CN114410583A - Nerve/blood vessel network based on animal adipose tissue-derived hydrogel and construction method and application thereof - Google Patents

Nerve/blood vessel network based on animal adipose tissue-derived hydrogel and construction method and application thereof Download PDF

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CN114410583A
CN114410583A CN202111628952.4A CN202111628952A CN114410583A CN 114410583 A CN114410583 A CN 114410583A CN 202111628952 A CN202111628952 A CN 202111628952A CN 114410583 A CN114410583 A CN 114410583A
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蒋兴宇
刘晓艳
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Southwest University of Science and Technology
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Abstract

The invention relates to a nerve/blood vessel network based on animal adipose tissue-derived hydrogel as well as a construction method and application thereof, wherein the nerve/blood vessel network comprises a three-dimensional nerve network and an artificial blood vessel, the three-dimensional nerve network is a neuron network which is constructed in a stacking mode and has at least two layers of cell structures by taking hydrogel prepared from acellular matrixes of animal adipose tissues as a substrate, and as the preparation material of the hydrogel can be taken from autologous adipose tissues, the nerve/blood vessel network based on the animal adipose tissue-derived hydrogel constructed by the invention can be transplanted to an autologous body so as to avoid immune rejection reaction. The nerve/blood vessel network based on the hydrogel derived from the animal adipose tissues can accurately simulate the microenvironment for the growth of the nerve network, can monitor the change of electrical signals of neuron activity in real time, and has important significance for promoting brain science research.

Description

Nerve/blood vessel network based on animal adipose tissue-derived hydrogel and construction method and application thereof
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a nerve/blood vessel network based on animal adipose tissue-derived hydrogel as well as a construction method and application thereof.
Background
In the related art, although there are some advances in the research related to the construction of three-dimensional neuron networks, due to the complexity of the brain environment, the research on the growth and development of nerves and signal transduction in the brain is not very clear. In order to visually monitor the activity of cerebral neurons, a regular neural network is constructed in vitro, and has great significance in exerting normal functions of signal transduction, regulation and control feedback and the like. The method has the advantages that the growth of the neural network is induced by utilizing the electrical stimulation, the three-dimensional neural network of the autologous brain is simulated, and the electrical signal change of the neuron activity is monitored in real time, so that the brain science research is greatly promoted.
At present, a three-dimensional neuron network is mainly constructed by directionally inducing and differentiating neural stem cells or by means of a microfluidic technology and synthesized high-molecular hydrogel. Wherein, the neural stem cells are directionally differentiated into neural tissues to induce and generate neurons, astrocytes and oligodendrocytes. The three-dimensional neural network constructed by the micro-fluidic technology is characterized in that neural cells are planted on a three-dimensional microsphere bracket, and the cells are connected to form the neural network after being cultured, so that the growth of neurites is controlled. The three-dimensional neural network constructed in the synthetic high-water-separation gel is mainly characterized in that neuron cells and a high-molecular hydrogel (such as polyethylene glycol or sodium alginate) system are mixed and cultured.
However, the related art has certain defects that the neural tissue formed by the directional induction and differentiation of the stem cells is complicated and long in induction on one hand, and the formed neural network has a simple structure on the other hand, so that the complicated neural network needs to be reproduced by other methods. The three-dimensional neural network constructed by purely utilizing the microfluidic technology only provides a bracket for the construction of the neural network, and cannot completely copy the microenvironment for the growth of the neural tissue. However, the three-dimensional neural network constructed by using the synthetic polymer hydrogel has a limited ability in simulating the biological composition of the original tissue because the mechanical rigidity of the polymer hydrogel is too different from the brain tissue environment, and the polymer hydrogel usually contains relatively simple biological components and cannot supply nutrients to the neural network.
In order to visually monitor the activity of cerebral neurons, a regular three-dimensional neural network is constructed in vitro, sufficient nutrient supply is provided, and the neural network has great significance in exerting normal functions of signal transduction, regulation and control feedback and the like. Based on this, we developed a neural/vascular network based on animal adipose tissue-derived hydrogels.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a hydrogel-based three-dimensional neural network which can simulate the three-dimensional neural network of an autologous brain and monitor the change of electrical signals of neuron activity in real time.
The invention also provides a hydrogel-based nerve/blood vessel network.
The invention also provides a construction method of the hydrogel-based neural/vascular network.
The invention also provides application of the three-dimensional neural network in monitoring the formation of the neural network.
The invention also provides an application of the neural/vascular network in monitoring the formation of the neural network.
