Vascular plant-based scaffolds and in vitro methods for culturing neural stem cells

Abstract

A cellulose scaffold based on vascular plants is provided for the in vitro production or proliferation of neural stem cells. The scaffold can be obtained by decellularizing a vascular plant or a portion thereof and can support the proliferation of neural stem cells. The scaffold can be coated with any conventional coating agent known in the art, such as biomolecules, synthetic biomolecules (poly-L-ornithine), ligands, proteins, amino acids, antibodies, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-L-lysine, small molecules, hydrogels, or Matrigels. An in vitro method for proliferation or production of neural stem cells is also provided.

Description

【Technical Field】 【0001】 The present invention relates to the production or proliferation of neural stem cells for regenerative medicine or stem cell-based therapies. More specifically, the present invention relates to a vascular plant-based scaffold that acts as a substrate for the in vitro production or proliferation of neural stem cells. The present invention also provides a method for the in vitro production or proliferation of neural stem cells. 【Background Art】 【0002】 Neural stem cells (NSCs) are self-renewing cells that can proliferate in vitro and maintain the ability to differentiate into neurons, astrocytes, and oligodendrocytes. NSC transplantation has been investigated as a treatment strategy for numerous central nervous system disorders, and many preclinical studies have reported promising results. 【0003】 NSC-based therapies are at the forefront of regenerative medicine strategies for various neurological abnormalities and injuries such as stroke, traumatic brain injury, and spinal cord injury. However, currently, there are significant limitations to the effectiveness of NSC therapies, such as low graft survival and low efficiency of neural differentiation. To overcome these problems, biocompatible scaffolds for supporting cell survival and directing differentiation have emerged and are used as a medium for engrafting NSCs while supporting the survival of transplanted cells. 【0004】 Mammalian spinal cords have limited regenerative capacity after spinal cord injury (SCI). The non-regenerative capacity of the adult central nervous system may be partly due to the limited ability of damaged neurons to re-establish functional axons beyond the lesion. Mechanical trauma to tissue triggers various cellular responses, including increased proliferation of ependymal cells that line the central canal. Upon activation, ependymal cells re-express the characteristics of neural stem cells, migrate to the injury site, and spontaneously differentiate into oligodendrocytes and astrocytes there. While ependymal cells can differentiate into neurons in vitro, this differentiation has not been demonstrated in vivo after SCI, which is likely due to the specific characteristics of the post-SCI environment. Complex pathological mechanisms create an unsuitable microenvironment at the injury site, which inhibits axonal regrowth and damages surrounding tissue. For example, oligodendrocyte death induced by SCI leads to the accumulation of myelin breakdown products at the injury site. Molecules such as Nogo-A and myelin-associated glycoproteins are released from damaged myelin into the extracellular matrix after SCI and are important axon growth inhibitors. Many therapeutic strategies aim to establish a more favorable environment for regeneration by removing inhibitory molecules, providing nutritional support, directing stem cell fate, remyelination of axons, or transplanting biomaterial scaffolds that can induce axon growth. In recent years, multidisciplinary therapy has attracted considerable attention because it addresses multiple SCI pathological aspects and often yields greater benefits than their individual components. Such therapeutic strategies combine cell grafts, biomaterials, migratory training, and neurotrophic factors with the aim of modulating the injury microenvironment to promote repair. 【0005】 The development of biocompatible scaffolds for bridging spinal cord injury (SCI) is at the forefront of tissue engineering strategies. Transplantable biomaterials can be used not only to scaffold structures to induce cell attachment and migration, but also to modulate inflammatory responses or deliver other therapeutic substances such as stem cells and growth factors. Regenerative biomaterials for SCI are designed to replicate the properties of spinal cord tissue, and therefore possess the mechanical strength, porosity, and internal microstructure suitable for neurite outgrowth beyond the injury site. Furthermore, the surface chemistry of the biomaterial is particularly important, as it can be utilized to influence cell migration and differentiation. 【0006】 For the repair of spinal cord infections (SCIs), both degradable and non-degradable biomaterials are being investigated. For degradable scaffolds to function as substitutes for the extracellular matrix, their degradation rate must match that of axonal regeneration. Synthetic materials are attractive candidates for spinal cord tissue engineering due to their controllable mechanical properties and high batch-to-batch consistency. For example, polyethylene glycol is a synthetic polymer that can be crosslinked to form injectable hydrogels, which have been shown to provide a framework for tissue regeneration and promote motility recovery in rat models of SCIs. In addition, scaffolds based on poly(glycolic acid lactate copolymer) have resulted in significant improvements in motility and increased tissue remodeling in African green monkeys with incomplete SCIs. However, many synthetic biomaterials are limited by their hydrophobicity, which can weaken cell adhesion, or by the possibility that they release toxic byproducts during degradation. [Overview of the Initiative] [Means for solving the problem] 【0007】 This invention teaches a cellulose scaffold based on vascular plants for the in vitro production or proliferation of neural stem cells. In some embodiments, the plant-based scaffold is suitable for in vitro culture or proliferation of neural stem cells. The scaffold can be obtained by decellularizing a vascular plant or a portion thereof. In some embodiments, the scaffold may be obtained by mercing a vascular plant or a portion thereof. In some embodiments, the scaffold can support the proliferation of neural stem cells. 【0008】 The scaffold can be coated with one or more coating agents known in the art. For example, the scaffold can be coated with biomolecules, synthetic biomolecules, ligands, proteins, amino acids, antibodies, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-l-lysine, low molecular weight materials, hydrogels, or Matrigels. In certain embodiments, the scaffold is coated with a synthetic biomolecule such as poly-L-ornithine (PLO). 