Hydrogel-based bionic brain micro-tissue as well as construction method and application thereof

Abstract

The invention belongs to the technical field of organoid culture, and discloses a hydrogel-based bionic brain micro-tissue as well as a construction method and application thereof. The hydrogel-based bionic brain micro-tissue is prepared by controlling the proportion of methylacryloylated gelatin to methylacryloylated hyaluronic acid, the proportion of a photoinitiator, the proportion of graphene oxide and key parameters of photocuring treatment. The hydrogel-based bionic brain micro-tissue has the advantages of definite components, controllable structure and form, low cost, easiness in standardization and the like, and meanwhile, the hydrogel-based bionic brain micro-tissue shows the biological properties of high cell activity, excellent biocompatibility, good safety and outstanding physiological-like functions. The method can be applied to the fields of brain-like tissue construction, nervous system disease modeling and mechanism research, neurodrug pharmacodynamics research, drug neurotoxicity evaluation or developmental biology research and the like.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically relating to a hydrogel-based biomimetic brain microtissue, its construction method, and its application. Background Technology

[0002] Currently, the construction of in vitro brain tissue models mainly relies on technologies such as 3D bioprinting and Matrigel embedding. However, while 3D bioprinting can achieve precise control of model structure, the shear forces generated during the printing process can easily damage cells, especially the inherently fragile primary nerve cells. Matrigel, represented by Matrigel, has many limitations due to its main components being animal-derived substances: not only are there large variations in composition between different batches, posing potential risks of pathogen contamination and ethical controversies; its complex composition system can also cause specific interference with cellular physiological behavior, and its unadjustable mechanical properties make it difficult to efficiently adapt to the specific brain tissue microenvironment. Furthermore, its high cost limits its large-scale application.

[0003] Therefore, there is an urgent need to develop a biomimetic brain micro-tissue that can accurately simulate the mechanical microenvironment of brain tissue, and also has the advantages of clear composition, high biocompatibility, low preparation cost, and controllable morphology and structure. Summary of the Invention

[0004] In view of the aforementioned problems in the prior art, this invention provides a hydrogel-based biomimetic brain microtissue, its construction method, and its applications. By controlling the ratio of methacrylamide gelatin and methacrylamide hyaluronic acid, the ratio of photoinitiator, the ratio of graphene oxide, and key parameters of the photocuring process, this invention creatively prepares a hydrogel-based biomimetic brain microtissue suitable for industrial production and possessing excellent biological functions. This hydrogel-based biomimetic brain microtissue not only exhibits properties such as well-defined composition, high cell activity and biocompatibility, controllable structure and morphology, low cost, and ease of standardization, but also possesses excellent physiological functions and high safety. This invention can be applied to fields such as brain-like tissue construction, modeling and mechanism research of nervous system diseases, pharmacodynamic research of neuropharmaceuticals, drug neurotoxicity assessment, and developmental biology research.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a method for constructing hydrogel-based biomimetic brain microtissues, the flowchart of which is shown below. Figure 1 As shown, it includes the following steps: S1. Methacrylamide gelatin and methacrylamide hyaluronic acid are added to the photoinitiator solution and a pre-crosslinking reaction is carried out at 75℃-85℃ to obtain a composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is mixed with graphene oxide to obtain a graphene oxide-enhanced composite hydrogel precursor solution. S3. The graphene oxide-enhanced composite hydrogel precursor solution is subjected to photocuring treatment to obtain hydrogel-based biomimetic brain micro-tissue; The mass ratio of the methacrylated gelatin to the methacrylated hyaluronic acid is 5:1.8-5:2.2.

[0006] This invention employs a photocrosslinking method to prepare hydrogels. During preparation, a photoinitiator, methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid (HAMA), and graphene oxide (GO) are added. Upon light exposure, the photoinitiator cleaves to generate free radicals. These free radicals attack the double bonds of GelMA and HAMA, causing crosslinking. Simultaneously, the carboxyl and hydroxyl groups of GO crosslink with the amino and carboxyl groups of GelMA and HAMA, forming a stable physical crosslinking system. The surface and edges of GO have numerous hydrophilic functional groups, exhibiting exceptional stability, biocompatibility, adsorption capacity, and a large specific surface area. This provides suitable sites for hydrogel synthesis and neural stem cell proliferation, thereby forming a 3D hydrogel scaffold.

[0007] In this invention, GelMA, HAMA, and GO together constitute the substrate of the hydrogel scaffold. The mechanical properties of the matrix, especially viscoelasticity, play a central role in regulating stem cell fate and organoid development. Accordingly, the present invention optimizes the mass ratio of GelMA to HAMA so that the elastic modulus of the prepared hydrogel is closest to that of mouse brain tissue. GelMA retains the inherent amino acid motifs of gelatin and its biological functions supporting cell adhesion and tissue remodeling, while the modified form formed a stable covalent cross-linked network, ensuring the mechanical integrity of the material. HAMA inherits the excellent biocompatibility of hyaluronic acid and can effectively simulate the physiological characteristics of the extracellular matrix (ECM) of brain tissue, providing a more suitable mechanical microenvironment for nerve cells. On this basis, the introduction of GO endows the hydrogel with the ability to actively guide the behavior of nerve cells and can also simulate the biological characteristics of the natural microenvironment of neural tissue, thereby significantly improving nerve cell activity; at the same time, the well-defined composition of the hydrogel system avoids the immune risks of animal-derived components.

