A hydrogel scaffold for three-dimensional culture of primary neural cells in vitro and its preparation method and application

Abstract

The present invention provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro, and a preparation method and application thereof. The hydrogel scaffold is formed by cross-linking acrylic polymer, polyvinyl alcohol and polyethylene glycol. The present invention aims to develop a scaffold for long-term 3D culture of neuronal cells in vitro based on hydrogel, which can culture primary neuronal cells, differentiate three-dimensional neural networks in vitro, construct brain disease models such as epilepsy, and be used for accurate analysis of neural circuit functions and mechanism research of neuropsychiatric diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and specifically relates to a hydrogel scaffold and a preparation method and application thereof, and in particular relates to a hydrogel scaffold for three-dimensional culture of primary nerve cells in vitro and a preparation method and application thereof. Background Art

[0002] Traditional cell culture is usually two-dimensional, such as using culture dishes, tissue culture flasks and microplates. Cells usually form a monolayer only on the contact surface with the support, and interactions with environmental stimuli occur only at the edge of the cells. In a two-dimensional culture mode, only 50% of the cell surface can contact the culture medium. Cells tend to flatten along the surface of the support and undergo morphological changes relatively early, which may induce changes in the biochemical function of the cells.

[0003] In the study of neurological disorders, injuries, toxicity and drug efficacy, there are high requirements for in vitro models of the central nervous system. A three-dimensional in vitro model that can highly simulate the in vivo microenvironment can bridge the gap between traditional two-dimensional culture and animal models. Hydrogels are favored by researchers because of their extremely hydrophilic three-dimensional network structure, good biocompatibility and suitable mechanical strength, and are widely used in the research and development of three-dimensional cell culture scaffolds. In existing studies, hydrogel scaffolds prepared from natural hydrogel materials such as chitosan, sodium alginate, and hyaluronic acid are used for in vitro three-dimensional culture of tumor cells as drug screening models. Some researchers also use sodium alginate and chitosan as 3D printing inks, and use biological 3D printing to manufacture three-dimensional scaffolds for in vitro cell culture to cultivate PC12 cells.

[0004] CN104548208A discloses a method for preparing a cell-loaded three-dimensional scaffold, characterized in that autologous cells are mixed evenly with a high-temperature intermittently sterilized hydrogel solution to obtain a mixed solution, which is sprayed into a crosslinker solution, allowed to stand, and then the crosslinker solution is removed, and the cell-loaded microspheres are obtained after rinsing; the cell-loaded microspheres are mixed with a matrix material to obtain a cell blend; a bioprinter is used to extrude the cell blend onto a three-dimensional micro-motion platform carrying a sterilized glass bottom plate, and the cell-loaded three-dimensional scaffold is formed by stacking layers. The present invention ensures high-activity cell printing by reducing the damage to cell activity and biological activity caused by the cell printing process.

[0005] CN110055221A discloses a brain-like disease treatment tissue model based on cell 3D printing technology and its preparation method and application. The preparation method includes the following steps: mixing primary nerve cells extracted from the affected part of the brain lesion with a hydrogel material and a cross-linking agent to obtain a printing bio-ink; based on the mechanical characteristics of the layered structure and elastic modulus of the natural cerebral cortex, the printing bio-ink is printed and formed using biological 3D printing technology, and then tissue culture is performed to obtain a brain-like disease treatment tissue model based on cell 3D printing technology.

[0006] CN108888803A discloses a biological scaffold and its hydrogel system. The hydrogel system comprises: a first gel, a second gel and a nano bone substitute material or a nano nerve substitute material, the first gel is formed by polyvinyl alcohol and water, and the second gel is formed by a photoinitiator, a photocrosslinker and water. The biological scaffold is formed by the hydrogel system. The application also provides a preparation method and use of the biological scaffold, and the biological scaffold can provide a good growth environment for bone cells and nerve cells, and can be used for bone repair or nerve repair.