【0009】 The vascular plant-based scaffolds specified above may be used or employed in regenerative medicine or for the production or propagation of biopharmaceuticals. The scaffolds may also be used or employed in neurotissue engineering or in the treatment of nerve disorders, abnormalities, or injuries. Vascular plant-based scaffolds may be used or employed in in vitro drug production, propagation, or in vitro drug testing. The scaffolds may also be used or employed in in vitro production or propagation of neuronal cell proteins, or in vitro harvesting of neuronal cell proteins. The scaffolds may also be used in the production or propagation of nerve grafts or nerve cell banks, or in the production or propagation of nerve constructs for in vitro disease modeling. 【0010】 A method for in vitro production or proliferation of neural stem cells is provided. This method involves seeding a culture medium containing neural stem cells onto a cellulose scaffold based on a vascular plant as defined above in a container, and allowing the neural stem cells to attach to and proliferate on the cellulose scaffold for a predetermined period of time. The predetermined period of time may range from at least 24 hours to 5 days. 【0011】 This method may involve a step of coating the container with a native biomolecule, synthetic biomolecule, ligand, protein, amino acid, antibody, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-L-lysine, low molecular weight, hydrogel, or Matrigel before the step of seeding onto the vascular scaffold. For example, this method may involve coating the container with poly-L-ornithine and laminin. This method may involve a pretreatment step before the step of seeding onto the scaffold. The pretreatment step may be a step of sterilizing the scaffold with 70% ethanol, a step of washing the scaffold with physiological saline solution, surfactant solution, physiological buffer, or salt solution, or a step of pre-treating the surface of the scaffold. The washing step may further involve an incubation step of incubating the scaffold overnight in a salt solution. 【0012】 The surface of the scaffold can be pre-treated by chemical treatment, physical treatment, functionalization, succinylation, or a combination thereof. For example, the surface can be pre-treated with biomolecules, synthetic biomolecules, ligands, proteins, amino acids, antibodies, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-L-lysine, small molecules, hydrogels, Matrigels, or pharmaceutically acceptable compounds. The surface can also be chemically treated with synthetic biomolecules such as poly-L-ornithine. [Brief explanation of the drawing] 【0013】 [Figure 1]This figure shows cross-sections of decellularized asparagus (Asparagus officinalis) scaffolds. Figure 1A provides an overview of the cellulose scaffold (scale bar = 1 mm). Figure 1B provides an SEM of the scaffold cross-section (scale bar = 200 μm). Figure 1C provides an SEM of the vascular bundles within the scaffold cross-section (scale bar = 100 μm). Figure 1D provides an SEM of the cross-section of a scaffold coated with poly-l-ornithine (scale bar = 100 μm). [Figure 2] This figure provides confocal microscopy images (maximum projection images) of adult rat NSCs cultured on a decellularized asparagus scaffold. F-actin was stained with fluorescein phalloidin, the nucleus was stained with Hoechst 33342 (blue), and cellulose was stained with Congo red. Figure 2A shows NSCs grown for 72 hours on a poly-l-ornithine (PLO) coated scaffold (scale bar = 500 μm). Figure 2B shows NSCs grown for 14 days on an uncoated scaffold (scale bar = 300 μm). Figure 2C shows a higher magnification sagittal section of an NSC on a PLO coated scaffold (scale bar = 100 μm). Figure 2D shows a higher magnification cross-section of an NSC on a PLO coated scaffold (scale bar = 100 μm), where white arrows indicate neurite-like projections. [Figure 3] This figure shows the percentage reduction of AlamarBlue reagent by NSCs grown on a 3D cellulose scaffold (green) compared to a 2D control (gray) after 5 days of culture. (Error bars represent the standard deviation, with N=3 for each condition). [Figure 4]This figure provides an analysis of NSC lineages by immunohistochemistry. The figure shows representative confocal microscopy images (maximum projection images) of adult rat NSCs after 7 days of culture in differentiation medium. The nuclei were stained with Hoechst 33342 (blue). Figure 4A shows NSCs grown on a PLO-coated culture plate (2D) stained with GFAP (green). Figure 4B shows NSCs grown on a PLO-coated scaffold (3D) stained with GFAP (green). Figure 4C shows NSCs grown on a PLO-coated culture plate (2D) stained with βIII tubulin (red). Figure 4D shows NSCs grown on a PLO-coated scaffold (3D) stained with βIII tubulin (red). (Scale bar = 50 μm). [Figure 5] This figure shows an in vitro SEM of an asparagus scaffold coated with 20 μg / ml PLO. [Figure 6] This figure shows an in vitro SEM of an asparagus scaffold coated with 40 μg / ml PLO. [Figure 7] This figure shows an in vitro SEM image of an asparagus scaffold coated with 100 μg / ml PLO. [Figure 8] This figure shows an asparagus scaffold coated with 20 μg / ml PLO, on which fetal rat neural stem cells were seeded. [Figure 9] This figure shows a confocal microscope image (maximum projection image) of in vitro cell cultures of fetal rat neural stem cells. F-actin was stained with fluorescein phalloidin, the nuclei were stained with Hoechst 33342 (blue), and cellulose was stained with Congo red. [Figure 10] This figure shows a confocal microscope image (maximum projection image) of in vitro cell cultures of rat adult hippocampal neural stem cells. F-actin was stained with fluorescein phalloidin, the nucleus was stained with Hoechst 33342 (blue), and cellulose was stained with Congo red. [Figure 11]This figure shows the results of Fourier transform infrared spectroscopy (FTIR) analysis of asparagus scaffolds coated with PLO. [Figure 12] This figure shows H&E-stained in vivo subcutaneous grafts (20 μg / ml PLO) at 1 week, 4 weeks, 8 weeks, and 12 weeks, indicating cell infiltration into the scaffold. [Figure 13] This figure shows CD45-stained in vivo subcutaneous grafts (20 μg / ml PLO) demonstrating minimal foreign body response at 12 weeks. [Figure 14] This figure shows CD31-stained in vivo subcutaneous grafts (20 μg / ml PLO) demonstrating scaffold vascularization at 1, 4, 8, and 12 weeks. [Figure 15] This figure shows that NSC adheres to Matrigel, and although the adhesion is clearly visible, it is not optimal. Figure 15A shows adhesion to Matrigel 1:25, Figure 15B shows adhesion to uncoated scaffolding, and Figure 15C shows adhesion to scaffolding coated with PLO. [Figure 16]This figure shows plant-derived cellulose biomaterials