CN113699109A - Construction method of in-vitro modular neuron network - Google Patents
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Abstract
The invention relates to a method for constructing an in-vitro modular neuron network, which is characterized in that patterns covering all structural parameter combinations to be researched are generated based on a digital image processing technology and are spliced into a single pattern, and a micro-printing plate containing all the patterns to be researched can be prepared by combining a soft lithography process based on laser direct writing and a surface modification method, so that a modular protein pattern containing any different parameter combinations can be generated by single printing, and the in-vitro construction of the modular neuron network consisting of real neurons and different parameter combinations is realized.
Description
Technical Field
The invention relates to the technical field of tissue engineering, in particular to a method for constructing an in-vitro modular neuron network.
Background
A number of studies have shown that in the brains of many animals including humans, neurons are not uniformly distributed to form an overall neural network, but rather, in a modular, distributed configuration, form individual integrated units with very high local neuron density and synapse density, with relatively sparse axon projections and synapse connections between units. Therefore, the research on the modular neuron network plays a very important role in researching the memory and learning behaviors of the advanced animals so as to understand the consciousness generation of human beings. However, most of the current studies on the modular neuron network focus on the study of the neural circuits formed between specific brain regions in a living rodent, and these circuits have important research values because they carry important sensory or motor functions and inherently have neural encoding and decoding functions. However, also because of its internal complete codec function, many studies can only link the behavior level and the firing level of some neurons to try to understand its codec mode. Such studies are limited by the inability to change or customize important parameters of the modular neuronal network, and therefore have been slow in research on the codec mechanism of the modular neuronal network. Therefore, the in vitro construction of the modular neuron network with different parameter combinations formed by real neurons is very important for researching the encoding and decoding modes of the modular neuron network, the influence of information transmission between the structural parameters and the network and the dynamic characteristics of the modular neuron network, and the mutual relation between the parameters and the pharmacological action.
Disclosure of Invention
The invention aims to provide a method for constructing an in-vitro modular neuron network, so as to realize the in-vitro construction of the modular neuron network composed of real neurons and different parameter combinations.
In order to achieve the purpose, the invention provides the following scheme:
a method of constructing an in vitro modular neuronal network, the method comprising the steps of:
generating a mask pattern covering all structural parameters to be researched by adopting a digital image processing method;
preparing a PDMS micro printing plate according to the mask pattern by adopting a soft photoetching process of laser direct writing;
generating a protein pattern in a surface-modified culture dish according to the PDMS micro printing plate printing, and obtaining the culture dish printed with the protein pattern;
and (3) performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in-vitro modular neuron network.
Optionally, the generating a mask pattern covering all the structural parameters to be studied by using a digital image processing method specifically includes:
programming and drawing a modular pattern with adjustable parameters;
splicing a plurality of modular patterns with adjustable parameters to obtain spliced patterns;
and adjusting the parameters of each parameter-adjustable modular pattern in the spliced patterns to generate a mask pattern covering all the parameters of the structure to be researched.
Optionally, the programming of the modular pattern with adjustable drawing parameters specifically includes:
generating a Containment prow×pcolA module value matrix of individual elements; wherein p isrowAnd pcolRepresenting the number of horizontal elements and vertical elements of the module numerical matrix;
resetting all elements in a square area with the upper left corner ((i-1) × 2n +1, (j-1) × 2n +1) and the lower right corner ((i-1) × 2n + n, (j-1) × 2n + n) in the module numerical matrix to be 255, and finishing the drawing of the module area of the modular pattern with adjustable parameters; where i and j traverse from 1 to N, respectivelyrowAnd NcolN is the side length of the module, NrowNumber of modules contained in transverse direction for modular pattern with adjustable parameters, NcolThe number of modules contained in the longitudinal direction for the modular pattern whose parameters are adjustable;
the upper left corner of the module value matrix is ((i-1) × 2n + round (n/(X)con+1))×(k-1)-xwidth(j-1). times.2 n + n/2), and coordinates of elements in the lower right corner are ((i-1). times.2 n + round (n/(X)con+1))×(k-1)+xwidth(j-2) all elements in the (j-1) x 2n +3n/2) region are set to be 255, and the jth and j +1 th elements in the ith row are completedDrawing transverse connecting bands among the modules; where k traverses from 1 to Xcon，XconThe number of the horizontal connecting bands between the jth and the j +1 th modules of the ith row is, round () is the operator of rounding, xwidthThe bandwidth of a transverse connection band between the jth module and the jth +1 module in the ith row is set;
the upper left corner is ((j-1). times.2 n + round (n/(Y)con+1))×(k'-1)-ywidth(i-2) × 2n + n/2), and coordinates of bottom-right corner elements are ((j-1) × 2n + round (n/(Y))con+1))×(k'-1)+ywidthSetting all elements in the area of (i-1) multiplied by 2n +3n/2) as 255, and completing drawing of the longitudinal connecting band between the ith module and the (i +1) th module in the jth column; wherein k' is traversed from 1 to Ycon，YconThe number of longitudinal connected bands between the ith and the (i +1) th modules in the jth column, round () is the operator of rounding, ywidthThe bandwidth of the vertical connecting band between the ith and (i +1) th modules in the jth column.