The invention also provides application of the construction method of the neural/vascular network in monitoring the formation of the neural network.
In a first aspect of the invention, there is provided a hydrogel-based three-dimensional neural network comprising at least two layers of a neuron-hydrogel network;
the neuron-hydrogel network comprises a hydrogel and a neural cell;
the preparation raw material of the hydrogel is acellular matrix of adipose tissue;
the neural cells are seeded on the hydrogel.
Preferably, the three-dimensional neural network is obtained by stacking at least two layers of neuron-hydrogel networks.
Preferably, the neuron-hydrogel network is obtained by culturing nerve cells on the basis of hydrogel through a microfluidic chip.
In some embodiments of the invention, the neural cells comprise at least one of primary neuronal cells, cortical neuronal cells and hippocampal neuronal cells.
In some embodiments of the invention, the adipose tissue comprises animal adipose tissue.
In some embodiments of the invention, the adipose tissue further comprises autologous adipose tissue.
In some embodiments of the invention, the adipose tissue is porcine adipose tissue.
In some embodiments of the present invention, the method for constructing the three-dimensional neural network comprises:
(1) taking adipose tissues for decellularization treatment to obtain acellular matrix hydrogel;
(2) culturing neuron cells by taking the acellular matrix hydrogel in the step (1) as a substrate to obtain a neuron-hydrogel network;
(3) and (3) stacking the neuron-hydrogel network in the step (2) to obtain a three-dimensional neural network.
In some embodiments of the invention, the decellularization process comprises, after delipidizing the adipose tissue, collecting the cytoplasmic matrix, and adding a pepsin salt solution to obtain a cytoplasmic matrix solution.
In some embodiments of the invention, the solvent of the pepsin salt solution is 0.005-0.02mol/L aqueous HCl.
In some embodiments of the invention, the solvent of the pepsin salt solution is 0.01mol/L HCl in water.
In some embodiments of the invention, the pepsin salt solution has a mass concentration of 0.5-2 mg/mL.
In some embodiments of the invention, the pepsin salt solution has a mass concentration of 1 mg/mL.
In some embodiments of the invention, the culturing of the cultured neuronal cells comprises microfluidic chip culturing.
In some embodiments of the invention, the neuronal cell is at 1 × 107The density of the cell/mL is injected into the hydrogel prepared by the animal adipose tissue acellular matrix in the microfluidic chip.
In some embodiments of the invention, the medium in which the neuronal cells are cultured is Neurobasal medium.
In some embodiments of the invention, the Neurobasal medium comprises 10% by weight of B27 additive and 1% by weight of penicillin-streptomycin.
In some embodiments of the invention, the neuronal cells are cultured at a temperature of 28 ℃ to 38 ℃.
In some embodiments of the invention, the neuronal cells are cultured at a temperature of 37 ℃.
In some embodiments of the invention, the neuronal cells are cultured at a temperature of 37 ℃ to facilitate hydrogel gelation and growth of the neuronal cells.
In some embodiments of the invention, the hydrogel-based three-dimensional neural network is suitable for use in non-disease diagnostic and therapeutic methods.
In a second aspect of the invention, there is provided a hydrogel-based neural/vascular network comprising a blood vessel and the three-dimensional neural network; the blood vessel comprises a blood vessel cell;
preferably, the vascular cells include at least one of vascular endothelial cells, smooth muscle cells, and fibroblasts.
In some embodiments of the invention, the vascular cells comprise vascular endothelial cells, smooth muscle cells, and fibroblasts.
In some embodiments of the invention, the blood vessel is an artificial blood vessel.
In some embodiments of the present invention, the culture medium used for culturing the vascular cells is DMEM medium.
In some embodiments of the invention, the DMEM medium further comprises 20% by mass fetal bovine serum and 1% by mass penicillin-streptomycin.
In some embodiments of the invention, the neural cell is at least one of a primary neuronal cell, a cortical neuronal cell, and a hippocampal neuronal cell.
In some embodiments of the invention, the hydrogel-based neural/vascular network is suitable for use in non-disease diagnostic and therapeutic methods.