in a rodent model of complete spinal cord transection. Figure 16A shows a decellularized plant-derived scaffold composed of vascular bundles and parenchyma (scale bar = 1 mm). Figure 16B shows a SEM of the microarchitecture of the cellulose scaffold (scale bar = 100 μm). Figure 16C shows hematoxylin and eosin staining of a cross-section of the cellulose scaffold after 12 weeks in vivo. Host cells are infiltrating the vascular bundles of the scaffold (scale bar = 200 μm). Figure 16D shows the exposed complete spinal cord transection (left box) and the scaffold after transplantation (right box). Figure 16E shows hematoxylin and eosin staining of a sagittal section of spinal cord tissue with a cellulose scaffold (outlined) after 12 weeks in vivo (scale bar = 2 mm). Figure 16F shows immunohistochemical staining (brown) for CD31 in sagittal sections at the biomaterial tissue interface (representative image of the ASP group). CD31-positive blood vessels are indicated by arrows (scale bar = 200 μm). Figure 16G shows immunohistochemical staining (brown) for CD31 in sagittal sections at the biomaterial tissue interface (representative image of the PLO group). CD31-positive blood vessels are indicated by arrows (scale bar = 200 μm). [Figure 17] This figure shows the evaluation of locomotion after complete spinal cord transection. Figure 17A shows the mean BBB score for each experimental group at 2 weeks post-SCI compared to 11 weeks post-SCI (two-way ANOVA, unadjusted Fisher's LSD method, ****P < 0.0001, *P = 0.0158, mean ± sem, n=11 PLO animals, n=9 ASP animals, n=8 untreated animals). Figure 17B shows the mean KSAT swimming evaluation score for each experimental group at 2 weeks post-SCI compared to 11 weeks post-SCI (two-way ANOVA, unadjusted Fisher's LSD method, ***P = 0.0001, mean ± sem, n=11 PLO animals, n=9 ASP animals, n=8 untreated animals). Figure 17C shows the average maximum angle achieved by each experimental group in the slope evaluation during the final week (mean ± sem, n=11 PLO animals, n=9 ASP animals, n=8 untreated animals). [Figure 18]This is a figure showing 5-HT immunohistochemical staining of a sagittal section of spinal cord injury at T8-T9. Figure 18A shows 5-HT staining of the spinal cord transplanted with an ASP scaffold (outlined). IHC was performed using DAB as the chromogen (brown) (scale bar = 2 mm). Figure 17B shows 5-HT staining of the non-transplanted spinal cord. IHC was performed using DAB as the chromogen (brown) (scale bar = 2 mm). [Figure 19] This is a figure showing retrograde neural tract tracing of the ascending sensory pathway by CTb injection into the sciatic nerve. Figure 19A shows axons traced with CTb at the most distal rostral. Each point represents the distance (mm) between the injury site and the axons traced with CTb at the most distal rostral in one animal. The bar represents the mean ± s.e.m of each group (one-way ANOVA, *P = 0.0264, n = 4 PLO animals, n = 2 ASP animals, n = 3 untreated animals). Figure 19B shows a hext (blue) stained cross-section of the T10 spinal cord (caudal to the injury), confirming the presence of CTb (red) in the spinal column and lamina 4 (scale bar 300um). Figure 19C shows a hext (blue) stained sagittal section of the spinal cord of an SCI animal without a scaffold. Axons traced with CTb (red) can be seen in the spinal column (scale bar 500um). Figure 19D shows a hext (blue) stained sagittal section of the spinal cord of an SCI animal with an uncoated scaffold. Axons traced with CTb (red) can be seen in the spinal column (scale bar 500um). Figure 19E shows a hext (blue) stained sagittal section of the spinal cord of an SCI animal transplanted with a cellulose scaffold coated with PLO. Axons traced with CTb (red) can be seen in the spinal column (scale bar 500um). [Figure 20]This figure shows the anterograde marking of the corticospinal tract by injection of dextranamine into the hindlimb motor cortex. Figure 20A shows the axon traced with dextranamine at the most distal caudal end. Each point represents the distance (mm) between the site of injury and the axon traced with dextranamine at the most distal caudal end in one animal. The bars represent the mean ± sem for each group (one-way ANOVA, P=0.0903, n=4 PLO animals, n=4 ASP animals, n=3 untreated animals). Figure 20B shows a Hoechst (blue) stained cross section of the T6 spinal cord (rostral to the injury), confirming the presence of dextranamine (green) in the corticospinal tract (scale bar 300um). Figure 20C shows a Hoechst (blue) stained sagittal section of the spinal cord of a scaffoldless SCI animal. The axon traced with dextranamine (green) can be seen in the corticospinal tract (scale bar 500um). Figure 20D shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal transplanted with an uncoated cellulose scaffold. Axons traced with DA (green) can be seen in the corticospinal tract (scale bar 500 μm). Figure 20E shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal transplanted with a PLO-coated cellulose scaffold. Axons traced with DA (green) can be seen in the corticospinal tract (scale bar 500 μm). [Figure 21]Figure showing that immunostaining of β-III tubulin and neurofilament-200 reveals that neurons adhere to the scaffold and migrate along the channels. Figure 21A shows β-III tubulin (red) and Hoechst (blue) staining of a sagittal section within the biomaterial coated with PLO. The cell body (identified by the arrow) can be seen inside the scaffold (scale bar 50 μm). Figure 21B shows β-III tubulin (red) and Hoechst (blue) staining of a sagittal section within the biomaterial coated with PLO. It can be seen that axons (identified by the arrow) are sprouting inside the scaffold (scale bar 50 μm). Figure 21C shows neurofilament 200 (green) and Hoechst (blue) staining of a sagittal section at the interface (dashed line) between the biomaterial coated with PLO and the spinal cord (scale bar 200 μm). Figure 21D shows neurofilament 200 (green) and Hoechst (blue) staining of a sagittal section at the interface (dashed line) between the biomaterial coated with PLO and the spinal cord. It can be seen that NF200-positive cells are infiltrating the biomaterial (asterisk), and NF200 axonal projections extend from the dorsal to the ventral surface of the biomaterial (arrow) (scale bar 50 μm). [Figure 22] Figure showing Luxol fast blue (LFB) staining of sagittal tissue sections for myelin assessment at the spinal cord injury site. Figure 22A shows representative LFB staining at the scaffold-tissue interface in an animal transplanted with a cellulose scaffold coated with PLO. The contour indicates the cellulose scaffold (scale bar = 200 μm). Figure 22B shows LFB staining at the scaffold-tissue interface in an animal transplanted with an uncoated cellulose scaffold (contour drawn) (scale bar = 200 μm). Figure 22C shows LFB staining at the