Optionally, the splicing a plurality of modular patterns with adjustable parameters to obtain a spliced pattern specifically includes:
generating a Containment Prow×PcolA concatenation number matrix of individual elements;
wherein, ProwRepresenting the number of transverse elements, P, in the concatenated numerical matrixcolRepresenting the number of vertical elements in the data matrix,
frowand fcolRespectively representing the number of transverse and longitudinal arrangements of the parameter-adjustable modular patterns to be stitched, growAnd gcolThe horizontal spacing and the vertical spacing between the modular patterns with adjustable parameters to be spliced are respectively; p is a radical ofrowAnd pcolRepresenting the number of horizontal elements and vertical elements of the module numerical matrix;
copying the element values of the modular pattern with adjustable (x, y) th parameter to the upper left corner of the splicing numerical value matrix to be (p)row×(x-1)+1,pcolX (y-1) +1) and lower right corner is (p)row×(x-1)+prow,pcol×(y-1)+pcol) Wherein x and y traverse from 1 to f, respectivelyrowAnd fcol。
Optionally, the adjustable parameters in the modular pattern with adjustable parameters include: the length of sides of the modules, the distance between the centers of the modules, the number of the communicating belts between the modules and the width of the communicating belts are used for adjusting the number of the neurons, and the distance between the centers of the modules, the number of the communicating belts between the modules and the width of the communicating belts are used for adjusting the communicating strength between the neurons.
Optionally, the preparing the PDMS micro-printing plate according to the mask pattern by using the soft lithography process of laser direct writing specifically includes:
loading the mask pattern into a mask generator to perform laser exposure masking on the chromium plate to generate a masked chromium plate;
developing and etching the masked chrome plate to obtain a mold;
and pouring a mixed solution of PDMS prepolymer and a curing agent on the mold to solidify and glue to obtain the PDMS micro printing plate.
Optionally, the protein pattern is generated in the surface-modified culture dish according to the printing of the PDMS micro-printing plate, so as to obtain the culture dish printed with the protein pattern, and the method specifically includes:
dripping 0.2% agarose aqueous solution into a circular area with the central diameter of 25mm of a 35mm polypropylene culture dish, and carrying out ultraviolet irradiation and natural air drying to obtain a culture dish with modified surface;
placing the PDMS micro printing plate at the bottom of the surface modified culture dish with the pattern surface facing downwards;
and pressing the balance weight on the PDMS micro printing plate for a preset time period, and then taking down the balance weight and the PDMS micro printing plate to obtain the culture dish printed with the protein pattern.