[0008] The method for constructing hydrogel-based biomimetic brain microtissues provided by this invention allows for the controllable adjustment of the material's mechanical properties through precise control of the hydrogel component ratio; and the final microscopic morphology and macroscopic structure of the material can be controlled by adjusting key parameters such as wavelength and time during photocuring. Ultimately, this results in in vitro models with superior performance, better meeting the needs of related research and applications.

[0009] Preferably, the final concentration of graphene oxide in the biomimetic brain microtissue is 0.125 mg / mL to 1 mg / mL.

[0010] For example, the final concentration of graphene oxide in the biomimetic brain microtissue is 0.125 mg / mL-0.91 mg / mL, and it is added to the system in the form of a graphene oxide solution.

[0011] Preferably, the photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0012] More preferably, the final concentration of the photoinitiator in the composite hydrogel precursor solution is 1 mg / mL-2 mg / mL.

[0013] More preferably, the mass ratio of the photoinitiator to methacrylamide gelatin is 1:50-2:50.

[0014] More preferably, the solvent of the photoinitiator solution includes at least one of deionized water, PBS buffer, or Neurobasal complete culture medium.

[0015] Preferably, the pre-crosslinking reaction time is 1.5h-2.5h.

[0016] Preferably, the photocuring process includes irradiation at a wavelength of 403nm-407nm for 20s-40s.

[0017] More preferably, in the method for constructing hydrogel-based biomimetic brain microtissue provided by the present invention, The photocuring process includes irradiation at a wavelength of 403nm-407nm for 20s-40s; The mass ratio of the methacrylated gelatin to the methacrylated hyaluronic acid is 5:2.

[0018] The mass ratio of the methacrylated gelatin to the methacrylated hyaluronic acid is 5:2.

[0019] In a second aspect, the present invention provides a hydrogel-based biomimetic brain microtissue, which is prepared by the method for constructing hydrogel-based biomimetic brain microtissue described in the first aspect.

[0020] The hydrogel-based biomimetic brain microtissue provided by this invention not only possesses advantages for industrialization, such as well-defined composition, controllable structure and morphology, low cost, and ease of standardization, but also exhibits advantages such as high cell activity, excellent biocompatibility, good safety, and prominent physiological functions. Compared with simple 2D culture, cells cultured in the hydrogel-based biomimetic brain microtissue provided by this invention show better cell activity, and the hydrogel-based biomimetic brain microtissue exhibits drug screening characteristics similar to the blood-brain barrier.

[0021] Thirdly, the present invention provides the application of the hydrogel-based biomimetic brain microtissue provided in the second aspect in brain-like tissue construction, modeling and mechanism research of nervous system diseases, pharmacodynamic research of neuropharmaceuticals, assessment of drug neurotoxicity, or developmental biology research.

[0022] Preferably, the neurological disease includes at least one of cerebrovascular disease or central nervous system disease.

[0023] Given the comprehensive advantages of the hydrogel-based biomimetic brain microtissue provided by this invention, such as high industrial adaptability and excellent biological performance, it has broad application prospects and huge industrialization potential in many cutting-edge fields such as brain tissue construction, modeling and mechanism research of nervous system diseases, pharmacodynamics research of neuropharmaceuticals, drug neurotoxicity assessment or developmental biology research. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A flowchart of the method for constructing hydrogel-based biomimetic brain microtissue provided by the present invention; Figure 2 This is a SEM image of hydrogel-based biomimetic brain microtissue I in Example 1 of the present invention; Figure 3 This is a comparison diagram of the elastic modulus of hydrogel-based biomimetic brain micro-tissues I-IV and mouse brain tissue in Example 1 of the present invention; Figure 4 This is a comparison of the energy storage modulus G' and loss modulus G'' of different groups in Example 2 of the present invention; wherein, Figure 4 A represents a comparison diagram of hydrogel-based biomimetic brain microtissue I and GelMA / HAMA composite hydrogels I~III. Figure 4 B represents a comparison diagram of hydrogel-based biomimetic brain micro-tissue I and GelMA / HAMA composite hydrogels IV~VII; Figure 5 The swelling kinetics curve of hydrogel-based biomimetic brain micro-tissue I gel microspheres in Example 3 of the present invention; Figure 6 This is a microscopic image of primary NSCs 0-72h in Example 4 of the present invention; Figure 7 The growth of NSCs in gel microspheres prepared from hydrogel-based biomimetic brain microtissue I is shown in Example 4 of the present invention. Figure 8The effect of extracts with different numbers of gel beads on the survival rate of NSCs cells in Example 4 of the present invention; Figure 9 This is an example of the effect of different gel extraction days on NSC cell survival rate in Example 4 of the present invention; Figure 10 This is a comparison of the survival rates of NSCs cells between hydrogel-based biomimetic brain microtissue extract and composite hydrogel extract in Example 4 of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] The main reagents used in this invention are: GelMA, freeze strength of 300g Bloom, and grafting rate of 60%; purchased from Aladdin Reagent Co., Ltd., product number: M398240; HAMA, weight-average molecular weight (Mw) 300KD, grafting rate 40%; purchased from Aladdin Reagent Co., Ltd., product number: H398345; Graphene oxide solution, concentration 5 mg / mL, purchased from ACSmaterial; Neurobasal complete culture medium, purchased from Gibco, USA; Phosphate-buffered saline (PBS) was purchased from Hunan Bickman Biotechnology Co., Ltd.