cyst-tissue interface in an animal without a scaffold (scale bar = 200 μm). [Figure 23]This figure shows Luxor Fast Blue (LFB) staining of sagittal tissue sections for myelin evaluation at spinal cord injury sites. Figure 23A shows typical LFB staining at the scaffold-tissue interface in animals transplanted with PLO-coated cellulose scaffolds. The outline indicates the cellulose scaffold (scale bar = 200 μm). Figure 23B shows LFB staining at the scaffold-tissue interface in animals transplanted with uncoated cellulose scaffolds (outline shown) (scale bar = 200 μm). Figure 23C shows LFB staining at the cyst-tissue interface in animals without scaffolds (scale bar = 200 μm). [Figure 24] This figure shows the immunohistochemical staining of glial fibrillary acidic protein (GFAP) in sagittal spinal cord sections after SCI. Figure 24A shows a GFAP (green) stained spinal cord section from an animal transplanted with a PLO-coated cellulose scaffold. The nuclei were stained with DAPI (blue). The outline shows the cellulose scaffold (scale bar = 200 μm). Figure 24B shows a GFAP (green) stained spinal cord section from an animal transplanted with an uncoated cellulose scaffold (outline shown). The nuclei were stained with DAPI (blue). (Scale bar = 200 μm). Figure 24C shows a GFAP (green) stained spinal cord section from an animal without a scaffold. The nuclei were stained with DAPI (blue). (Scale bar = 200 μm). [Modes for carrying out the invention] 【0014】 The following description is merely an example of a preferred embodiment and is not limited to any combination of features necessary to carry out the present invention. 【0015】 All terms are intended to be understood in the same way as they are understood by those skilled in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure relates. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. 【0016】 While various features of this disclosure may be described in the context of a single embodiment, the features may also be provided individually or in any suitable combination. Conversely, while this disclosure may be described herein in the context of individual embodiments for clarity, this disclosure may also be implemented in a single embodiment. 【0017】 The following definitions are provided for the benefit of those skilled in the art and apply to this application. Accordingly, the technical terms used herein are intended solely to describe specific embodiments and are not intended to limit them. 【0018】 definition In this application, the use of the singular form includes the plural form unless otherwise specifically stated. It should be noted that, as used herein, the singular forms "a," "an," and "the" include the plural referent unless the context clearly indicates otherwise. 【0019】 In this application, the use of “or” means “and / or” unless otherwise specified. The terms “and / or” and “any combination thereof” as used herein, and their grammatical equivalents, may be used interchangeably. These terms may convey that any and all combinations are specifically considered. The term “or” may be used conjunctively or disjunctively unless the context specifically refers to a disjunctive use. 【0020】 Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is non-restrictive. 【0021】 In this specification, any references to “some embodiments,” “an embodiment,” “one embodiment,” “alternative embodiments,” or “other embodiments” mean that certain features, structures, or characteristics described in relation to an embodiment are included in at least some embodiments of this disclosure, but not necessarily in all embodiments. 【0022】 As used herein and in the claims, the terms “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are comprehensive or open-ended and do not exclude any additional undescribed elements or method steps. It is considered that any embodiment discussed herein may be carried out with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the compositions of the Disclosure may be used to carry out the methods of the Disclosure. 【0023】 As used herein, the term “about” and its grammatical equivalents in relation to a reference number may include the number itself and a range of values ​​plus or minus 10% from that number. The terms “about” or “approximately” mean within an acceptable margin of error of a particular value as determined by those skilled in the art, which in part depends on how the value was measured or determined, i.e., the limits of the measuring system. For example, “about” may mean a standard deviation of 1 or less or greater than 1 for each implementation in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another embodiment, the quantity “about 10” includes 10 and any quantity from 9 to 11. In yet another embodiment, the term “about” in relation to a reference number may also include a range of values ​​of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly in relation to biological systems or processes, the term “about” may mean within one order of magnitude of the value, preferably within five times, and more preferably within two times. Where a specific value is stated in the application and claims, unless otherwise stated, the term “approximately” should be assumed to mean within the acceptable margin of error of that value. 【0024】 Cell scaffolds can enhance the therapeutic efficacy of neural stem cells (NSCs) by promoting strong cell adhesion, inducing cell migration, and protecting transplanted NSCs from the cytotoxic damage environment resulting from central nervous system (CNS) injury. 【0025】 Because scaffolds provide three-dimensional physical support for cell proliferation, they can be considered a 3D cell culture system. This system exhibits a more accurate in vivo microenvironment compared to conventional 2D culture, and the behavior of cells within the 3D system is more physiologically relevant, making it a better cell culture method for tissue engineering and drug development. Therefore, various scaffold-based neural constructs are being developed for use in disease modeling, regenerative medicine, and stem cell niche research. 【0026】 In this invention, the inventors investigated decellularized plant tissue as a novel scaffold for three-dimensional in vitro culture, production, or proliferation of NSCs. The proposed plant cellulose scaffold was shown to support the attachment and proliferation of neural stem cells (NSCs) from the adult rat hippocampus, as shown in the accompanying drawings (Figure 10). Furthermore, NSCs differentiated on the cellulose scaffold showed significantly increased expression of neuron-specific beta-III tubulin and glial fibrillary acidic proteins compared to 2D culture, indicating that the scaffold can enhance the differentiation of NSCs into astrocyte and neuronal lineages (Figure 4). These findings suggest that plant-derived cellulose scaffolds have potential for use in neural tissue engineering and that their native surface topography can be used to guide NSC differentiation. 