In a third aspect of the present invention, there is provided a method for constructing a hydrogel-based neural/vascular network, comprising the steps of:
s1, preparation of hydrogel: adding pepsin salt solution into an animal adipose acellular matrix to obtain an acellular matrix solution A, adjusting the pH value and osmotic pressure to obtain an acellular matrix solution B, and adding the obtained acellular matrix solution B into a microfluidic chip to form gel to obtain an animal adipose tissue acellular matrix hydrogel;
s2, construction of a three-dimensional neuron network: culturing neuron cells by using the animal adipose tissue acellular matrix hydrogel in the step S1 as a substrate and adopting a microfluidic chip, and obtaining a three-dimensional neuron network by stacking layer by layer;
s3, construction of a three-dimensional nerve/blood vessel network: mixing the vascular cells with the acellular matrix solution B in the step S1, injecting the mixture into a mold for culture to form a hollow vascular structure wrapped by hydrogel, and injecting the three-dimensional neuron network in the step S2 into the outer layer to obtain the three-dimensional nerve/blood vessel network based on the acellular matrix hydrogel of the animal adipose tissue.
In some embodiments of the present invention, the solvent of the pepsin salt solution in step S1 is 0.005-0.02mol/L HCl aqueous solution.
In some embodiments of the present invention, the solvent of the pepsin salt solution in step S1 is 0.01mol/L HCl aqueous solution.
In some embodiments of the invention, the mass concentration of pepsin in the pepsin salt solution in step S1 is 0.5-2 mg/mL.
In some embodiments of the invention, the mass concentration of pepsin in the pepsin salt solution in step S1 is 1 mg/mL.
In some embodiments of the present invention, the solution used for adjusting the pH value in step S1 is 0.1mol/L NaOH aqueous solution.
In some embodiments of the invention, the pH is adjusted to 6.5 to 7.5 in step S1.
In some embodiments of the invention, the pH is adjusted to 7.0 in step S1.
In some embodiments of the present invention, the osmolality is adjusted to 260-320 mOsm/L using 10 XPBS as described in step S1.
In some embodiments of the invention, the osmolality is adjusted to 308mOsm/L using 10 XPBS as described in step S1.
In some embodiments of the present invention, the gel forming time in step S1 is 20-40 minutes.
In some embodiments of the present invention, the gel forming time in step S1 is 30 minutes.
In some embodiments of the invention, the temperature of the gel forming in step S1 is 35 to 38 ℃.
In some embodiments of the invention, the temperature of the gel forming in step S1 is 37 ℃.
In some embodiments of the invention, the neuronal cells in step S2 are derived from the brain of a suckling mouse.
In some embodiments of the invention, the culture temperature of the neuronal cells in step S2 is 28-38 ℃.
In some embodiments of the invention, the culture temperature of the neuronal cells in step S2 is 37 ℃.
In some embodiments of the invention, the medium of the neuronal cells in step S2 is Neurobasal medium.
In some embodiments of the invention, the Neurobasal medium in step S2 includes 10% by mass of B27 additive and 1% by mass of penicillin-streptomycin.
In some embodiments of the present invention, the culture medium of the vascular cells in step S3 is DMEM (Dulbecco' S modified eagle medium) medium.
In some embodiments of the present invention, the vascular cells are cultured in step S3 for 4 to 6 days.
In some embodiments of the invention, the mold in step S3 is a cylindrical mold.
In some embodiments of the invention, the artificial blood vessel is used to provide nutrients for the growth of neurons.
In some embodiments of the invention, at least the following benefits are achieved: the method is adopted to construct the hydrogel-based nerve/blood vessel network, can provide nutrient substances for the growth of neuron cells through artificial blood vessels, and has better mechanical properties.
In a fourth aspect of the invention, there is provided a use of a three-dimensional neural network in monitoring neuronal network formation.
In some embodiments of the invention, the use of the three-dimensional neural network in monitoring neuronal network formation for modeling diseases affecting neurons and/or neuronal network formation in the human brain.
In some embodiments of the invention, the use of the three-dimensional neural network in monitoring neuronal network formation for modeling neurotoxicity and/or changes in neuronal stem cell plasticity caused by disease-associated protein aggregation.
In some embodiments of the invention, the use of the three-dimensional neural network for monitoring neuronal network formation for the testing of molecules and/or active substances that affect neuronal activity and/or network formation.
In a fifth aspect of the invention, there is provided a use of a neural/vascular network for monitoring neuronal network formation.
In some embodiments of the invention, the use of the neural/vascular network in monitoring neuronal network formation for modeling diseases affecting neurons and/or neuronal network formation in the human brain.
In some embodiments of the invention, the use of the neural/vascular network in monitoring neuronal network formation for modeling neurotoxicity and/or changes in neuronal stem cell plasticity caused by disease-associated protein aggregation.
In some embodiments of the invention, the use of the neural/vascular network for monitoring neuronal network formation for the testing of molecules and/or active substances that affect neuronal activity and/or network formation.