[0028] Example 1 This invention provides a method for constructing hydrogel-based biomimetic brain microtissue, the method comprising the following steps: S1. Under 80℃ water bath conditions, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added and dissolved, and a pre-crosslinking reaction was carried out at 80℃ for 2h to obtain a composite hydrogel precursor solution. S2. Add 500 μL of graphene oxide solution with a concentration of 5 mg / mL to the composite hydrogel precursor solution and mix. Stir and mix at 80°C for 1 h to obtain graphene oxide-enhanced composite hydrogel precursor solution. The final concentration of graphene oxide in the graphene oxide-enhanced composite hydrogel precursor solution is 0.125 mg / mL. S3. The graphene oxide-enhanced composite hydrogel precursor solution was photocured at 405 nm for 20 s to obtain hydrogel-based biomimetic brain microtissue I.

[0029] The hydrogel-based biomimetic brain microtissue I prepared above was freeze-dried using a vacuum freeze dryer. After immersion in liquid nitrogen for 2 minutes, it brittlely broke into two halves that adhered to a conductive gel. The microstructure of the sample was observed using a scanning electron microscope (SEM). To enhance the conductivity of the sample, it was sputter-coated with gold before testing. The porosity structure was observed at a working voltage of 10 kV. The SEM image of the hydrogel-based biomimetic brain microtissue I is shown below. Figure 2 As shown, where Figure 2 a) and Figure 2 b) is the SEM image at magnification.

[0030] Depend on Figure 2 It is evident that the hydrogel-based biomimetic brain microtissue I possesses a uniformly distributed porous three-dimensional structure with pore sizes within an appropriate range. Furthermore, the pores are interconnected, forming a scaffold network conducive to material exchange and cell growth. This structure highly mimics the physical characteristics of the natural extracellular matrix, providing nerve cells with the necessary attachment interfaces, directional migration channels, and nutritional metabolic space, thereby fully meeting the microenvironmental conditions required for nerve cell survival and functional maintenance.

[0031] Example 2 This invention provides a method for constructing hydrogel-based biomimetic brain microtissue, the method comprising the following steps: S1. Under 80℃ water bath conditions, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added and dissolved, and a pre-crosslinking reaction was carried out at 80℃ for 2h to obtain a composite hydrogel precursor solution. S2. Add 1 mL of graphene oxide solution with a concentration of 5 mg / mL to the composite hydrogel precursor solution and mix. Stir and mix at 80°C for 1 h to obtain a graphene oxide-enhanced composite hydrogel precursor solution. The final concentration of graphene oxide in the graphene oxide-enhanced composite hydrogel precursor solution is 0.25 mg / mL. S3. The graphene oxide-enhanced composite hydrogel precursor solution was photocured at 405 nm for 20 s to obtain hydrogel-based biomimetic brain microtissue II.

[0032] Example 3 This invention provides a method for constructing hydrogel-based biomimetic brain microtissue, the method comprising the following steps: S1. Under 80℃ water bath conditions, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added, and a pre-crosslinking reaction was carried out at 80℃ for 2h to obtain a composite hydrogel precursor solution. S2. Add 2 mL of graphene oxide solution with a concentration of 5 mg / mL to the composite hydrogel precursor solution and mix. Stir and mix at 80°C for 1 h to obtain a graphene oxide-enhanced composite hydrogel precursor solution. The final concentration of graphene oxide in the graphene oxide-enhanced composite hydrogel precursor solution is 0.45 mg / mL. S3. The graphene oxide-enhanced composite hydrogel precursor solution was photocured at 405 nm for 20 s to obtain hydrogel-based biomimetic brain microtissue III.

[0033] Example 4 This invention provides a method for constructing hydrogel-based biomimetic brain microtissue, the method comprising the following steps: S1. Under 80℃ water bath conditions, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added, and a pre-crosslinking reaction was carried out at 80℃ for 2h to obtain a composite hydrogel precursor solution. S2. Add 4 mL of graphene oxide solution with a concentration of 5 mg / mL to the composite hydrogel precursor solution and mix. Stir and mix at 80°C for 1 h to obtain graphene oxide-enhanced composite hydrogel precursor solution. The final concentration of graphene oxide in the graphene oxide-enhanced composite hydrogel precursor solution is 0.91 mg / mL. S3. The graphene oxide-enhanced composite hydrogel precursor solution was photocured at 405 nm for 20 s to obtain hydrogel-based biomimetic brain microtissue IV.