【0027】 The inventors investigated the potential of generating plant-derived cellulose biomaterials that have been shown to support cell invasion and vascularization in vivo. In recent years, various plant tissues have been decellularized for preclinical applications, including skin tissue engineering, nerve tissue engineering, and bone tissue engineering, to create biocompatible scaffolds for mammalian cell culture. For example, Dickie et al. decellularized spinach leaves while maintaining their vascular architecture and then re-resident them with human dermal microvascular endothelial cells. Furthermore, the mushroom Flammulina velutipes has been successfully used as a nerve guide tube in a rat model of sciatic nerve disorder. 【0028】 As demonstrated above, plant-derived biomaterials are becoming increasingly attractive for biomedical applications, partly due to their improved cost-effectiveness, scalability, and lower immunogenicity compared to animal sources. 【0029】 This invention investigates the feasibility of plant-derived biomaterials as 3D in vitro culture systems for adult rat neural stem cells. In a typical embodiment, the physical characteristics and mechanical tests of an asparagus scaffold were examined by scanning electron microscopy. Those skilled in the art will readily understand and recognize that any vascular plant material or a portion thereof can be used in the preparation of the proposed scaffold. 【0030】 In addition, the scaffold's ability to support NSC attachment and migration was evaluated over various time periods. The differentiation potential of neural stem cells in this 3D culture system was also investigated by immunostaining for neuron-specific β-III tubulin and glial fibrillary acidic protein markers. 【0031】 Embodiment One embodiment of the present invention provides a cellulose scaffold based on a vascular plant for the in vitro production or proliferation of neural stem cells. In some embodiments, the plant-based scaffold is suitable for the in vitro culture or proliferation of neural stem cells. The scaffold can be obtained by decellularizing a vascular plant or a portion thereof. In some embodiments, the scaffold may be obtained by mercing a vascular plant or a portion thereof. In some embodiments, the scaffold can support the proliferation of neural stem cells. 【0032】 In this invention, any vascular plant known in the art can be used to guide a plant-based scaffold. In some embodiments, the vascular plant may be asparagus, celery, ferns, horsetail, conifers, flowering plants, or clubmosses. 【0033】 In some embodiments, the scaffold may be coated with one or more coating agents known in the art. In certain embodiments, the scaffold may be coated with biomolecules, synthetic biomolecules, ligands, proteins, amino acids, antibodies, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-l-lysine, low molecular weight, hydrogel, or Matrigel. In certain embodiments, the scaffold is coated with synthetic biomolecules. In certain embodiments, the scaffold is coated with poly-L-ornithine (PLO). 【0034】 In some embodiments, the scaffold may have a porous structure containing multiple vascular bundles of varying diameters and multiple parenchyma cells. In certain embodiments, the vascular bundles may be scattered among the parenchyma cells. In some embodiments, the vascular bundles may be unevenly spaced and extend along the length of the scaffold. The parenchyma cells form the surface of the scaffold. 【0035】 The porous structure of the scaffold may promote the attachment, proliferation, or differentiation of neural stem cells. In certain embodiments, the porous structure of the scaffold may allow neural stem cells to maintain their differentiation ability. In some other embodiments, the porous structure of the scaffold may promote the differentiation of neural stem cells into neurons, dopaminergic neurons, motor neurons, astrocytes, oligodendrocytes, or combinations thereof. In some embodiments, the porous structure of the scaffold may promote or support the differentiation of neural stem cells into neurons and astrocytes. 【0036】 In embodiments where the scaffold is coated with poly-L-ornithine, the coating agent increases the production or proliferation of neural stem cells by promoting the formation of filopodia. In some embodiments, the porous structure of the scaffold promotes or supports the increased production or proliferation of neural stem cells by enabling nutrient exchange, oxygen exchange, and removal of waste within the scaffold. 【0037】 The porosity of the scaffolding can range from 10% to 95%, and the scaffolding can have an elastic modulus ranging from 1 kPa to 1000 kPa. In certain embodiments, the scaffolding is biocompatible, biodegradable, or both. 【0038】 In certain embodiments, the surface of the scaffold can be modified by chemical or physical treatment. This modification may enhance the adhesion of neural stem cells to the scaffold surface. In certain embodiments, the surface of the scaffold can be treated by surface functionalization, gamma irradiation, radical treatment, oxidation treatment, or a combination thereof. 【0039】 In the embodiments described above, functionalization can be achieved by providing a functional group that generates an electric charge on the surface of the scaffold. Functionalization can be carried out by mixing the scaffold with the functional group, by allowing the functional group to interact with the scaffold for a predetermined amount of time, or by any other procedure known in the art. The functional group may be a primary amine, a tertiary amine, a quaternary compound, an alcohol group, a carboxylic acid group, an aldehyde group, a sulfonyl group, or a combination thereof. In certain embodiments, the functional group may be an alkene, an alkyne, an amine, a ketone, an amide, an ester, a nitrile group, or an ether. 【0040】 In some embodiments, the scaffold promotes or supports the three-dimensional proliferation of neural stem cells, in which case the neural stem cells form neurospheres on the scaffold. In certain embodiments, the scaffold may promote or support the growth of neurite-like projections by cells. In certain other embodiments, the scaffold may promote or support the growth of multiple neural stem cell layers. In some further embodiments, the scaffold may promote or support the production or proliferation of neuronal proteins. 