Optionally, performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in vitro modular neuron network, wherein the method further comprises the following steps:
placing a semi-hollow annular sleeve in the central region of a 35mm petri dish;
pouring a mixed solution of PDMS prepolymer and a curing agent into a 35mm culture dish to solidify and glue to obtain a PDMS annular structure, and stripping the PDMS annular structure from the 35mm culture dish;
punching two through holes at positions 180 degrees apart of the PDMS annular structure, and punching two through holes at positions 180 degrees apart on the side wall of the culture dish printed with the protein pattern;
placing the PDMS ring structure with the through holes in a culture dish with the through holes and printed with the protein patterns, wherein the two through holes of the PDMS ring structure respectively correspond to the two through holes of the culture dish with the protein patterns;
and sequentially passing one pipeline through one through hole of the culture dish printed with the protein pattern and one through hole of the PDMS annular structure, sequentially passing the other pipeline through the other through hole of the culture dish printed with the protein pattern and the other through hole of the PDMS annular structure, and fixing to obtain the culture dish printed with the protein pattern after the constraint of the PDMS annular structure is added.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects:
the invention provides a method for constructing an in-vitro modular neuron network, which comprises the following steps: generating a mask pattern covering all structural parameters to be researched by adopting a digital image processing method; preparing a PDMS micro printing plate according to the mask pattern by adopting a soft photoetching process of laser direct writing; generating a protein pattern in a surface-modified culture dish according to the PDMS micro printing plate printing, and obtaining the culture dish printed with the protein pattern; and (3) performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in-vitro modular neuron network. The invention can generate patterns covering all structural parameter combinations to be researched based on a digital image processing technology, and can splice the patterns into a single pattern, and can prepare the micro-printing plate containing all the patterns to be researched by combining a soft lithography process based on laser direct writing and a surface modification method, so that modular protein patterns containing any different parameter combinations can be generated by single printing, and the modular neuron network of different parameter combinations formed by real neurons can be constructed in vitro.
The invention also utilizes the microfluidic technology to carry out open constraint on the substrate which is subjected to surface modification and patterning, and has the function of supporting contact type electrophysiological detection on the basis of effectively improving the inoculation density and having a perfusion channel to support the accurate drug test function.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a flow chart of a method for constructing an in vitro modular neuron network according to the present invention;
FIG. 2 is a schematic diagram of a method for constructing an in vitro modular neuron network according to the present invention;
FIG. 3 is an example diagram of a modular pattern provided by the present invention;
FIG. 4 is a diagram illustrating an example of a mask pattern provided by the present invention;
FIG. 5 is a flow chart for preparing a PDMS micro-printing plate provided by the present invention;
FIG. 6 is a flow chart of a printing generation protein pattern provided by the present invention;
FIG. 7 is a flow chart of adding PDMS ring structural constraints provided by the present invention;
fig. 8 is a schematic structural view of a culture dish printed with a protein pattern after being constrained by adding a PDMS ring structure according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention aims to provide a method for constructing an in-vitro modular neuron network, so as to realize the in-vitro construction of the modular neuron network composed of real neurons and different parameter combinations.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
The invention combines the digital image generation and processing technology, the micro-contact printing technology, the numerical control processing technology and the microfluidic technology, has the advantages of high flux capable of covering all network structure parameters by single printing, adjustable inoculation density, high controllability capable of supporting drug testing and high expansibility for contact type electrophysiological measurement and the like, can be used for forming a reliable modular network with multiple structure parameters, and the covering parameters comprise the number and density of neurons in the module, the communication strength among the modules and the like. In addition, the network also comprises a perfusable channel and an open design, can simultaneously support contact electrophysiological measurement means such as patch clamp and the like and apply functions such as neurotransmitter or synapse antagonist and the like, and can be used as an in-vitro test model of a cranial nerve circuit.
As a preferred embodiment, as shown in fig. 1 and 2, the present invention has the following embodiments:
Further, step 101 specifically includes:
the programming program draws the modular pattern. Modular pattern moldA block region and a connected region, wherein the block region is a square region of (n × n) pixels 2, and the center distance of the block is nwidthThe connected region is BnumRoot bandwidth of xwidthA band-shaped region of pixels. The program compiling is divided into two steps: generating F from design parametersnumImages with different sheet parameters; all images were fused into one image so that a single print produced protein patterns of all parameters.
Wherein, the first step is to consider the connected region as BnumThe unpatterned spacing between modules is also N pixels in the root band region, the pattern comprising N in the transverse directionrowA module comprising N in the longitudinal directioncolAnd (4) a module. First, the horizontal and vertical directions are generated as prowAnd pcolElement, a uint8 (unsigned 8 bit integer) matrix with an element value of 0, where,
next, the module area is drawn. Nesting adopts two cyclic functions, all elements in a square area with the upper left corner ((i-1) × 2N +1, (j-1) × 2N +1) and the lower right corner ((i-1) × 2N + N, (j-1) × 2N + N) are reset to be 255, wherein i and j respectively traverse from 1 to NrowAnd Ncol。
Again, connected banded regions are drawn. And respectively adopting two cyclic functions to draw the transverse and longitudinal bands in sequence. Taking the transverse band as an example, consider the band width as xwidthX between the jth and jth +1 modules in the ith rowconThe coordinates of the elements of the upper left corner and the lower right corner of the root connecting band are ((i-1) × 2n + round (n/(X)con+1))×(k-1)-xwidth(j-1). times.2 n + n/2) and ((i-1). times.2 n + round (n/(X)con+1))×(k-1)+xwidth2, (j-1). times.2 n +3n/2) where k traverses from 1 to XconAnd round () is a rounding operator, and all elements of the above area are set to 255. The modular pattern obtained through the above steps is shown in fig. 3.