[0007] CN109054049A discloses a PEG-based thermosensitive hydrogel three-dimensional cell scaffold and a preparation method and application thereof. The preparation method of the three-dimensional cell scaffold comprises: mixing an acrylate-based water-soluble macromolecular polymerization monomer containing an oligoethylene glycol (PEG) side chain, an initiator, a surfactant, a cross-linking agent and / or a co-monomer to react and obtain a PEG-based thermosensitive nanogel; uniformly mixing the mixture with cells at room temperature, and embedding the cells in situ by heat induction to form a PEG-based thermosensitive hydrogel three-dimensional cell scaffold. The scaffold is an ideal tissue engineering material and can be applied to fields such as in vitro cell culture.

[0008] Currently, researchers have developed many scaffolds suitable for in vitro three-dimensional cell culture, which are used in tissue engineering or tumor models. However, due to the complexity of neural tissue, it is difficult to achieve long-term three-dimensional culture of primary neuronal cells in vitro, and the manufacturing process is complex and the cost is high. Therefore, it is still very challenging to develop neural scaffolds that are suitable for neural cell growth, three-dimensional network construction, microstructure observation and specific functional regulation. Summary of the invention

[0009] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro and its preparation method and application. The present invention aims to develop a scaffold for long-term 3D culture of neuronal cells in vitro based on hydrogel, which can culture primary neuronal cells, differentiate three-dimensional neural networks in vitro, construct brain disease models such as epilepsy, and be used for accurate analysis of neural circuit functions and mechanism research of neuropsychiatric diseases.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro, wherein the hydrogel scaffold is formed by cross-linking an acrylic polymer, polyvinyl alcohol and polyethylene glycol.

[0012] In the present invention, the porous hydrogel scaffold can be made of an acrylic polymer as the basic skeleton, and polyvinyl alcohol can be added to form a network by esterification and cross-linking with the acrylic polymer. The introduction of polyethylene glycol can further strengthen the cross-linking network of the hydrogel on the one hand, and improve the biocompatibility on the other hand. Polyethylene glycol also serves as a porogen. Polyethylene glycol that is not completely cross-linked during the heat treatment process will dissolve in water during the swelling process to form pores.

[0013] The patent of this invention is mainly to design a new type of effective hydrogel scaffold for three-dimensional culture of primary neuronal cells in vitro. It aims to manufacture a three-dimensional porous scaffold based on hydrogel materials to simulate and create an in vitro microenvironment similar to the physiological conditions in vivo, which can be suitable for the growth of nerve cells and the construction of three-dimensional neural networks in vitro. It can be used as a simplified model for researchers to accurately and quantitatively analyze the functions of neural circuits and comprehensively explore the cognition of the brain and the treatment of brain diseases.

[0014] In the present invention, the raw materials for preparing the hydrogel scaffold include, by mass percentage, 20-80% of acrylic polymer, 0.5%-79.5% of polyvinyl alcohol and 0.5%-79.5% of polyethylene glycol.

[0015] Taking the mass of the raw materials for preparing the hydrogel scaffold as 100%, the content of the acrylic polymer is 20-80%, for example, it can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, etc.

[0016] Taking the mass of the raw materials for preparing the hydrogel scaffold as 100%, the content of the polyvinyl alcohol is 0.5%-79.5%, for example, it can be 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 79.5%, etc.

[0017] Taking the mass of the raw materials for preparing the hydrogel scaffold as 100%, the content of the polyethylene glycol is 0.5%-79.5%, for example, it can be 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 79.5%, etc.

[0018] Preferably, the raw materials for preparing the hydrogel scaffold include, by mass percentage, 30-50% acrylic polymer, 20-40% polyvinyl alcohol and 20-40% polyethylene glycol.

[0019] Preferably, the acrylic polymer is selected from polyacrylic acid and / or derivatives of polyacrylic acid.

[0020] Preferably, the derivative of polyacrylic acid is selected from any one or a combination of at least two of carbomer, polymethacrylic acid, polyacrylamide or polyacrylonitrile.

[0021] Preferably, the number average molecular weight of the acrylic polymer is 400,000-500,000, for example, 400,000, 410,000, 420,000, 430,000, 440,000, 450,000, 460,000, 470,000, 480,000, 490,000, 500,000, etc.

[0022] Preferably, the number average molecular weight of the polyvinyl alcohol is 80,000-100,000, for example, it can be 80,000, 82,000, 84,000, 86,000, 88,000, 90,000, 92,000, 94,000, 96,000, 98,000, 100,000, etc.