In a sixth aspect of the invention, an application of the construction method of the nerve/blood vessel network in monitoring the formation of the neuron network is provided.
In some embodiments of the invention, the use of the method of constructing a neural/vascular network for monitoring neuronal network formation for modeling a disease affecting neurons and/or neuronal network formation in the human brain.
In some embodiments of the invention, the use of the method of constructing a neural/vascular network for monitoring neuronal network formation for modeling neurotoxicity and/or changes in neuronal stem cell plasticity caused by disease-related protein aggregation.
In some embodiments of the invention, the use of the method for constructing a neural/vascular network for monitoring neuronal network formation for the testing of molecules and/or active substances that influence neuronal activity and/or network formation.
The above methods and applications are applicable to non-disease diagnostic and therapeutic methods.
The invention has the beneficial effects that:
1) the nerve/blood vessel network of the invention takes hydrogel prepared by acellular matrix of animal adipose tissue as a substrate, the mechanical rigidity of the hydrogel has smaller difference with brain tissue environment, and the hydrogel has good surface three-dimensional structure and is more close to the microenvironment for nerve tissue growth, thus being beneficial to the adsorption and growth of nerve cells and the formation of three-dimensional nerve network. The presence of blood vessels may subsequently supply nutrients for the growth of neurons.
2) The adipose tissue can be obtained from the body through a liposuction operation as a loose connective tissue, and a three-dimensional neuron-blood vessel network constructed based on the hydrogel can be transplanted into the body, so that immune rejection is effectively avoided.
3) The nerve/blood vessel network based on the hydrogel derived from the animal adipose tissues accurately simulates the microenvironment for the growth of the nerve network, can monitor the electrical signal change of the neuron activity in real time, and has important significance for promoting brain science research.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a scanning electron microscope image of extracellular matrix before and after decellularization of porcine fat in example 1 of the present invention.
FIG. 2 is a scanning electron micrograph of the porcine fat extracellular matrix of example 3 of the present invention.
FIG. 3 is a scanning electron micrograph of neurons on hydrogel of example 3 of the present invention.
FIG. 4 is a three-dimensional neuron network immunofluorescence map based on porcine fat hydrogel of example 3 of the present invention.
FIG. 5 is a multilayered three-dimensional neuron immunofluorescence map based on porcine fat hydrogel of example 4 of the present invention.
FIG. 6 is a fluorescent image of a multilayered neuronal-vascular network based on a porcine fat hydrogel according to example 4 of the present invention.
Fig. 7 is a calcium imaging plot of a neuronal network based on a porcine fat hydrogel of example 5 of the present invention.
Fig. 8 is a graph of calcium signal response at 1 and 2 in the fig. 7 pig fat hydrogel based neuronal network calcium imaging graph.
FIG. 9 is an electrical signal diagram of the activity of a single layer neuronal network cultured in an anhydrous gel.
FIG. 10 is a graph of electrical signals of three-dimensional neuron network activity based on porcine fat hydrogel culture.
FIG. 11 is an electrical signal diagram of activity of three-dimensional neuron networks cultured on the basis of porcine fat hydrogel under ATP stimulation.
FIG. 12 is a raster pattern of electrical signals of single-layer neuron network cultured in anhydrous gel, three-dimensional neuron network cultured based on porcine fat hydrogel, and three-dimensional neuron network activity cultured based on porcine fat hydrogel under ATP stimulation.
FIG. 13 is a graph showing neuron discharge rate analysis in different channels for a single-layer neuron network cultured in an anhydrous gel (2D culture), a three-dimensional neuron network cultured on a hydrogel (3D hydrogel culture), and a three-dimensional neuron network cultured on a hydrogel under ATP stimulation (3D hydrogel + ATP culture).
FIG. 14 is graphs showing neuron burst analysis of a single-layer neuron network cultured in an anhydrous gel (2D culture), a three-dimensional neuron network cultured in a hydrogel (3D hydrogel culture), and a three-dimensional neuron network cultured in a hydrogel under ATP stimulation (3D hydrogel + ATP culture).
FIG. 15 is a graph showing the discharge correlation analysis of a single-layer neuron network cultured in an anhydrous gel (2D culture), a three-dimensional neuron network cultured in a hydrogel (3D hydrogel culture), and a three-dimensional neuron network cultured in a hydrogel under ATP stimulation (3D hydrogel + ATP culture).
