, it is characterised in that axon growth photomask（1-1）Rectangle printing opacity at
Length and width be respectively 1cm, 20 μm, cell capture point photomask（1-2）Circular printing opacity at a diameter of 150 μm.
A kind of 8. side for the single neuron culture for being used to study barometric gradient damage such as claim 1-4 any one of them mould
Method, it is characterised in that the described method comprises the following steps：
1）The single celled culture of neuron
Mould as described in claim 1-4 is placed in horizontal culture environment, neuron conditioned medium is added, positioned at cell
The neuron axon of capture point can be grown along the direction of axon growth gel, and aixs cylinder is grown after cultivating 3-4 weeks；
2）Barometric gradient loads
It is unicellular to prepare the trapezoidal hydrogel layer developing approach of different height difference, the trapezoidal hydrogel with neuron is placed in
In horizontal culture environment, the active force of vertical direction is loaded on the top, utilizes difference caused by the deformation difference of gel itself
Stress, there is provided the barometric gradient of long aixs cylinder neuron axon part, is studied under barometric gradient effect, long aixs cylinder neuron axis
The change of slurry stream transport and dependent interaction mechanism.
9. according to the method described in claim 8, it is characterized in that, the neuron conditioned medium is containing 1%B27, 10% serum
Neuronal culture.
10. method according to claim 8 or claim 9, it is characterised in that the method is thin suitable for including ganglia retinae
Neuronal cell including born of the same parents, Deiter's cells.
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Inventor after: Wu Shen Inventor after: Wang Ningli Inventor after: Zhang Jingxue Inventor after: Mao Yingyan Inventor after: Li Yuhui Inventor after: Xu Feng Inventor before: Li Yuhui Inventor before: Xu Feng Inventor before: Wu Shen Inventor before: Wang Ningli Inventor before: Zhang Jingxue Inventor before: Mao Yingyan |
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Effective date of registration: 20180118 Address after: 710049 Xianning West Road, Shaanxi, No. 28 Xi'an Jiao Tong University, Applicant after: Li Yuhui Applicant after: Xu Feng Address before: 100005 Beijing Hutong Hutong, Dongcheng District, No. 17 Applicant before: Beijing Institute of Ophthalmology |
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