[0023] Preferably, the number average molecular weight of the polyethylene glycol is 3000-5000, for example, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, etc.

[0024] Preferably, the swelling rate of the hydrogel scaffold in water is 500-3000%, for example, it can be 500%, 600%, 800%, 1000%, 1200%, 1400%, 1600%, 1800%, 2000%, 2200%, 2400%, 2600%, 2800%, 3000%, etc.

[0025] Preferably, the maximum water absorption rate of the hydrogel scaffold is 1500-4000%, for example, it can be 1500%, 1600%, 1800%, 2000%, 2200%, 2400%, 2600%, 2800%, 3000%, 3200%, 3400%, 3600%, 3800%, 4000%, etc.

[0026] Preferably, the porosity of the hydrogel scaffold is 70-95%, for example, it can be 70%, 75%, 80%, 85%, 90%, 95%, etc.

[0027] Preferably, the Young's modulus of the hydrogel scaffold is 0.3-20 kPa, for example, it may be 0.3 kPa, 2 kPa, 4 kPa, 6 kPa, 8 kPa, 10 kPa, 12 kPa, 14 kPa, 16 kPa, 18 kPa, 20 kPa, etc.

[0028] In a second aspect, the present invention provides a method for preparing the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to the first aspect, the preparation method comprising the following steps:

[0029] (1) mixing an acrylic polymer, polyvinyl alcohol and polyethylene glycol, and compressing the mixture into tablets to obtain tablets;

[0030] (2) heat treating the tablet obtained in step (1);

[0031] (3) Swelling the tablets after the heat treatment in step (2) to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0032] In the present invention, the preparation of the porous hydrogel scaffold for long-term culture of primary neuronal cells in vitro includes tableting, heat treatment, swelling (such as Figure 1 The preparation process is simple and low-cost, and primary neurons can be cultured in three dimensions for a long time in vitro to differentiate into neural networks. Optogenetics can be used in vitro to study cell functions and to construct in vitro models of brain diseases such as epilepsy.

[0033] Preferably, in step (1), the tableting comprises placing powdered acrylic polymer, polyvinyl alcohol and polyethylene glycol in a mold and tableting them using a tablet press.

[0034] Preferably, in the tableting, the total mass of the mixed powder of a single tablet is 250-300 mg, for example, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, etc.

[0035] Preferably, the diameter of the mold used for tableting is 5-20 mm, for example, it can be 5 mm, 6 mm, 8 mm, 10 mm, 12 mm, 14 mm, 15 mm, 16 mm, 18 mm, 20 mm, etc.

[0036] Preferably, the tableting pressure is 1-4 tons, for example, 1 ton, 1.5 tons, 2 tons, 2.5 tons, 3 tons, 3.5 tons, 4 tons, etc., and the tableting time is 20-40 s, for example, 20 s, 25 s, 30 s, 35 s, 40 s, etc.

[0037] Preferably, in step (2), the heat treatment is specifically: placing the tablet obtained in step (1) in an oven for heating.

[0038] Preferably, the heat treatment temperature is 120-140°C, for example, it can be 120°C, 125°C, 130°C, 135°C, 140°C, etc., and the heat treatment time is 20-60min, for example, it can be 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc.

[0039] Preferably, in step (3), the swelling treatment is specifically as follows: firstly, the heat-treated tablet is placed in water, and swelled at 60-85°C (for example, 60°C, 65°C, 70°C, 72°C, 74°C, 76°C, 78°C, 80°C, 82°C, 85°C, etc.) for 1-4h (for example, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, etc.), and then cooled to 25-40°C (for example, 25°C, 30°C, 35 ℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.) and swell for 24-48h (for example, it can be 24h, 26h, 28h, 30h, 35h, 40h, 45h, 48h, etc.); finally, the tablets are taken out and placed in PBS buffer, and immersed for 60-80h (for example, it can be 60h, 65h, 70h, 75h, 80h, etc.) at 20-40℃ (for example, it can be 20℃, 25℃, 30℃, 35℃, 40℃, etc.).