Secondly, on the basis of obtaining the modular patterns with different parameters, all the patterns are spliced into a pictureThe scheme can realize single printing to generate all protein patterns, and the splicing realization process is as follows. Consider a total of fnOpening pattern to be spliced, fn＝FnumThen the layout of the total pattern is frow×fcolI.e. transverse and longitudinal respectively contain frowAnd fcolAnd (5) a sheet pattern. Wherein
Then, first, a containment P is generatedrow×PcolA value matrix of uint8 with an element value of 0, wherein,
Second, nesting using two round functions, will fxThe element values of the images to be spliced are copied to the upper left element coordinate and the lower right element coordinate and are respectively (p)row×(i-1)+1,pcolX (j-1) +1) and (p)row×(i-1)+prow,pcol×(j-1)+pcol) Where x ═ 1,2, …, n), i and j traverse from 1 to f, respectivelyrowAnd fcol，x＝fcolX (i-1) + j. And finally, storing the matrix as a bmp format bitmap.
So far, the modular network generating any parameter can be covered only by changing the parameter value, and the adjustable parameter of the method comprises the following steps: width n of square module area, module center distance nwidthNumber of communicating zones B of communicating areanumAnd width xwidthAnd the width n is used for adjusting the number of neurons of the modular network, and the rest parameters are used for adjusting the communication strength between the modules. The mask pattern obtained through the above steps is shown in fig. 4.
And 102, preparing the PDMS micro printing plate according to the mask pattern by adopting a soft photoetching process of laser direct writing.
102, preparing the PDMS micro-printing plate according to the mask pattern by using the laser direct writing soft lithography process, specifically comprising: loading the mask pattern into a mask generator to perform laser exposure masking on the chromium plate to generate a masked chromium plate; developing and etching the masked chrome plate to obtain a mold; and pouring a mixed solution of PDMS prepolymer and a curing agent on the mold to solidify and glue to obtain the PDMS micro printing plate.
Further, as shown in fig. 5, step 102 specifically includes:
2-1) a uniform-thickness chrome plate coated with 12 μm thick glue is purchased, divided into small chrome plates, and the small chrome plates with proper size are placed on a vacuum clamping platform of a mask generator. Firstly, replacing a laser writing head with a 2mm writing head with the highest precision, wherein a single pixel corresponds to a 200nm line width, opening a software Exposure Wizard matched with a mask generator, changing an operation mode into a gray mode so as to expose through a bitmap, loading a mask pattern, then determining the optimal laser parameters including a laser duty ratio, laser intensity, an energy mode and the like, carrying out laser focusing by adopting a pneumatic and optical hybrid automatic focusing mode, and finally starting the mask generator;
2-2) after the exposure is finished, closing the vacuum clamping platform, taking out the chromium plate, putting the chromium plate into a 60mm polypropylene culture dish, adding 1:4 AZ400K aqueous solution, developing for 6 minutes, and keeping the culture dish to shake all the time during the development so as to avoid local formation of deposits and influence on the uniformity of the development process. Washing with deionized water for three times after the development is finished, and drying;
2-3) placing the developed and blow-dried chromium plate into a new culture dish, adding an etching agent, etching for 45-60 seconds, keeping shaking the culture dish in the etching process, washing with deionized water for three times after etching is finished, and blow-drying;
2-4) placing the etched chromium plate on a stage of an optical microscope, and selecting an objective lens with proper magnification to check the local details of the template pattern. If the pattern edge is clear and the structure is complete, the pattern can be used as a template of the micro-printing plate, otherwise, the above process is repeated until the ideal effect is obtained.