[0034] Example 5 This invention provides a method for constructing hydrogel-based biomimetic brain microtissue, the method comprising the following steps: S1. Under 75℃ water bath conditions, 30mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added, and a pre-crosslinking reaction was carried out at 75℃ for 2.5h to obtain a composite hydrogel precursor solution. S2. Add 500 μL of graphene oxide solution with a concentration of 5 mg / mL to the composite hydrogel precursor solution and mix. Stir and mix at 80°C for 1 h to obtain graphene oxide-enhanced composite hydrogel precursor solution. The final concentration of graphene oxide in the graphene oxide-enhanced composite hydrogel precursor solution is 0.125 mg / mL. S3. The graphene oxide-enhanced composite hydrogel precursor solution was photocured at 405 nm for 30 s to obtain hydrogel-based biomimetic brain microtissue V.

[0035] Example 6 This invention provides a method for constructing hydrogel-based biomimetic brain microtissue, the method comprising the following steps: S1. Under 85℃ water bath conditions, 25mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added, and a pre-crosslinking reaction was carried out at 85℃ for 1.5h to obtain a composite hydrogel precursor solution. S2. Add 500 μL of graphene oxide solution with a concentration of 5 mg / mL to the composite hydrogel precursor solution and mix. Stir and mix at 85°C for 0.5 h to obtain a graphene oxide-enhanced composite hydrogel precursor solution. The final concentration of graphene oxide in the graphene oxide-enhanced composite hydrogel precursor solution is 0.125 mg / mL. S3. The graphene oxide-enhanced composite hydrogel precursor solution was photocured at 405 nm for 20 s to obtain hydrogel-based biomimetic brain microtissue VI.

[0036] Comparative Example 1 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium. After it was completely dissolved, 1.4g of methacrylamide gelatin was added and dissolved. The pre-crosslinking reaction was carried out at 80℃ for 2h to obtain the composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405nm for 20s to form GelMA / HAMA composite hydrogel I.

[0037] Comparative Example 2 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium. After it was completely dissolved, 0.88g of methacrylamide gelatin and 0.52g of methacrylamide hyaluronic acid were added and dissolved. The mixture was then subjected to a pre-crosslinking reaction at 80℃ for 2h to obtain a composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405nm for 20s to form GelMA / HAMA composite hydrogel II.

[0038] Comparative Example 3 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium. After it was completely dissolved, 0.78g of methacrylamide gelatin and 0.62g of methacrylamide hyaluronic acid were added and dissolved. The mixture was then subjected to a pre-crosslinking reaction at 80℃ for 2h to obtain a composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405nm for 20s to form GelMA / HAMA composite hydrogel III.

[0039] Comparative Example 4 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 20mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium. After it was completely dissolved, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added and dissolved. The pre-crosslinking reaction was carried out at 80℃ for 2h to obtain the composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405 nm for 60 s to form GelMA / HAMA composite hydrogel IV.

[0040] Comparative Example 5 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 0.5 mg of photoinitiator LAP was heated and dissolved in 20 mL of Neurobasal complete medium. After it was completely dissolved, 1 g of methacrylamide gelatin and 0.4 g of methacrylamide hyaluronic acid were added and dissolved. The pre-crosslinking reaction was carried out at 80℃ for 2 h to obtain the composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405nm for 60s to form GelMA / HAMA composite hydrogel V.

[0041] Comparative Example 6 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 10mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium. After it was completely dissolved, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added and dissolved. The pre-crosslinking reaction was carried out at 80℃ for 2h to obtain the composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405 nm for 60 s to form GelMA / HAMA composite hydrogel VI.

[0042] Comparative Example 7 This comparative example provides a GelMA / HAMA composite hydrogel and its preparation method, which includes the following steps: Under the conditions of S1.80℃ water bath, 100mg of photoinitiator LAP was heated and dissolved in 20mL of Neurobasal complete medium. After it was completely dissolved, 1g of methacrylamide gelatin and 0.4g of methacrylamide hyaluronic acid were added and dissolved. The pre-crosslinking reaction was carried out at 80℃ for 2h to obtain the composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is photocured at 405 nm for 60 s to form GelMA / HAMA composite hydrogel VII.

[0043] Example 1 This invention examines the elastic modulus of the hydrogel-based biomimetic brain microtissues prepared in Examples 1-6 and compares them with mouse brain tissue. The effects are illustrated using hydrogel-based biomimetic brain microtissues I-IV as examples. The hydrogel-based biomimetic brain microtissues prepared in Examples 5-6 can also achieve technical effects comparable to hydrogel-based biomimetic brain microtissue I.