【0041】 In some embodiments, the vascular plant-based scaffolds defined above may be used or employed in regenerative medicine or for the production or propagation of biopharmaceuticals. In certain embodiments, the scaffolds may be used or employed in neurotissue engineering or in the treatment of nerve disorders, abnormalities, or injuries. In certain specific embodiments, the vascular plant-based scaffolds may be used or employed in in vitro drug production, propagation, or in vitro drug testing. In some other embodiments, the scaffolds may be used or employed in the in vitro production or propagation of neuronal cell proteins, or in the in vitro harvesting of neuronal cell proteins. The scaffolds may also be employed in the production or propagation of nerve grafts or nerve cell banks, or in the production or propagation of nerve constructs for in vitro disease modeling. 【0042】 In certain embodiments, a method for in vitro production or proliferation of neural stem cells is provided. This method involves seeding a culture medium containing neural stem cells onto a cellulose scaffold based on a vascular plant as defined above in a container, and allowing the neural stem cells to adhere to and proliferate on the cellulose scaffold for a predetermined amount of time. In certain embodiments, the predetermined amount of time is in the range of at least 24 hours to 5 days. In certain specific embodiments, the predetermined amount of time is at least 48 hours. 【0043】 In certain embodiments, the culture medium is injected with growth-promoting nutrients or growth factors, such as enzymes, antigens, specific proteins, inhibitors, stem cell factors, binders, and stimulants, which promote, facilitate, or increase the attachment and proliferation of neural stem cells to the scaffold. 【0044】 In certain embodiments, the culture medium is selected from any cell culture medium known in the art. In some embodiments, the culture medium is selected from any medium known to promote the proliferation of neural stem cells. In some embodiments, the culture medium is DMEM MEM / F12, Aplha MEM, knockout DMEM / F12, FBS, FCS, goat serum, or horse serum. In some other embodiments, the culture medium is A: 1× knockout DMEM / F-12 supplemented with 2% B27 (catalog 17504044, ThermoFisher), 2 mM GlutaMAX-I (ThermoFisher), B: serum-free medium (1× knockout D-MEM / F-12) having 2% StemPro Neural Supplement (ThermoFisher, A1050801), 20 ng / mL bFGF, 20 ng / mL EGF, or 2 mM GlutaMAX-I. 【0045】 In certain embodiments, the method can be carried out at temperatures in the range of 30°C to 45°C. In some embodiments, the method can be carried out at temperatures in the range of 32°C to 42°C. In some embodiments, the method can be carried out at pH ranges known in the art to support the proliferation of neural stem cells. In certain embodiments, the method can be carried out at pH in the range of 6.8 to 7.2. 【0046】 In some embodiments, the method can be carried out in any growth chamber known in the art. In certain embodiments, the method can be carried out in a container such as a flask, culture vessel, bioreactor, petri dish, multiwell plate, or glass chamber. 【0047】 In certain embodiments, the method may involve a step of coating the container with a native biomolecule, synthetic biomolecule, ligand, protein, amino acid, antibody, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-L-lysine, low molecular weight, hydrogel, or Matrigel prior to the step of seeding onto a vascular scaffold. In some embodiments, the container is coated with a native biomolecule, a synthetic biomolecule, or both. In some embodiments, the method involves coating the container with poly-L-ornithine and laminin. Poly-L-ornithine coating can increase the production or proliferation of neural stem cells by promoting filopodia formation. 【0048】 In some embodiments, the method involves a pretreatment step before the step of sowing seeds on the scaffold. The pretreatment step may include sterilizing the scaffold with 70% ethanol, washing the scaffold with physiological saline solution, surfactant solution, physiological buffer, or salt solution, or pre-treating the surface of the scaffold. In certain embodiments, the washing step may further involve an incubation step in which the scaffold is incubated overnight in a salt solution. 【0049】 In certain embodiments, the surface of the scaffold can be pre-treated by chemical treatment, physical treatment, functionalization, succinylation, or a combination thereof. In some embodiments, the surface can be pre-treated with biomolecules, synthetic biomolecules, ligands, proteins, amino acids, antibodies, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-L-lysine, small molecules, hydrogels, Matrigels, or pharmaceutically acceptable compounds. In one embodiment, the surface is chemically treated with a synthetic biomolecule for chemical treatment using poly-L-ornithine. The pre-treatment allows for modification of the scaffold surface and enhances cell production or proliferation by increasing adhesion to the scaffold surface. 【0050】 In some embodiments, the surface of the scaffold is functionalized by providing a functional group that generates an electric charge on the surface of the scaffold. The functional group can be selected from any functional group known in the art. The functional group may be a primary amine, a tertiary amine, a quaternary compound, an alcohol group, a carboxylic acid, an aldehyde, a sulfonyl, or a combination thereof. In some embodiments, the functional group may be an alkene, an alkyne, an amine, a ketone, an amide, an ester, a nitrile group, or an ether. 【0051】 In some embodiments, the method includes a sterilization step prior to the seeding step on the scaffold, in which the scaffold is sterilized for a predetermined time. In some embodiments, the sterilization step can be performed by autoclaving, treatment with 70% ethanol, gamma irradiation, or treatment with ethylene oxide. The sterilization step may be performed for 20 minutes to several days. 【0052】 In some embodiments, the production or proliferation of neural stem cells by the above method can be increased by modulating the rigidity of the scaffold. In some embodiments, the production or proliferation of neural stem cells can be increased by modulating the anisotropy of the scaffold. 【0053】 In certain embodiments, the method further includes a quantification step for quantifying the production or proliferation of neural stem cells. The quantification step can be performed by any cell quantification assay known in the art. In certain embodiments, quantification can be performed by a biomolecular quantification assay, a cell quantification assay, a cell viability assay, image analysis, immunohistochemistry, or electrophysiology. In some embodiments, after the quantification step, an optimization step may be performed to optimize the growth conditions in the container, to optimize the amount of scaffold, to optimize the size of the scaffold, to optimize the structure of the scaffold, or a combination thereof. 