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1: pig adipose tissue decellularization treatment
1. Obtaining of pig fat acellular matrix
Extracting adipose tissues of pigs under the aseptic condition, cleaning blood clots by using deionized water, cutting the adipose tissues into small blocks with the size of 1mm multiplied by 1mm, washing the small blocks with the deionized water for 2-3 times, and placing the cut adipose tissues into a place with the temperature of minus 80 ℃ and 37 ℃ for repeated freeze thawing for 3 times.
Adding 2 volumes of deionized water into the fat tissue after freeze thawing, homogenizing for 10min, centrifuging for 10min at the rotating speed of 1200rpm at 4 ℃, demixing the fat tissue after centrifugation, taking the white precipitate at the lowest layer, adding 1% Triton-X100, shaking for 1h at the temperature of 25 ℃, then washing for 30min by using the deionized water, repeating for 3 times, centrifuging for 10min at the speed of 1200rpm at 4 ℃, collecting the precipitate, adding 100% isopropanol, shaking overnight, and centrifuging to remove the residual lipid component. Then, 2 times the volume of 1mol/L NaCl solution (containing 100U/ml DNase + 100. mu.g/ml RNase) was added, shaken in a shaker at 37 ℃ overnight, centrifuged, washed with deionized water for 30min, repeated 3 times, and centrifuged to collect flocs. Freezing the flocculent precipitate in a refrigerator at-80 deg.C overnight, lyophilizing in a lyophilizer to obtain pig fat acellular matrix, grinding the acellular matrix into powder, packaging, and sterilizing.
2. Scanning electron microscope observation of pig fat acellular matrix
Observing extracellular matrixes before and after the decellularization of the pig fat through a scanning electron microscope, specifically as shown in figure 1, wherein A in figure 1 is a scanning electron microscope image of the extracellular matrixes before the decellularization of the pig fat, B in figure 1 is a scanning electron microscope image of the extracellular matrixes after the decellularization of the pig fat, and as can be seen from figure 1, cells in the pig fat are basically removed completely, and extracellular matrix components are fully reserved; the pig fat acellular matrix hydrogel prepared by the method can effectively reduce host rejection caused by cell components.
3. Preparation of acellular matrix solution A
Preparing 1mg/mL pepsin salt solution by using sterile 0.01mol/L HCl solution, weighing 10mg of the acellular matrix powder, adding 1mL pepsin salt solution, fully stirring for 72h at 4 ℃ until no macroscopic powdery substance exists, obtaining acellular matrix solution A, sealing and storing at 4 ℃ for later use.
Example 2: acquisition and culture of neuronal and vascular cells
1. Extraction and culture of neurons
SD-line suckling mice (purchased from Guangdong medical animal center) born for 1-2 days were decapitated and brains were peeled off on ice, placed in PBS pre-cooled at 4 ℃, corpus callosum was dissected under a dissecting scope with ophthalmologic forceps, two hemispheres of the brains were spread out to both sides, cortical tissues and hippocampal tissues were peeled off respectively with ophthalmologic forceps, placed in DMEM/F12 serum-free medium (Gibco, cat # C11330500BT) pre-cooled at 4 ℃, the tissues were cut into 1mm × 1mm pieces with forceps and transferred to 15mL centrifuge tubes respectively, 1mL trypsin solution with a mass fraction of 2.5% was added, and digested at 37 ℃ for 15 min. After digestion was complete, F12 medium was added to stop digestion and gently rinsed three times to remove trypsin solution. The tissue was blown with a sterile pipette 5-10 times, the cell suspension was placed in a centrifuge, centrifuged at 1000rpm for 5min at 4 ℃ and the supernatant was discarded, and the obtained cortical and hippocampal neurons were resuspended in neuronal medium (Gibco, cat # 21103049) (Neurobasal medium + 10% B27 additive + 1% penicillin-streptomycin), respectively.
2. Culture of vascular cells
The invention adopts three vascular cells, including vascular endothelial cells, smooth muscle cells and fibroblasts, the cells are purchased from American Type Culture Collection (ATCC), and the culture environment is as follows: DMEM medium (Gibco, cat # 11965092) + 20% fetal bovine serum + 1% penicillin-streptomycin.
Example 3: construction of three-dimensional neuron network based on porcine adipose tissue-derived hydrogel
1. Preparation of hydrogel derived from porcine adipose tissue
The acellular matrix solution A obtained in example 1 was added with 0.1mol/L NaOH solution to adjust pH to 7 at 4 ℃ and the osmotic pressure was adjusted with 10 XPBS to obtain an acellular matrix solution B. And adding the acellular matrix solution B into a square microfluidic chip, and forming hydrogel at 37 ℃ for about 30 min.