[0044] The specific method for determining the viscoelasticity of GelMA / HAMA composite hydrogels or hydrogel-based biomimetic brain microtissues in this invention is as follows: Fresh, intact mouse brains were randomly extracted from three C57Bl / 6J mice. The extracted brains were placed in cold PBS to prevent tissue dehydration and then placed on ice at 4°C before mechanical testing. All samples were tested immediately after extraction and completed within 2 hours. The average value was used to obtain the storage modulus G' or tissue modulus G'' of the mouse brain. Viscoelasticity was determined using an AntonPaar MCR302 rheometer. A 25 mm diameter parallel plate (PP25) was used, the test temperature was 37°C, and the gap between the sample and the plate was 1 mm to prevent uneven deformation and flow. Samples were allowed to equilibrate for five minutes before testing to ensure complete relaxation. After pre-strain, the viscosity of the sample was measured at amplitude scan to eliminate the effects of air bubbles and uneven distribution. Viscoelasticity tests were also performed on different hydrogels to compare the differences in viscoelasticity between the hydrogels and the mouse brain.

[0045] The elastic modulus comparison diagram of hydrogel-based biomimetic brain micro-tissues I-IV and mouse brain tissue is shown below. Figure 3 As shown.

[0046] Figure 3 This reflects the variation of storage modulus G' and loss modulus G'' of the hydrogel-based biomimetic brain microtissues I-IV prepared in this invention with shear strain. At the same time, the mechanical properties of this system are highly matched with those of biological tissues by comparison with mouse brain modulus.

[0047] Example 2 Following the measurement method described in Example 1, this invention investigated the differences in storage modulus G' and loss modulus G'' between the hydrogel-based biomimetic brain microtissue I prepared in Example 1 and the GelMA / HAMA composite hydrogels prepared in Comparative Examples 1-7. The comparison results of storage modulus G' and loss modulus G'' between different groups are as follows: Figure 4 As shown; where, Figure 4 A represents a comparison diagram of hydrogel-based biomimetic brain microtissue I and GelMA / HAMA composite hydrogels I~III. Figure 4 B represents a comparison image of hydrogel-based biomimetic brain microtissue I and GelMA / HAMA composite hydrogels IV~VII.

[0048] Depend on Figure 4 It can be seen that the storage modulus G' and loss modulus G'' of the GelMA / HAMA composite hydrogels prepared in Comparative Examples 1-7 differ significantly from those of the hydrogel-based biomimetic brain microtissue prepared in the embodiments of this invention. Specifically, the elastic modulus of the hydrogel-based biomimetic brain microtissue I provided by this invention has been significantly optimized, and is closer to the value of the real rat brain. This matching of key mechanical properties lays a reliable foundation for subsequent applications in the fields of neurodevelopment research, disease model construction, and high-throughput drug screening.

[0049] Example 3 The swelling properties of hydrogels are closely related to their further applications. Swelling properties reflect the hydrogel's ability to absorb and retain water. As a biomaterial for tissue engineering applications, the swelling rate is an important indicator, influencing cell adhesion and proliferation, providing a suitable environment for cell growth, and controlling the exchange of nutrients and cellular metabolic waste. While swelling improves cell adhesion, it can also negatively affect the hydrogel's mechanical properties.

[0050] This invention uses hydrogel-based biomimetic brain microtissue I as an example to investigate its swelling properties in PBS buffer (pH 7.4) and Neurobasal medium. Hydrogel-based biomimetic brain microtissue I was placed in a 0.5 mL EP tube cap and freeze-dried to obtain hydrogel-based biomimetic brain microtissue I gel beads, each gel bead containing approximately 20 mg. The swelling kinetics curve of the hydrogel-based biomimetic brain microtissue I gel beads is shown below. Figure 5 As shown.

[0051] Depend on Figure 5 It is evident that the swelling behavior of hydrogel-based biomimetic brain microtissue I is similar in different media, exhibiting a rapid initial swelling followed by a stabilization. The swelling rate reaches approximately 7500% in PBS and approximately 3500% in Neurobasal medium. The high swelling rate and rapid swelling characteristics of hydrogel-based biomimetic brain microtissue I allow it to absorb a large amount of water and reach a balanced swelling state in a short time. This characteristic is particularly important in brain-like organoid culture, ensuring that it can quickly match the mechanical requirements of the development process. During the development of brain-like organs, the mechanical properties of the microenvironment continuously change. The rapid swelling of the material allows it to adjust its stiffness and volume in real time, providing dynamic and matched mechanical support for the developing tissue. Simultaneously, it effectively avoids the accumulation of mechanical stress and the internal stress caused by asynchronous swelling of the inner and outer layers. Rapid and uniform swelling greatly reduces this stress accumulation, maintaining the integrity and stability of the material structure, minimizing physical damage to cells, and providing a gentler and safer physical environment for embedded cells.