2-5) mixing the PDMS prepolymer and the curing agent in a proper proportion, fully stirring, and keeping out of the sun and placing in a low-temperature environment to remove bubbles. A60 mm polypropylene culture dish is used as a vessel, and double-layer tin foil paper and an attached culture dish are cut and used for containing mixed liquid of a template and PDMS. And (3) putting the template with the right side facing upwards into a culture dish attached with double-layer tinfoil paper, adding the PDMS mixed solution with the bubbles removed, putting the whole culture dish into a constant-temperature incubator at 65 ℃ for two hours, taking out the culture dish, cutting out the solidified PDMS micro-printing plate, storing the PDMS micro-printing plate in deionized water, and taking out the PDMS micro-printing plate before use.
In the invention, after the mask is generated in the step 102 through the mask generator, the mask pattern is not required to be transferred to the silicon wafer, but the mask pattern is directly used as a mold, PDMS is poured on the mold, and the mold is solidified into glue.
And 103, generating a protein pattern in the surface-modified culture dish according to the PDMS micro printing plate printing, and obtaining the culture dish printed with the protein pattern.
103, printing the surface-modified culture dish with the protein pattern according to the PDMS micro printing plate to obtain the culture dish printed with the protein pattern, wherein the method specifically comprises the following steps: dripping 0.2% agarose aqueous solution into a circular area with the central diameter of 25mm of a 35mm polypropylene culture dish, and carrying out ultraviolet irradiation and natural air drying to obtain a culture dish with modified surface; placing the PDMS micro printing plate at the bottom of the surface modified culture dish with the pattern surface facing downwards; and pressing the balance weight on the PDMS micro printing plate for a preset time period, and then taking down the balance weight and the PDMS micro printing plate to obtain the culture dish printed with the protein pattern.
Further, as shown in fig. 6, step 103 specifically includes:
3-1) preparing 0.2% agarose aqueous solution. Firstly, pouring deionized water into a beaker, placing the beaker on a magnetic stirrer, adding a magnetic stirrer, starting a hot plate heating and stirring switch, and pouring the whole body after the deionized water is boiled. This operation was repeated three times to ensure a beaker with high cleanliness. Secondly, 50mL of deionized water and a magnetic stirrer were added, 100mg of agarose powder was added after the water was boiled, and after about ten minutes, the agarose dissolved in the water and the solution was seen to be clear. 0.15mL of agarose aqueous solution is taken by a Pasteur dropper and evenly covered on a circular area with the central diameter of 25mm of a 35mm polypropylene culture dish, so that the condition that the agarose concentration at the bottom of the dish is too low due to the fact that the dish wall is contacted to prevent a large amount of solution from being adsorbed by the dish wall is avoided. Standing in a fume hood, standing overnight under ultraviolet irradiation, naturally air drying, covering with a dish, and preserving in dry environment.
3-2) preparing a protein solution. The protein solution contained ECM gel and Poly-D-lysine (Poly-D-lysine) in PBS solution.
3-3) before microcontact printing, taking out the PDMS micro printing plate from deionized water, putting the PDMS micro printing plate into a 50mL centrifuge tube filled with 10% SDS (sodium dodecyl sulfate) aqueous solution, ultrasonically cleaning for five minutes, and standing for ten minutes to uniformly plate a layer of SDS thin film on the micro printing plate, wherein the layer of SDS thin film can improve the protein separation efficiency. The microplate was removed from the SDS solution, rinsed with deionized water, and blow dried, placed right on the bottom of a new petri dish, 200 μ L of protein solution was dropped onto the pattern area, the dish lid was closed, the petri dish was transferred to a 37 ℃ incubator, and left for 20 minutes. After 20 minutes, the flexographic printing plate was removed, rinsed with deionized water, and blow dried.
3-4) simple protein printing based on centrifugal tube balance weight. The microplate was turned over using tweezers, pattern down, and placed at the bottom of a 35mm petri dish modified with an agarose surface. The weight is prepared, the weight is a 50mL centrifuge tube, 3 weights of 50g and the balance of water are added into the centrifuge tube, and the total weight is 200 g. Finally, the centrifuge tube was tightened, placed upside down over the flexographic plate, held for 2 minutes, then removed and the flexographic plate was removed using tweezers. And (3) standing the printed culture dish in a fume hood, covering the dish lid after drying, wrapping the dish lid by using double-layer tin foil paper, storing the dish lid in a dry environment, and avoiding ultraviolet irradiation.
In step 103, the surface of the culture dish is modified before printing, so that the surface is not suitable for cell attachment. After printing, only the pattern area printed with the protein is suitable for cell attachment.