2. Scanning electron microscope observation of hydrogel derived from porcine adipose tissue
The microstructure of the surface of the porcine fat extracellular matrix hydrogel was observed by a scanning electron microscope, and the result is shown in FIG. 2. Wherein, B in figure 2 is an enlarged view of a white dotted line frame in figure 2A, and it can be known from figure 2 that the porcine fat acellular matrix hydrogel prepared by the invention has a better surface three-dimensional structure and a microfiber structure, which is beneficial to the adsorption of neuron cells and the connection of a neural network.
3. Construction of neuron-hydrogel network based on pig adipose tissue-derived hydrogel
The neuron cells are divided into 1 × 107Injecting cell/mL density into hydrogel of microfluidic chip to make neuron cell grow on the pig fat extracellular matrix hydrogel substrate, and adding 1-2mL neuron culture medium (Neurobasal medium + 10% N27+ 1% penicillin-streptomycin) into the microfluidic chip to culture at 37 deg.CCulturing in a box, wherein the culture medium is changed for half every 3 days.
The growth morphology of hippocampal neurons on the porcine fat extracellular matrix hydrogel was observed by a scanning electron microscope, and the results are shown in fig. 3. Wherein, B in figure 3 is an enlarged view of the white dotted line frame A in figure 3. As can be seen from figure 3, the hippocampal neurons grow well and can be effectively adsorbed on the surface of the hydrogel.
4. Construction of three-dimensional neuron network based on pig fat hydrogel and immunofluorescence staining observation
And after 2 weeks of culture, removing the microfluidic chip, stacking the cortical neuron-hydrogel and the hippocampal neuron-hydrogel layer by layer, and constructing a three-dimensional neuron network based on the porcine fat hydrogel.
The neuron network and the multi-layer neuron (including cortical neuron and hippocampal neuron) structure formed are characterized by immunofluorescence staining, cell membrane dye and other fluorescence means, as shown in fig. 4 and fig. 5, wherein the 1 st and 2 nd layers in fig. 5 are cortical neurons, and the 3 rd layer is hippocampal neurons.
From fig. 4, it can be seen that: the growth state of the neurons on the hydrogel is good, and a tight neural network connection can be formed; as can be seen from FIG. 5, the multilayer neural network constructed by cortical neurons and hippocampal neurons can be controlled layer by layer, and a multilayer three-dimensional neural network is successfully constructed.
Example 4: construction of three-dimensional neuron-blood vessel network based on pig adipose tissue-derived hydrogel
Mixing the three vascular cells with the acellular matrix solution B in example 3, injecting into a cylindrical mold, standing at 37 ℃ for 30min to form a hollow vascular structure wrapped by hydrogel, and adding a DMEM medium to culture for 5 days. Injecting hydrogel wrapping the neuron into the outer layer of the hydrogel, culturing for 5 days by using a culture medium (DMEM: Neurobasal mass ratio is 1:1), and slowly extracting the cylindrical mold from the blood vessel-nerve network hydrogel to construct a three-dimensional neuron-blood vessel network based on the hydrogel derived from the animal adipose tissues. The neuron culture medium is introduced into the blood vessel by using an injection pump to provide nutrient substances for the growth of the neurons.
The three-dimensional neuron-blood vessel network structure is characterized and formed by immunofluorescence staining, cell membrane dye and other fluorescence means, and is specifically shown in fig. 6, wherein the left diagram of fig. 6 is the three-dimensional neuron network structure, the middle diagram is the bionic artificial blood vessel structure, and the right diagram is the hollow bionic artificial blood vessel structure wrapped in the three-dimensional neuron network, and can be known from fig. 6: the neuron network is wrapped on the periphery of the hollow bionic blood vessel, and the nerves can be tightly connected with the blood vessel, so that the three-dimensional neuron-blood vessel network is successfully constructed.
Example 5: functional verification of neuron-vessel network based on porcine fat extracellular matrix hydrogel
1. Calcium imaging and calcium signal response of neuronal networks
Calcium imaging characterization of the three-dimensional neuronal network was performed using calcium ion fluorescent probe (Fluo-4 AM), and calcium signal response of the neuronal network was detected, and the results are shown in fig. 7 and 8, where the calcium signal response sites of the "# 1 (Fluo-4)" and "# 2 (Fluo-4)" neurons in fig. 8 were taken from "1" and "2" in fig. 7.