[0052] In summary, the hydrogel-based biomimetic brain microtissue I provided by this invention has the characteristics of high swelling rate and short swelling time, and can become a smart material that can actively adapt to the dynamic process of biological development, providing timely, mild and stable mechanical support for complex three-dimensional culture systems such as brain-like organs.

[0053] Example of effect 4 This invention investigated the growth of neural stem cells in the hydrogel-based biomimetic brain microtissue prepared above, and explored its biocompatibility, cell adhesion, and safety. The specific details are as follows: The primary neural stem cells extracted in this invention are derived from C57BL / 6 strain pregnant mice. Primary NSCs were extracted from the forebrain region of the fetal mice at 13.5 days of gestation. The pregnant mice were purchased from Beijing Sibefore Biotechnology Co., Ltd.

[0054] 1. Extraction and culture of primary neural stem cells (1) Modification of four-well plates for cell culture: Take out the unopened sterile four-well plates from the biosafety cabinet and place them in a laminar flow hood. Add 0.3 mL of 0.1% poly-L-lysine solution to each well for coating (the volume should be enough to completely cover the bottom of the well), seal the edges of the plate with sealing film, and let it stand at 4°C for 16 h. Discard the poly-L-lysine solution from the wells and gently rinse each well with PBS solution. Then add 0.1 μg / mL of laminin solution and let it stand at room temperature for 2 h. After washing the wells with PBS solution, the plates are ready for cell seeding.

[0055] (2) C57BL / 6 mice at 13.5 days of gestation were euthanized by vertebral dislocation and their abdomens were disinfected with 75% alcohol swabs.

[0056] (3) Use microdissecting forceps and fine scissors to open the abdominal cavity and expose the uterus containing the embryo by presenting the entire abdominal cavity in a "Y" shape.

[0057] (4) Quickly dissect and separate the uterus containing the embryo, and rinse it three times with pre-cooled PBS.

[0058] (5) Transfer to a clean bench, remove the fetal mouse on ice and extract the forebrain tissue under a stereomicroscope, taking care to remove the meninges and other tissue structures as much as possible.

[0059] (6) Transfer the tissue to a 15 mL centrifuge tube containing 0.25% trypsin solution (0.5 mL of fetal rat brain tissue per cell) and digest at 37°C for 10 min.

[0060] (7) Repeatedly and gently blow and digest until no obvious tissue blocks form a cell suspension, centrifuge at 1000 rpm for 3 min; discard the supernatant.

[0061] (8) Add Neurobasal medium and wash 3 times (1000r / min, 3min / time), discard the supernatant, and obtain primary neural stem cells (primary NSCs).

[0062] (9) Add NSCs proliferation medium and gently pipette, then filter through a 70µm cell sieve into a 15mL centrifuge tube.

[0063] (10) Seed the cells into modified four-well plates and change half of the medium daily. After one week of culture, passage the cells and co-culture them with hydrogel-based biomimetic brain micro-tissue I.

[0064] Morphological changes of primary NSCs from 0 to 72 hours were observed under a microscope. Specific microscopic images of primary NSCs from 0 to 72 hours are shown below. Figure 6 As shown.

[0065] like Figure 6As shown, neurospheres (NSCs) extracted from fetal rat forebrain tissue appear as aggregated spherical or ellipsoidal structures under a microscope, with some exhibiting a beaded appearance due to adhesion between the spheres. The intercellular connections within the neurospheres are tight with minimal gaps, forming a compact spherical structure that is uniform in size, highly refractive, and exhibits a tendency to aggregate rather than adhere to the culture wall. Primary NSC extraction primarily involves digestion of cells using trypsin and mechanical tube blowing. At 0 h, a few undigested cells are visible under the microscope. During culture, some cells divide to form 2-4 cell clusters. Subsequently, at 24 h, 48 h, and 72 h, cells continuously proliferate, generating neurospheres composed of hundreds of spherical cells. Incompletely digested brain tissue is removed during daily half-volume medium changes. After 7 days of cell culture, when the cell density reaches 80%, passage is performed. Passage still yields a large number of suspended neurospheres, similar to the primary culture. This indicates that NSCs exhibit a strong intercellular adhesion and aggregation pattern, with few individual cells scattered between the spheres.

[0066] 2. Hydrogel-based biomimetic brain microtissue I culture of NSCs Following the method in Example 1, hydrogel-based biomimetic brain microtissue I was freeze-dried to prepare gel microspheres. The gel microspheres were then sterilized by high temperature and high pressure. NSCs cultured for 7 days were then distributed at 4 × 10⁻⁶ cells per well. 5 Cells / mL were seeded into 8 gel microspheres and cultured at 37°C and 5% CO2 for one week. The growth of NSCs in the hydrogel-based biomimetic brain microtissue I gel microspheres was observed under a microscope. Figure 7 As shown.