【0054】 In some embodiments, the method further includes an incubation step of incubating the scaffold and neural stem cells at a predetermined temperature. In certain embodiments, the incubation step may be carried out at a temperature in the range of 25°C to 50°C, preferably 30°C to 45°C. 【0055】 In some embodiments, the scaffold and neural stem cells may be incubated with a predetermined concentration of CO2, at a predetermined temperature, and for a predetermined duration. In some specific embodiments, the scaffold and neural stem cells may be incubated with 5% CO2 at 37°C. 【0056】 The attachment, proliferation, growth, and differentiation of neural stem cells on a scaffold can be carried out using the methods and various embodiments described above. The produced neural stem cells, after attachment to the scaffold, may differentiate into neurons, astrocytes, oligodendrocytes, or a combination thereof. In certain embodiments, neural stem cells differentiate into neurons and astrocytes after attachment to the scaffold. The differentiation of neural stem cells can be determined by any known assay in the art. For example, differentiation can be determined using immunohistochemical assays, cell imaging, confocal microscopy, flow cytometry, or electrophysiology. 【0057】 Consideration of the experiment Spinal cord injury (SCI) is a debilitating neurological condition that results in patients with a wide range of consequences, including loss of motor function and significant limitation of quality of life. Implantable biomaterials are emerging as a therapeutic strategy to modulate the SCI microenvironment and facilitate axonal regeneration. 【0058】 Recently, plant-derived scaffolds have been investigated in vivo for various biomedical applications, including tissue engineering of skin, tendons, nerves, and bone. Natural materials such as collagen, fibrin, alginates, chitosan, and hyaluronic acid are widely studied as regenerative scaffolds for SCI due to their excellent biocompatibility and cell adhesion properties. Collagen-based scaffolds, in particular, are a promising SCI treatment and are being investigated in diverse forms, including collagen hydrogels, collagen sponges, or collagen scaffolds, which deliver therapeutic substances to the injury. Current human clinical trials are reaffirming the therapeutic capabilities of collagen scaffolds, particularly their ability to promote tissue regeneration and functional recovery. One common problem associated with scaffolds made from natural polymers is insufficient mechanical strength and in vivo durability, to which several studies are attempting to solve by adding crosslinking agents or combining natural and synthetic biomaterials. 【0059】 To overcome these challenges, many different plant species can be decellularized using surfactants, sonication, enzymatic digestion, or freeze-thaw methods to produce cellulose-based scaffolds composed of β(1→4)-linked D-glucose units. Plant cellulose scaffolds have been shown to be biocompatible in vivo and supported extracellular matrix deposition when subcutaneously transplanted. In addition, these scaffolds vascularized as quickly as one week after transplantation. Because cellulose is not biodegradable in humans, scaffolds made from this material are designed to provide long-term durability in vivo. Furthermore, plant cellulose has proven to be a versatile material that can be combined with hydrogels to obtain customizable macroscopic structures. For example, cellulose scaffolds can be functionalized with chemical coatings to promote cell adhesion. 【0060】 In this invention, the inventors propose the use of a plant-derived cellulose scaffold to support the recovery of locomotion and the repair of nerve tissue in a rat model of spinal cord injury. In some cases, the plant-derived cellulose scaffold can be coated with various coating agents. In specific cases, the scaffold can be coated with poly-L-ornithine, a positively charged amino acid chain that has been shown to promote the differentiation of neural stem cells into neurons and enhance myelin regeneration. 【0061】 In typical experiments, after complete spinal cord transection, animals were implanted with plant-derived scaffolds coated with poly-L-ornithine (PLO). Motor function recovery was then assessed using the Basso, Beattie, and Bresnahan (BBB, Basso, Beattie, and Bresnahan) locomotion scale and the Karolinska Institute Swim Assessment Tool (KSAT). Retrograde tracing of ascending sensory pathways revealed enhanced regeneration in animals implanted with PLO-coated scaffolds. Immunostaining for β-III tubulin and NF200 indicated axonal sprouting within the cellulose biomaterial, and LFB staining highlighted myelinization around the PLO-coated scaffolds. 【0062】 For example, a cellulose scaffold made from asparagus stalks coated with poly-L-ornithine may support the attachment and proliferation of neural stem cells in vitro. In culture, rat neural stem cells form neurospheres, which attach to cellulose biomaterials and readily infiltrate their channels. Increased expression of neuron-specific beta-III tubulin and glial fibrillary acidic proteins in cells grown on these scaffolds suggests that these promote the differentiation of NSCs into astrocytes and neurons in vitro. 【0063】 For example, the ability of poly-L-ornithine-coated cellulose biomaterials to support the repair and functional recovery of nerve tissue after traumatic spinal cord injury (SCI). To demonstrate the present invention, the inventors transplanted scaffolds coated with poly-L-ornithine and uncoated scaffolds immediately after complete spinal cord transection in rats housed in an enriched environment. Motor recovery was monitored by BBB open-field evaluation and KSAT. Spinal sprouting and regeneration were analyzed by nerve pathway tracing after injecting the retrograde tracer CTb into the sciatic nerve. These results indicate enhanced sensory pathway regeneration in animals transplanted with coated grafts compared to controls. In addition, visualization of descending nerve fibers of the corticospinal tract in the motor cortex by dextranamine injection revealed that CST axons project along the cellulose biomaterial. Finally, spinal cord tissue was immunostained for various markers to identify key cell types interacting with the biomaterial. In short, the inventors have demonstrated the ability of a surface-modified plant-derived scaffold to promote axonal sprouting in lesions and induce motor recovery after SCI. 【0064】 1.1. Characterization of plant cellulose scaffolds Figure 1 shows a cross-section of a decellularized asparagus scaffold. Figure 1A provides an overview of the cellulose scaffold (scale bar = 1 mm). Figure 1B provides an SEM of the scaffold cross-section (scale bar = 200 μm). Figure 1C provides an SEM of the vascular bundles within the scaffold cross-section (scale bar = 100 μm). Figure 1D provides an SEM of the cross-section of a scaffold coated with poly-l-ornithine (scale bar = 100 μm). 