As can be seen from fig. 7 and 8: the three-dimensional neural network constructed by the invention has a normal signal conduction function, and the membrane potential of the neuron can generate depolarization so as to change calcium ions in the neuron.
2. Neuronal network activity electrical signals
The single-layer neuron network cultured in the anhydrous gel (2D culture) was used as a control group, and was placed on a designed flexible circuit (liquid metal-high polymer conductor MPC) together with the three-dimensional neuron network based on the hydrogel derived from porcine adipose tissue, and the culture was continued for 4 to 5 days, and four site-activity electric signals in the single-layer neuron network cultured in the anhydrous gel (2D culture), the three-dimensional neuron network cultured in the hydrogel (3D hydrogel culture) and the three-dimensional neuron network cultured in the hydrogel under ATP stimulation (3D hydrogel + ATP culture) were recorded by the electrophysiological system, respectively, with the results shown in fig. 9 to 12.
Wherein FIG. 9 is a diagram of electric signals of single layer neuron activity in anhydrous gel culture; FIG. 10 is a graph of electrical signals of three-dimensional neuron activation based on porcine fat hydrogel culture; FIG. 11 is a diagram of electrical signals for three-dimensional neuron activation based on porcine fat hydrogel culture under ATP stimulation, and 4 sites of the neural network are randomly selected from the four diagrams in FIGS. 9-11; FIG. 12 is a raster pattern of electrical signals of three-dimensional neuron activation in the absence of hydrogel culture of single layer neurons, in the presence of three-dimensional neurons cultured on the basis of porcine fat hydrogel, and under ATP stimulation.
As can be seen from FIGS. 9-11, the three-dimensional neuron networks cultured by adding hydrogel were significantly more active in discharge than the single-layer neuron networks cultured in the absence of hydrogel, and showed an increase in action potential amplitude (as shown in FIG. 10), while the three-dimensional neuron networks cultured in the presence of ATP (as shown in FIG. 11) were significantly decreased in action potential amplitude relative to the three-dimensional neuron networks cultured in the absence of ATP; as can be seen from the grating graph of the neuron activity electrical signals of fig. 12, the three-dimensional neuron network discharge frequency based on hydrogel culture was increased (as shown in B of fig. 12) compared to the single-layer neuron network discharge frequency based on hydrogel culture (a of fig. 12), and the three-dimensional neuron network discharge frequency based on hydrogel culture after stimulation with ATP (as shown in C of fig. 12) was decreased relative to the three-dimensional neuron network discharge frequency based on hydrogel culture without ATP stimulation.
FIG. 13 is a graph showing neuron discharge rate analysis in different channels for a single-layer neuron network cultured in an anhydrous gel (2D culture), a three-dimensional neuron network cultured on a hydrogel (3D hydrogel culture), and a three-dimensional neuron network cultured on a hydrogel under ATP stimulation (3D hydrogel + ATP culture). As can be seen from fig. 13, the discharge rate of the three-dimensional neuron network cultured based on hydrogel was enhanced (the discharge rate was stronger the deeper the color tone was), compared to the discharge rate of the two-dimensional neuron network cultured without hydrogel, while the discharge rate of the entire neuron network was decreased after ATP stimulation was added.
FIG. 14 is a diagram showing the analysis of neuron burst of a single-layer neuron cultured in an anhydrous gel (2D culture), a three-dimensional neuron cultured in a hydrogel (3D hydrogel culture), and a three-dimensional neuron network cultured in a hydrogel under ATP stimulation (3D hydrogel + ATP culture). The burst number of the neurons refers to the burst frequency of the neuron network, and as can be seen from fig. 14, compared with the two-dimensional neuron electrical signal cultured in the anhydrous gel, the burst number of the neurons of the three-dimensional neuron network cultured in the hydrogel is increased, the burst duration is increased, and the number of peak action potentials per burst is increased, which indicates that the porcine fat hydrogel can support the formation of the neuron network and enhance the activity of the neurons. After ATP is added, the number of neuron bursts of the whole neuron network is reduced, the burst duration is reduced, and the number of peak action potentials under each burst is reduced, so that the ATP can inhibit the electrical activity of neurons to a certain extent.