[0040] Preferably, step (4) is further performed after step (3): sterilizing the hydrogel scaffold obtained by the swelling treatment.

[0041] Preferably, in step (4), the sterilization treatment is specifically: placing the hydrogel scaffold in PBS buffer, and sterilizing it at a high pressure of 0.05-0.2MPa (for example, it can be 0.05MPa, 0.06MPa, 0.08MPa, 0.1MPa, 0.15MPa, 0.2MPa, etc.) and a high temperature of 110-130°C (for example, it can be 110°C, 115°C, 120°C, 125°C, 130°C, etc.) for 10-30min (for example, it can be 10min, 15min, 20min, 25min, 30min, etc.).

[0042] In a third aspect, the present invention provides a use of a hydrogel scaffold for three-dimensional culture of primary nerve cells in vitro as described in the first aspect in three-dimensional cell culture in vitro or in constructing an in vitro model of epilepsy.

[0043] The hydrogel scaffold provided by the present invention is not only used for culturing primary neuronal cells, but can also be applied to the in vitro three-dimensional culture of other cell lines and the construction of other in vitro disease models or drug screening models.

[0044] In a fourth aspect, the present invention provides a method for three-dimensional culture of primary neural cells in vitro, the method comprising the following steps: inoculating cells into the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro as described in the first aspect, culturing, and differentiating to form a three-dimensional neural network in vitro.

[0045] Preferably, the cell inoculation amount is 1×10 5 -1×10 7 pcs / hole, for example, 1×10 5 pcs / hole, 5×10 5 pcs / hole, 1×10 6 pcs / hole, 5×10 6 pcs / hole, 1×10 7 Pieces / holes, etc.

[0046] Preferably, the culture medium is Neurobasal medium.

[0047] Preferably, the neural matrix culture medium also includes B27 additive (B-27 TM supplement) and dual antibody (penicillin-streptomycin).

[0048] Preferably, the added amount of the B27 additive accounts for 1-3% of the total mass of the neural matrix culture medium, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, etc.

[0049] Preferably, the added amount of the bispecific antibody accounts for 0.5-2% of the total mass of the neural matrix culture medium, for example, it can be 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, etc.

[0050] Preferably, the culture temperature is 36-38°C, for example, it can be 36°C, 36.5°C, 36.8°C, 37°C, 37.2°C, 37.5°C, 38°C, etc., and the culture time is more than 10 days, for example, it can be 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, etc.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The hydrogel scaffold of the present invention has a simple preparation process and low cost, and can be used to culture primary neuronal cells in three dimensions for a long time in vitro and differentiate into neural networks. Optogenetics can be used in vitro to study cell functions and to construct in vitro models of brain diseases such as epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The present invention provides a flow chart of the preparation process of the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro and cell seeding.

[0054] Figure 2 This is a diagram of the appearance of the hydrogel scaffold provided in Example 1 after swelling.

[0055] Figure 3 An in vitro three-dimensional neural network differentiated and formed in the hydrogel scaffold provided in Application Example 1.

[0056] Figure 4 An in vitro three-dimensional neural network differentiated and formed in the hydrogel scaffold provided in Application Example 2.

[0057] Figure 5 An in vitro three-dimensional neural network differentiated and formed in the hydrogel scaffold provided in Application Example 3.

[0058] Fig. 6A Raw signal images before and after light stimulation for in vitro optogenetic applications.

[0059] Figure 6B Waveform diagram of a type of neuron used for in vitro optogenetics.

[0060] Figure 7 Construct a diagram for an in vitro epilepsy model. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0062] The components and equipment sources of the following examples and comparative examples are as follows:

[0063]

[0064]

[0065] Example 1

[0066] This embodiment provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro. The hydrogel scaffold is formed by cross-linking polyacrylic acid, polyvinyl alcohol and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include, by mass percentage, 50% polyacrylic acid, 30% polyvinyl alcohol and 20% polyethylene glycol.