[0067] Depend on Figure 7 It was observed that after one week of culture, NSCs exhibited significant cell stacking and aggregation. This indicates that the hydrogel-based biomimetic brain microtissue I prepared in this invention possesses excellent biocompatibility, cell adhesion-promoting properties, and is non-toxic. First, cells can only effectively adhere, spread, and further migrate and interact after recognizing and adapting to the chemical and physical properties of the material surface. The aggregation phenomenon is the result of cells actively seeking and approaching each other, proving that the material itself is non-toxic and can support basic cellular life activities. Second, the experimental results also show a significant enhancement in intercellular interactions. In traditional two-dimensional culture, cells typically grow in a monolayer. However, in the three-dimensional hydrogel-based biomimetic brain microtissue provided in this application, NSCs cells can migrate, recognize, and aggregate in all directions, representing a crucial initial step in simulating cell self-assembly during in vivo development or tissue regeneration.

[0068] 3. CCK-8 assay for the safety and cell growth-promoting properties of hydrogel-based biomimetic brain microtissue This invention employs cytotoxicity assays to determine the biosafety and cell growth-promoting properties of hydrogels. The hydrogel extract is co-cultured with cells for a period of time before testing. One to eight gel beads are placed in 1 mL of NSCs proliferation medium and incubated at 4°C for 24 hours. The gel beads are then removed to obtain the hydrogel extract.

[0069] Eight gel beads were placed in 1 mL of NSCs proliferation medium and stored in a 4°C refrigerator. The liquid was collected on days 1, 2, 3, 5, 7, 14 and 21 to obtain hydrogel extracts for different number of days.

[0070] CCK-8 assay method: Healthy NSCs were mechanically pipetted and collected into centrifuge tubes, and the cell concentration was adjusted to 2×10⁻⁶ using a cell counter. 5 To determine the cell density (cells / mL), seed each well of a 96-well plate with 100 μL of the above cell suspension and incubate overnight in a cell culture incubator set at 37°C and 5% CO2. After cell attachment, add 100 μL of hydrogel extraction buffer to each well. After 24 h of co-culture, add 10 μL of CCK-8 solution to each well and continue culturing for 1 h–4 h. Detect the absorbance at 450 nm every h using a microplate reader. Terminate the experiment when the OD value is close to 1. This experiment included 6 replicate wells. Cell viability was calculated using Formula 1.

[0071] Formula 1 The effect of extracts with different numbers of gel beads on NSC cell viability (n=6) Figure 8 As shown. The effect of different gel extraction days on NSC cell survival (n=6) is shown below. Figure 9 As shown.

[0072] To evaluate the feasibility of the hydrogel-based biomimetic brain microtissue prepared in this invention as a long-term effective in vitro cell culture model, its biocompatibility was assessed through in vitro cell experiments. In this invention, noncellular spore cells (NSCs) responsible for constructing the brain-like tissue were selected for cytotoxicity experiments. Cell number and cell viability were used as indicators of cell compatibility to evaluate the biocompatibility of the hydrogel-based biomimetic brain microtissue prepared in this invention. A blank group containing only cell culture medium was included in this invention. The viability of NSCs was assessed using a CCK-8 cell proliferation assay kit. The CCK-8 reagent contains water-soluble tetrazolium salt WST-8, which turns yellow under the action of mitochondrial dehydrogenases and exhibits absorbance at 450 nm, and can be used for cell viability analysis.

[0073] Depend on Figure 8It was found that after 24 hours of culture, the cell viability of the hydrogel extracts obtained with different numbers of gel beads all exceeded 100%. International standards consider a cell viability rate higher than 80% for tissue engineering materials to indicate good biocompatibility, which meets national standards (ISO 10993). 5). Compared with other groups, the extract of 8 gel beads showed the best effect on promoting the proliferation of NSCs, so this group was selected for subsequent experiments.

[0074] Depend on Figure 9 It can be seen that, compared with the control group, the extract of the hydrogel-based biomimetic brain microtissue of this invention can increase the cell survival rate several times in the early stage of culture (e.g., 1 day), and this advantage continues to expand with the extension of culture time, showing a significant time-dependent growth characteristic. This data strongly proves that the hydrogel-based biomimetic brain microtissue provided by this invention not only has excellent biocompatibility, but also significantly enhances the metabolic activity and proliferation capacity of cells. During the period from day 3 to day 28 of culture, compared with the control group, the hydrogel-based biomimetic brain microtissue extract has a significant effect on improving the survival rate of NSCs, with a survival rate increase of 350%~480% compared with the control group.

[0075] To investigate the effect of GO addition to GelMA / HAMA composite hydrogel on NSC cell viability, the present invention further conducted the following experiments: A composite hydrogel system with a mass ratio of GelMA and HAMA of 5:2 was prepared. The preparation method was basically the same as that in Example 1, except that graphene oxide solution was not added in step S2. All other operations and parameters were the same as in Example 1. Finally, GelMA / HAMA composite hydrogel-1 was obtained and lyophilized to obtain composite hydrogel-1 gel microspheres. Following the method described in this example, eight composite hydrogel-1 gel beads were placed in 1 mL of NSCs proliferation medium and placed in a 4°C refrigerator for 24 h. The gel beads were then removed to obtain composite hydrogel extract-1 day.