FIG. 15 is a graph showing the discharge correlation analysis of a single-layer neuron network cultured in an anhydrous gel (2D culture), a three-dimensional neuron network cultured in a hydrogel (3D hydrogel culture), and a three-dimensional neuron network cultured in a hydrogel under ATP stimulation (3D hydrogel + ATP culture). As can be seen from the correlation analysis of neural network firing in fig. 15, compared with the single-layer neuron network electrical signal cultured in the absence of hydrogel, the neuron firing correlation of the three-dimensional neuron network cultured on the basis of hydrogel is enhanced, which indicates that the functional connection formed between the neuron networks is tighter, and further proves that the hydrogel of the present invention can make the three-dimensional neuron network more tightly connected; after ATP is added, the neuron discharge correlation of the whole neuron network is obviously reduced, and the ATP can inhibit the functional connection of the neuron network to a certain extent.
In conclusion, the acellular matrix hydrogel derived from animals is used as a substrate, the three-dimensional neuron-blood vessel network is constructed by stacking layer by layer, the microenvironment for the growth of the neural network is accurately simulated, and the electrophysiological activity of the neural network is verified, and the result shows that compared with a single-layer neuron cultured by anhydrous gel, the three-dimensional neuron cultured by adding the hydrogel has obviously stronger discharge activity, and shows that the action potential amplitude is increased and the neuron discharge frequency is increased; the action potential amplitude of the three-dimensional neuron cultured based on the hydrogel after the ATP stimulation is obviously reduced compared with the action potential amplitude of the three-dimensional neuron cultured based on the hydrogel without the ATP stimulation, and the discharge frequency of the neuron is reduced. In addition, because the adipose tissue derived from the pig for preparing the pig fat acellular matrix hydrogel is a loose connective tissue and can be obtained from a self body through liposuction surgery, a three-dimensional neuron-blood vessel network constructed based on the hydrogel can be transplanted into the body, and immune rejection reaction is avoided.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims (10)

1. A hydrogel-based three-dimensional neural network, characterized in that,
the three-dimensional neural network comprises at least two layers of the neuronal hydrogel network;
the neuronal hydrogel network comprises a hydrogel and a neural cell;
the preparation raw material of the hydrogel is acellular matrix of adipose tissue;
the neural cells are seeded on the hydrogel.
2. The hydrogel-based three-dimensional neural network of claim 1, wherein the neural cells comprise at least one of primary neuronal cells, cortical neuronal cells and hippocampal neuronal cells.
3. The hydrogel-based three-dimensional neural network of claim 1 or 2, wherein the adipose tissue comprises animal adipose tissue; preferably, the adipose tissue is porcine adipose tissue.
4. A hydrogel-based neural/vascular network comprising a blood vessel and the three-dimensional neural network of any one of claims 1-3;
the blood vessel comprises a blood vessel cell;
preferably, the vascular cells include at least one of vascular endothelial cells, smooth muscle cells, and fibroblasts.
5. A method of constructing a hydrogel-based neural/vascular network as claimed in claim 4, comprising the steps of:
s1, preparation of hydrogel: adding pepsin salt solution into an animal adipose acellular matrix to obtain an acellular matrix solution A, adjusting the pH value and osmotic pressure to obtain an acellular matrix solution B, and adding the obtained acellular matrix solution B into a microfluidic chip to form gel to obtain an animal adipose tissue acellular matrix hydrogel;
s2, construction of a three-dimensional neuron network: culturing neuron cells by using the animal adipose tissue acellular matrix hydrogel in the step S1 as a substrate and adopting a microfluidic chip, and obtaining a three-dimensional neuron network by stacking layer by layer;
s3, construction of a three-dimensional nerve/blood vessel network: mixing the vascular cells with the acellular matrix solution B in the step S1, injecting the mixture into a mold for culture to form a hollow vascular structure wrapped by hydrogel, and injecting the three-dimensional neuron network in the step S2 into the outer layer to obtain the three-dimensional nerve/blood vessel network based on the acellular matrix hydrogel of the animal adipose tissue.
6. The method for constructing the hydrogel-based neural/vascular network according to claim 5, wherein the temperature of the microfluidic chip for culturing the neuronal cells is 28-38 ℃; preferably, the temperature is 37 ℃.
Use of any one of (1) - (3) for monitoring neuronal network formation:
(1) the three-dimensional neural network of any one of claims 1-3;
(2) the nerve/vessel network of claim 4;
(3) the method of constructing a neural/vascular network as claimed in claim 5 or 6.
8. Use according to claim 7, for modeling a disease affecting the formation of neurons and/or neuronal networks in the human brain.
9. Use according to claim 7 for modeling neurotoxicity and/or changes in neuronal stem cell plasticity caused by disease-related protein aggregation.
10. Use according to claim 7 for the testing of molecules and/or active substances that influence neuronal activity and/or network formation.
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