[0067] The preparation method of the hydrogel scaffold comprises the following steps:

[0068] (1) Powdered polyacrylic acid, polyvinyl alcohol and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 250 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0069] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0070] (3) The tablets heat-treated in step (2) were swollen in water at 80°C for 2 h, and then swollen at 37°C for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reached a constant value, and a PAA-PVA-PEG hydrogel (such as Figure 2 shown);

[0071] (4) The swollen PAA-PVA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0072] Example 2

[0073] This embodiment provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro. The hydrogel scaffold is formed by cross-linking polyacrylic acid, polyvinyl alcohol and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include, by mass percentage, 40% polyacrylic acid, 40% polyvinyl alcohol and 20% polyethylene glycol.

[0074] The preparation method of the hydrogel scaffold comprises the following steps:

[0075] (1) Powdered polyacrylic acid, polyvinyl alcohol and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 270 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0076] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0077] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PVA-PEG hydrogel;

[0078] (4) The swollen PAA-PVA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. for 20 min in a reagent bottle containing PBS solution to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0079] Example 3

[0080] This embodiment provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro. The hydrogel scaffold is formed by cross-linking polyacrylic acid, polyvinyl alcohol and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include, by mass percentage, 30% polyacrylic acid, 40% polyvinyl alcohol and 30% polyethylene glycol.

[0081] The preparation method of the hydrogel scaffold comprises the following steps:

[0082] (1) Powdered polyacrylic acid, polyvinyl alcohol and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 300 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0083] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0084] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PVA-PEG hydrogel;

[0085] (4) The swollen PAA-PVA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0086] Example 4

[0087] This embodiment provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro. The hydrogel scaffold is formed by cross-linking polyacrylic acid, polyvinyl alcohol and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include, by mass percentage, 60% polyacrylic acid, 15% polyvinyl alcohol and 15% polyethylene glycol.

[0088] The preparation method of the hydrogel scaffold comprises the following steps:

[0089] (1) Powdered polyacrylic acid, polyvinyl alcohol and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 250 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0090] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0091] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PVA-PEG hydrogel;

[0092] (4) The swollen PAA-PVA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0093] Example 5

[0094] This embodiment provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro. The hydrogel scaffold is formed by cross-linking polyacrylic acid, polyvinyl alcohol and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include, by mass percentage, 25% polyacrylic acid, 50% polyvinyl alcohol and 25% polyethylene glycol.

[0095] The preparation method of the hydrogel scaffold comprises the following steps:

[0096] (1) Powdered polyacrylic acid, polyvinyl alcohol and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 250 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0097] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0098] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PVA-PEG hydrogel;

[0099] (4) The swollen PAA-PVA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0100] Example 6

[0101] This embodiment provides a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro. The hydrogel scaffold is formed by cross-linking carbomer, polyvinyl alcohol and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include, by mass percentage, 40% carbomer, 40% polyvinyl alcohol and 20% polyethylene glycol.

[0102] The preparation method of the hydrogel scaffold comprises the following steps:

[0103] (1) Powdered carbomer, polyvinyl alcohol and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 250 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the compression time is 30 s to obtain tablets;

[0104] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0105] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PVA-PEG hydrogel;

[0106] (4) The swollen PAA-PVA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro.

[0107] Comparative Example 1

[0108] This comparative example provides a hydrogel scaffold, which is formed only by cross-linking polyacrylic acid and polyvinyl alcohol. The raw materials for preparing the hydrogel scaffold include: 50% polyacrylic acid and 50% polyvinyl alcohol in terms of mass percentage.

[0109] The preparation method of the hydrogel scaffold comprises the following steps:

[0110] (1) Powdered polyacrylic acid and polyvinyl alcohol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 250 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0111] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0112] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then soaked in PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PVA hydrogel;

[0113] (4) The swollen PAA-PVA hydrogel was cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold.

[0114] Comparative Example 2

[0115] This comparative example provides a hydrogel scaffold, which is formed by cross-linking polyacrylic acid and polyethylene glycol. The raw materials for preparing the hydrogel scaffold include: 50% polyacrylic acid and 50% polyethylene glycol in terms of mass percentage.