And 104, performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in-vitro modular neuron network.
Step 104, performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in vitro modular neuron network, wherein the method also comprises the following steps: placing a semi-hollow annular sleeve in the central region of a 35mm petri dish; pouring a mixed solution of PDMS prepolymer and a curing agent into a 35mm culture dish to solidify and glue to obtain a PDMS annular structure, and stripping the PDMS annular structure from the 35mm culture dish; punching two through holes at positions 180 degrees apart of the PDMS annular structure, and punching two through holes at positions 180 degrees apart on the side wall of the culture dish printed with the protein pattern; placing the PDMS ring structure with the through holes in a culture dish with the through holes and printed with the protein patterns, wherein the two through holes of the PDMS ring structure respectively correspond to the two through holes of the culture dish with the protein patterns; and sequentially passing one pipeline through one through hole of the culture dish printed with the protein pattern and one through hole of the PDMS annular structure, sequentially passing the other pipeline through the other through hole of the culture dish printed with the protein pattern and the other through hole of the PDMS annular structure, and fixing to obtain the culture dish printed with the protein pattern after the constraint of the PDMS annular structure is added.
Further, as shown in fig. 7, the specific step of adding the PDMS ring structure constraint includes:
4-1) machining the semi-hollow annular sleeve based on a numerical control machine tool. Because the method adopts the purchase of commercial cortical neurons, the quantity of the commercial cortical neurons is limited, in order to improve the neuron inoculation density and simultaneously improve the experimental times of purchasing neurons at a time, a 35mm culture dish needs to be subjected to structural constraint, and the area of a non-patterned area is reduced as much as possible. Firstly, a semi-hollow annular sleeve is processed by a numerical control machine tool and is used as a template for subsequently preparing a PDMS annular structure. The annular sleeve is close to the annular structure, but one end of the annular sleeve is solid, so that the annular sleeve is considered as a balance weight on one hand, and PDMS is prevented from permeating on the other hand. The main parameter of the sleeve is its outer diameter D, which determines the total area S of the constraining zone, where S ═ pi D2/4。
4-2) preparing a PDMS ring structure based on the semi-hollow ring sleeve. And (3) standing the sleeve in the central area of a 35mm culture dish, and dripping a small amount of deionized water in the central area of the sleeve for temperature measurement of subsequent hot plate heating. Mixing the PDMS prepolymer and the curing agent in a proper proportion, fully stirring, and placing in a low-temperature environment in a dark place to remove bubbles. And after the bubbles are completely removed, pouring the PDMS mixed solution into a 35mm culture dish, slightly standing until the surface of the solution is horizontal. And then putting the culture dish on a hot plate, inserting a temperature measuring sensor of the hot plate below the liquid level in the annular sleeve, heating the hot plate to 65 ℃, keeping for 30 minutes, slightly solidifying the PDMS mixed liquid, transferring the culture dish to a constant-temperature incubator at 65 ℃ after fixing the position of the sleeve, taking out after two hours, sequentially taking out the central annular sleeve, and stripping the culture dish from the PDMS annular structure to obtain the PDMS annular structure with a complete structure.
4-3) processing the perfusable channel based on the PDMS ring structure. And respectively drilling holes in 180-degree areas adjacent to the side wall of the PDMS annular structure by using a drilling gun with a 2.5mm drill bit, washing with a 70% ethanol solution, and drying. And similarly, drilling holes on the corresponding positions of the side walls of the 35mm culture dishes with the agarose surface modified and the protein patterns printed on the side walls by using a 4mm drill, stopping drilling when the holes are nearly drilled, pricking the side walls from inside to outside by using tweezers, and blowing gas from inside to outside by using nitrogen to blow away fine impurities adhered to the side walls. PDMS annular structure after will holing presses the corresponding position of fixing to 35mm culture dish, later to wherein insert the rubber pipeline that the external diameter is 2mm, the pipeline extends from the extroversion inwards, stop after stretching out the annular structure inner wall slightly, with the Pasteur dropper toward pipeline and inner wall kneck dropwise add the dissolved 3% agarose solution of preheating, equally with the Pasteur dropper toward pipeline and annular structure outer wall kneck dropwise add the 3% agarose solution of preheating, to include the 35mm culture dish of PDMS annular structure and 2mm pipeline in arranging the 100mm culture dish in, cover the culture dish, it in order to cool down to stew in the fume hood, can fix the 2mm pipeline after the agarose solution solidifies. If perfusion culture is not added, the outlet of the pipeline extending out of the culture dish is clamped by a clip, the clip is soaked in 70% ethanol for 60 minutes before use, and is disinfected by ultraviolet irradiation overnight. When perfusion culture is added, the clip at two ends is taken down, the tail end of a 2mm pipeline at one end is connected into a 1mL injector through a luer valve, a culture solution containing a medicine with a specific concentration is filled in the injector, the injector is fixed on an injection pump, the tail end of the pipeline at the other end is connected into a 5mL injector containing waste liquid, the injector is open, the tail end of the pipeline is lower than the liquid level in the culture dish, the tail end of the pipeline is connected with a valve, when liquid needs to be drained, the valve is opened, the liquid in the culture dish is drained out of the pipeline under the action of gravity, the height of the tail end of the pipeline can be adjusted, the drainage speed can be adjusted, the structure of the culture dish printed with the protein pattern after the PDMS annular structure is restrained is obtained is shown in figure 8, and the 35mm petri dish comprises an Agarose coating, the protein pattern is printed, and the PDMS annular restraint and a perfusion channel.