[0076] Eight composite hydrogel-1 gel beads were placed in 1 mL of NSCs proliferation medium and placed in a 4℃ refrigerator for 72 h. The gel beads were then removed to obtain the composite hydrogel extract - 3 days.

[0077] Meanwhile, eight gel microspheres prepared from hydrogel-based biomimetic brain microtissue I were placed in 1 mL of NSCs proliferation medium and kept in a 4°C refrigerator for 24 h. The gel microspheres were then removed to obtain hydrogel-based biomimetic brain microtissue extract - 1 day.

[0078] The effects of the composite hydrogel extract and the hydrogel-based biomimetic brain microtissue extract on the viability of NSCs were investigated using the CCK-8 assay method described above. A comparison of the NSC cell viability effects of the hydrogel-based biomimetic brain microtissue extract and the composite hydrogel extract is shown in the figure below. Figure 10 As shown.

[0079] Depend on Figure 10 It can be seen that, compared with the control group, the composite hydrogel extract-1 day group and the composite hydrogel extract-3 day group slightly improved the survival rate of NSCs, with the survival rate improvement range of 17.4%~21%. Compared with the control group, the hydrogel-based biomimetic brain microtissue extract-1 day group significantly improved the survival rate of NSCs, with the survival rate improvement range of 262%.

[0080] Furthermore, the three-dimensional porous structure of the hydrogel-based biomimetic brain microtissue provided by this invention provides cells with an adhesion and growth space that is closer to the physiological state. The introduction of GO may create a microenvironment that is more conducive to the long-term survival, expansion and function of cells by promoting the transmission of intercellular electrical signals or regulating specific biological pathways.

[0081] In summary, given that the hydrogel-based biomimetic brain microtissue provided by this invention not only has well-defined components, controllable structure and morphology, low cost and easy standardization, but also exhibits advantages such as high cell activity, excellent biocompatibility, good safety and outstanding physiological functions, it has broad application prospects in many cutting-edge fields such as brain tissue construction, modeling and mechanism research of nervous system diseases, pharmacodynamics research of neuropharmaceuticals, drug neurotoxicity assessment or developmental biology research.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing hydrogel-based biomimetic brain microtissue, characterized in that, The construction method includes the following steps: S1. Methacrylamide gelatin and methacrylamide hyaluronic acid are added to the photoinitiator solution and a pre-crosslinking reaction is carried out at 75℃-85℃ to obtain a composite hydrogel precursor solution. S2. The composite hydrogel precursor solution is mixed with graphene oxide to obtain a graphene oxide-enhanced composite hydrogel precursor solution. S3. The graphene oxide-enhanced composite hydrogel precursor solution is subjected to photocuring treatment to obtain hydrogel-based biomimetic brain micro-tissue; The mass ratio of the methacrylated gelatin to the methacrylated hyaluronic acid is 5:1.8-5:2.

2.

2. The method for constructing hydrogel-based biomimetic brain microtissue as described in claim 1, characterized in that, The final concentration of graphene oxide in the hydrogel-based biomimetic brain microtissue is 0.125 mg / mL to 1 mg / mL.

3. The method for constructing hydrogel-based biomimetic brain microtissue as described in claim 1, characterized in that, The photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

4. The method for constructing hydrogel-based biomimetic brain microtissue as described in claim 1, characterized in that, The final concentration of the photoinitiator in the composite hydrogel precursor solution is 1 mg / mL-2 mg / mL; and / or The mass ratio of the photoinitiator to methacrylamide gelatin is 1:50-2:50; and / or The solvent for the photoinitiator solution includes at least one of deionized water, PBS buffer, or Neurobasal complete culture medium.

5. The method for constructing hydrogel-based biomimetic brain microtissue as described in claim 1, characterized in that, The pre-crosslinking reaction takes 1.5h-2.5h.

6. The method for constructing hydrogel-based biomimetic brain microtissue as described in claim 1, characterized in that, The photocuring process involves irradiation at a wavelength of 403nm-407nm for 20s-40s.

7. The method for constructing hydrogel-based biomimetic brain microtissue as described in claim 1, characterized in that, The photocuring process includes irradiation at a wavelength of 403nm-407nm for 20s-40s; and / or The mass ratio of the methacrylated gelatin to the methacrylated hyaluronic acid is 5:

2.

8. The hydrogel-based biomimetic brain microtissue prepared by the method for constructing hydrogel-based biomimetic brain microtissue according to any one of claims 1-7.

9. The application of the hydrogel-based biomimetic brain microtissue as described in claim 8 in brain-like tissue construction, modeling and mechanism research of nervous system diseases, pharmacodynamic research of neuropharmaceuticals, assessment of drug neurotoxicity, or developmental biology research.

10. The application as described in claim 9, characterized in that, The neurological diseases include at least one of cerebrovascular diseases or central nervous system diseases.

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