[0116] The preparation method of the hydrogel scaffold comprises the following steps:

[0117] (1) Powdered polyacrylic acid and polyethylene glycol are mixed in the above proportions, placed in a mold, and compressed using a tablet press. The total mass of the mixed powder for a single tablet is 250 mg, the mold diameter is 13 mm, the pressure is 2 tons, and the tableting time is 30 s to obtain tablets;

[0118] (2) placing the tablet obtained in step (1) in an oven and heat treating at 130° C. for 30 min;

[0119] (3) The tablets heat-treated in step (2) are swollen in water at 80° C. for 2 h, and then swollen at 37° C. for about 24 h to reach the maximum water absorption degree, and then infiltrated with PBS solution at room temperature. After about 72 h, the water absorption mass and volume expansion reach a constant value, thereby obtaining a PAA-PEG hydrogel;

[0120] (4) The swollen PAA-PEG hydrogel is cut into small cylinders of appropriate size according to the size of the well plate, and sterilized at a high pressure of 0.11 MPa and a high temperature of 121° C. in a reagent bottle containing PBS solution for 20 min to obtain the hydrogel scaffold.

[0121] Test Example 1

[0122] Test sample: hydrogel scaffolds provided in Examples 1-6 and Comparative Examples 1-2;

[0123] Test method:

[0124] (1) Swelling rate in water: The initial mass is recorded as W0, and the final mass reaching the equilibrium point in water is recorded as W1. The swelling rate is calculated according to the formula SI% = (W1-W0) / W0×100%;

[0125] (2) Maximum water absorption in PBS: The initial mass is recorded as W0, the final mass reaching the equilibrium point in PBS is recorded as W2, and the swelling rate is calculated according to the formula SI% = (W2-W0) / W0×100%;

[0126] (3) Porosity: Based on the improvement of the liquid displacement method, the hydrogel sample was cut into cylinders with a punch, freeze-dried, and the thickness and diameter of the cylinders were measured to calculate the volume V. 总 , weigh the sample and record the mass as W, then immerse the sample in mineral oil of known density, and take it out and weigh it after the mineral oil completely penetrates the pores of the sample and record it as W wet , through the formula V 孔 =(W wet -W) / ρ 矿物油 Calculate the pore volume, so the porosity ε = V 孔 / V 总 ×100%;

[0127] (4) Young's modulus: A universal testing machine is used to perform compression mode testing to obtain a stress-strain curve, and the Young's modulus is calculated based on the slope of the curve.

[0128] The specific test results are shown in Table 1:

[0129] Table 1

[0130]

[0131] From the test data in Table 1, it can be seen that the swelling rate of the hydrogel prepared by the present invention in water is 500-3000%, and the maximum water absorption rate after PBS infiltration can reach 1500-4000%; the porosity is 70-95%; and the Young's modulus is 0.3-20kPa. The preparation process of the hydrogel scaffold in the present invention is simple and low-cost, and primary neuronal cells can be cultured in vitro for a long time in three dimensions to differentiate into neural networks. Optogenetics can be used in vitro to study cell functions and to build in vitro models of brain diseases such as epilepsy.

[0132] Application Example 1

[0133] This application example provides a method for three-dimensional culture of primary neural cells in vitro, the method comprising the following steps: inoculating cells into the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro provided in Example 1, culturing, and differentiating to form a three-dimensional neural network in vitro;

[0134] The inoculation amount of the cells was 1×10 6 pcs / well (24-well plate);

[0135] Wherein, the culture medium is Neurobasal medium + 2% B-27 TM supplement(50X)+1%penicillin-streptomycin(10,000U·mL -1 ), fresh culture medium was replaced daily, the culture temperature was 37° C., and the culture time was 10 days.

[0136] Application Example 2

[0137] This application example provides a method for three-dimensional culture of primary neural cells in vitro, the method comprising the following steps: inoculating cells into the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro provided in Example 2, culturing, and differentiating to form a three-dimensional neural network in vitro;

[0138] The inoculation amount of the cells was 1×10 6 pcs / well (24-well plate);

[0139] Wherein, the culture medium is Neurobasal medium + 2% B-27 TM supplement(50X)+1%penicillin-streptomycin(10,000U·mL -1 ), fresh culture medium was replaced daily, the culture temperature was 37° C., and the culture time was 10 days.