The present invention adds constraints to reduce the exposed area of the culture dish substrate prior to step 104, so that fewer neurons can be seeded to meet the requirements; and a pipeline is added to realize the perfusion function.
104, performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in-vitro modular neuron network, which specifically comprises:
5-1) purchase of cortical neurons from E18 fetal rats in 1 million, dry ice transport. Storing in a refrigerator at minus 80 ℃ after the hand comes, and storing in liquid nitrogen after two days.
5-2) preparing a primary neuron culture solution. It contains 2% B27, 1% GlutaMAX, the balance being Neurobasal solution.
5-3) 4 35mm petri dishes with PDMS ring structures, which were agarose modified at the bottom of the dishes and printed with protein patterns, were prepared in advance. Before the inoculation experiment, the freezing tube is taken out of the liquid nitrogen, is rapidly placed in a water bath box at 37 ℃, and is rotated clockwise until a small amount of ice blocks are left in the freezing tube. Taking out from the water bath box, spraying alcohol for disinfection, and placing in a fume hood. About 1mL of the frozen stock solution was contained in the tube. The vial was opened and the frozen stock solution was removed into a 15mL centrifuge tube containing 5mL of primary nerve culture medium. The centrifuge tube now contains 6mL of the neuron suspension. Taking 1, 1.35, 1.65 and 2mL of suspension into a 35mm culture dish respectively, andtransferring the culture dish into an incubator at 37 deg.C under 5% carbon dioxide and nominal inoculation densities of 653, 882, 1078 and 1307/mm respectively2. In order to increase the actual inoculation density, after inoculation, the culture dish is taken out every 30 minutes, shaken for 10 times along the front-back direction and the left-right direction respectively, and then placed in the culture dish. This operation can be repeated 3-5 times, after which the dish is left to stand in the incubator and after one day the culture medium is changed to remove non-adherent neurons. Half of the culture broth was replaced every three days thereafter.
5-4) cortical neurons self-assemble to form modular neuronal networks. After day 3, neurons began to extend axons in large scale. After the seventh day, synapses began to form in large scale. After the tenth day, the neuron network is basically mature and can be used for functional tests such as calcium imaging or patch clamp and the like so as to analyze the influences of the number of neurons in different modules, the neuron density and the neuron communication strength among the modules on the information transmission and the network dynamic characteristics of the network. In combination with the perfusion channel, it can also be used to test the interaction between the above variables and different drug stimuli.
The invention has the characteristics and beneficial effects that:
1) the method is based on digital image processing technology, can generate patterns covering all structural parameter combinations to be researched, and can splice the patterns into a single pattern, and combines a soft lithography process based on laser direct writing and a surface modification method to prepare the micro-printing plate containing all the patterns to be researched so as to realize that a single printing can generate the modular protein pattern containing all the parameter combinations.
2) The method utilizes the microfluidic technology to carry out open type constraint on the substrate which is subjected to surface modification and patterning, can effectively improve the inoculation density, and has the function of supporting contact type electrophysiological detection on the basis of having a perfusion channel to support the accurate drug testing function.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The principles and embodiments of the present invention have been described herein using specific examples, which are provided only to help understand the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.
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