[0140] Application Example 3

[0141] This application example provides a method for three-dimensional culture of primary neural cells in vitro, the method comprising the following steps: inoculating cells into the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro provided in Example 3, culturing, and differentiating to form a three-dimensional neural network in vitro;

[0142] The inoculation amount of the cells was 1×10 6 pcs / well (24-well plate);

[0143] Wherein, the culture medium is Neurobasal medium + 2% B-27 TM supplement(50X)+1%penicillin-streptomycin(10,000U·mL -1 ), fresh culture medium was replaced daily, the culture temperature was 37° C., and the culture time was 10 days.

[0144] Test Example 2

[0145] Differentiation of three-dimensional neural networks in vitro

[0146] Test samples: in vitro three-dimensional neural networks differentiated in the hydrogel scaffolds of Application Examples 1-6 and Comparative Application Examples 1-3;

[0147] Test method: Cultivate the hippocampal neuron cells of mice within 24 hours of birth for 10 days, and obtain differentiated neural network images under a confocal microscope through immunohistochemical staining.

[0148] Test results:

[0149] Figure 3 The in vitro three-dimensional neural network is differentiated and formed in the hydrogel scaffold provided in Application Example 1. Figure 3 As shown, confocal microscopy images of mouse hippocampal neurons cultured in a hydrogel scaffold for 10 days were taken, and the 3D image reconstructed by iMaris software showed that neuronal cells can be cultured in the scaffold for more than 10 days and differentiated into a three-dimensional neural network in vitro.

[0150] Figure 4 The in vitro three-dimensional neural network is differentiated and formed in the hydrogel scaffold provided in Application Example 2. Figure 4 As shown, the morphology of mouse hippocampal neurons cultured in a hydrogel scaffold for 10 days was captured by confocal microscopy, indicating that neuronal cells can be cultured in the scaffold for more than 10 days and differentiated into a three-dimensional neural network in vitro.

[0151] Figure 5 The in vitro three-dimensional neural network is differentiated and formed in the hydrogel scaffold provided in Application Example 3. Figure 5As shown, the morphology of mouse hippocampal neurons cultured in a hydrogel scaffold for 10 days was captured by confocal microscopy, indicating that neuronal cells can be cultured in the scaffold for more than 10 days and differentiated into a three-dimensional neural network in vitro.

[0152] Test Example 3

[0153] In vitro optogenetic applications

[0154] Test sample: the hydrogel scaffold provided in Example 1;

[0155] Test method: After mouse hippocampal neurons were cultured in the scaffold for 10 days, electrophysiological signals were recorded using a customized multi-channel optode array through a 64-channel neural acquisition processor (Plexon, USA). The sampling rate of electrophysiological signals was 40kHz, and the filter range was 300-5000Hz. The sampling frequency of LFP was 1kHz, and the filter range was 1-500Hz. The blue light (473nm) stimulation parameters were power 10mW, 20Hz, 5ms.

[0156] Test results:

[0157] Figure 6A-6B For in vitro optogenetics applications, Fig. 6A As shown, the original signal diagram before and after light stimulation is as follows Figure 6B As shown, a waveform diagram of a type of neuron indicates that the hydrogel scaffold provided by the present invention can be used to study cell functions in vitro using optogenetics.

[0158] Test Example 4

[0159] In vitro epilepsy model construction

[0160] Test sample: the hydrogel scaffold provided in Example 1;

[0161] Construction method: After the hippocampal neurons of Vgat-ChR2 transgenic mice were cultured in the scaffold for ten days, 40 μg / mL of potassium channel blocker 4-aminopyridine (4-AP) was added to induce the neuronal cells to release epileptic waves to construct an external epilepsy model. After the epileptic waves were induced, blue light (473 nm) was used to activate Vgat-ChR2. The stimulation parameters were 10 mW, 130 Hz, 5 ms, 60 s on, 120 s off, and cyclic stimulation to inhibit the release of epileptic waves. Finally, MATLAB (MathWorks, USA) and Neuroexplorer (Plexon, USA) were used for data analysis.

[0162] Test results: Figure 7 Constructing diagrams for in vitro epilepsy models, e.g. Figure 7As shown, the energy density diagrams before epilepsy modeling, during seizure and after light inhibition of seizure indicate that the scaffold shown in the present invention can realize the construction of in vitro models of brain diseases such as epilepsy.

[0163] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro and its preparation method and application, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro, characterized in that: The preparation method comprises the following steps: (1) mixing an acrylic polymer, polyvinyl alcohol and polyethylene glycol, and compressing the mixture into tablets to obtain tablets; (2) heat treating the tablet obtained in step (1); (3) subjecting the tablets heat-treated in step (2) to swelling treatment to obtain the hydrogel scaffold for three-dimensional culture of primary neural cells in vitro; The hydrogel scaffold is formed by cross-linking acrylic polymer, polyvinyl alcohol and polyethylene glycol; The raw materials for preparing the hydrogel scaffold include, by weight percentage, 30-50% of acrylic polymer, 20-40% of polyvinyl alcohol and 20-40% of polyethylene glycol; The acrylic polymer is selected from polyacrylic acid and / or polyacrylic acid derivatives; The derivative of polyacrylic acid is selected from carbomer and / or polymethacrylic acid; The heat treatment temperature is 120-140°C, and the heat treatment time is 20-60min; In step (3), the swelling treatment is specifically as follows: firstly, the heat-treated tablets are placed in water, and swelled at 60-85°C for 1-4 hours, and then cooled to 25-40°C and swelled for 24-48 hours; finally, the tablets are taken out and placed in PBS buffer, and immersed at 20-40°C for 60-80 hours.

2. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The number average molecular weight of the acrylic polymer is 400,000-500,000.

3. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The number average molecular weight of the polyvinyl alcohol is 80,000-100,000.

4. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The number average molecular weight of the polyethylene glycol is 3000-5000.

5. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The swelling rate of the hydrogel scaffold in water is 500-3000%.

6. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The maximum water absorption rate of the hydrogel scaffold is 1500-4000%.

7. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The porosity of the hydrogel scaffold is 70-95%.

8. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The Young's modulus of the hydrogel scaffold is 0.3-20 kPa.

9. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: In step (1), the tableting specifically comprises placing powdered acrylic polymer, polyvinyl alcohol and polyethylene glycol in a mold and tableting them using a tablet press.

10. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 9, characterized in that: In the tableting, the total mass of the mixed powder of a single tablet is 250-300 mg.

11. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 9, characterized in that: The diameter of the die used for tabletting is 5-20 mm.

12. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: The tabletting pressure is 1-4 tons, and the tabletting time is 20-40 seconds.

13. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: In step (2), the heat treatment specifically comprises: placing the tablet obtained in step (1) in an oven for heating.

14. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 1, characterized in that: After step (3), step (4) is required: sterilizing the hydrogel scaffold obtained by the swelling treatment.

15. The method for preparing a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro according to claim 14, characterized in that: In step (4), the sterilization treatment is specifically as follows: placing the hydrogel scaffold in a PBS buffer solution, and sterilizing it at a high pressure of 0.05-0.2 MPa and a high temperature of 110-130° C. for 10-30 min.

16. Use of a hydrogel scaffold for three-dimensional culture of primary neural cells in vitro prepared by the preparation method according to any one of claims 1 to 15 in three-dimensional cell culture in vitro or in constructing an in vitro model of epilepsy.

17. A method for three-dimensional culture of primary neural cells in vitro, characterized in that: The method comprises the following steps: inoculating cells into a hydrogel scaffold for in vitro three-dimensional culture of primary neural cells obtained by the preparation method according to any one of claims 1 to 15, culturing, and differentiating to form an in vitro three-dimensional neural network.

18. The method according to claim 17, characterized in that The inoculation volume of the cells was 1×10 5 -1×10 7 Pieces / hole.

19. The method according to claim 17, characterized in that The culture medium is a neural matrix medium.

20. The method according to claim 19, characterized in that The neural matrix culture medium also includes B27 additive and double antibody.

21. The method according to claim 20, characterized in that The added amount of the B27 additive accounts for 1-3% of the total mass of the neural matrix culture medium.

22. The method according to claim 20, characterized in that The added amount of the dual antibody accounts for 0.5-2% of the total mass of the neural matrix culture medium.

23. The method according to claim 17, characterized in that The culture temperature is 36-38° C., and the culture time is more than 10 days.

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