【0065】 Raw asparagus stalks were cut into discs 4 mm in diameter and 1.2 mm in height, and then decellularized to remove all native cells. The resulting scaffolds consisted of vascular bundles (VB) scattered among parenchyma cells (shown in Figure 1A). The native structure inside the scaffolds was characterized by scanning electron microscopy (shown in Figures 1B-D). The majority of the scaffold surface consisted of parenchyma with an average diameter of 39 ± 15 μm. In addition, each scaffold was found to contain 14 ± 2 vascular bundles, which were aligned channels extending along the length of the scaffold with an average spacing of 602 ± 61 μm. 【0066】 The distribution of channel diameters observed in these scaffolds may be linked to cell survival, as it has been previously demonstrated that such porous networks allow for nutrient exchange and waste removal within the scaffold. The Young's modulus of the cellulose scaffolds was found to be 430 ± 139 kPa (n=12). Furthermore, the scaffolds were coated overnight with poly-l-ornithine, and adhesion was confirmed by SEM (Figure 1D) and Fourier transform infrared spectroscopy (FTIR). Figure 11 shows the FTIR of asparagus scaffolds coated with PLO. 【0067】 1.2. Cellulose scaffolding supports the attachment and proliferation of rat NSCs. Figure 2 shows confocal microscope images (maximum projection images) of adult rat NSCs cultured on a decellularized asparagus scaffold. F-actin was stained with fluorescein phalloidin, the nuclei were stained with Hoechst 33342 (blue), and cellulose was stained with Congo red. 【0068】 Figure 2A shows NSCs grown for 72 hours on a poly-l-ornithine (PLO) coated scaffold (scale bar = 500 μm). Figure 2B shows NSCs grown for 14 days on an uncoated scaffold (scale bar = 300 μm). Figure 2C shows a higher magnification sagittal section of an NSC on a PLO coated scaffold (scale bar = 100 μm). Figure 2D shows a higher magnification cross-section of an NSC on a PLO coated scaffold (scale bar = 100 μm), where white arrows indicate neurite-like processes. 【0069】 When a single-cell suspension of NSCs was seeded onto a scaffold, neurospheres were attached to the cellulose after 72 hours (Figure 2). Examination of cell attachment by F-actin staining revealed substantial cell-ECM and intercellular interactions. Many neurospheres with a diameter of up to 300 μm were found to be attached to the uncoated scaffold (Figure 2B), and neurite-like projections were observed to be projected from these neurospheres (Figure 2D). When a coating agent of poly-l-ornithine was added, cells migrated from the attached neurospheres and formed a pseudomonolayer on the scaffold surface (Figure 2A). Furthermore, it was observed that many neurospheres and individual cells migrated along channels in the scaffold (Figure 2C). 【0070】 In addition, cell proliferation on the scaffold was quantified using the AlamarBlue assay, thereby monitoring the chemical reduction of the culture medium and detecting the metabolic activity of the cells. Figure 3 shows the percentage reduction of the AlamarBlue reagent by NSCs grown on a 3D cellulose scaffold (green) compared to a 2D control (gray) after 5 days of culture (the error bars represent the standard deviation, with N=3 for each condition). 【0071】 Over 5 days of culture (Figure 3), the percentage of reduced AlamarBlue reagent gradually increased, indicating continuous proliferation of NSCs on the scaffold. Compared to NSCs grown as a monolayer (2D control), cells on the scaffold showed slightly reduced metabolic activity during the first 5 days of proliferation. 【0072】 1.3. Plant cellulose scaffolds enhance neuronal and astrocyte differentiation. To determine the effect of this 3D culture system on the differentiation potential of NSCs, the inventors monitored the expression of lineage-specific markers by NSCs. Single-cell suspensions of NSCs were simultaneously seeded at equal seeding densities on PLO-coated scaffolds and PLO-coated culture plates. After 7 days in differentiation medium, the cells were fixed and immunostained for GFAP (astrocytocyte marker) or neuron-specific βIII tubulin (immature neuron marker). 【0073】 Figure 4 shows the analysis of the NSC lineage by immunohistochemistry. Representative confocal microscopy images (maximum projection images) of adult rat NSCs after 7 days of culture in differentiation medium are provided. The nuclei were stained with Hoechst 33342 (blue). 【0074】 Figure 4A shows NSCs grown on a GFAP-stained (green) PLO-coated culture plate (2D). Figure 4B shows NSCs grown on a GFAP-stained (green) PLO-coated scaffold (3D). Figure 4C shows NSCs grown on a βIII-tubulin-stained (red) PLO-coated culture plate (2D). Figure 4D shows NSCs grown on a βIII-tubulin-stained (red) PLO-coated scaffold (3D) (scale bar = 50 μm). 【0075】 Interestingly, a significantly higher GFAP+ cell fraction was observed in the cellulose scaffold (shown in Figure 4B) compared to the 2D monolayer culture (shown in Figure 4A) (P < 0.0001, n=5). Similarly, immunohistochemistry revealed enhanced βIII tubulin expression on the 3D scaffold. As shown in Figure 4C, cells under 2D conditions were 0.78 ± 0.7% βIII tubulin+, while cells grown on the 3D scaffold were 16.4 ± 4.5% βIII tubulin+, as shown in Figure 4D, indicating a significant increase in the expression of this early neuronal marker (P < 0.0001, n=4). Both findings demonstrate that NSCs retained their ability to differentiate into various lineages when cultured on this 3D cellulose scaffold. 【0076】 Additional microscopic image data is provided in the attached drawings. For example, Figure 5 shows an in vitro SEM of an asparagus scaffold coated with 20 μg / ml PLO, Figure 6 shows an in vitro SEM of an asparagus scaffold coated with 40 μg / ml PLO, and Figure 7 shows an in vitro SEM of an asparagus scaffold coated with 100 μg / ml PLO. 【0077】 Figure 8 shows an asparagus scaffold coated with 20 μg / ml PLO, on which fetal rat neural stem cells were seeded. Figure 9 shows a confocal microscope image (maximum projection) of an in vitro cell culture of fetal rat neural stem cells. F-actin was stained with fluorescein phalloidin, the nuclei with Hoechst 33342 (blue), and cellulose with Congo red. 【0078】 Figure 10 shows confocal microscope images (maximum projection images) of in vitro cell cultures of adult rat hippocampal neural stem cells. F-actin was stained with fluorescein phalloidin, the nuclei were stained with Hoechst 33342 (blue), and cellulose was stained with Congo red. 【0079】 In vivo subcutaneous grafts are shown in Figures 12-14. Figure 12 shows H&E-stained in vivo subcutaneous grafts (20 μg / ml PLO) showing cell infiltration into the scaffold at 1, 4, 8, and 12 weeks. Figure 13 shows CD45-stained in vivo subcutaneous grafts (20 μg / ml PLO) showing minimal foreign body response at 12 weeks. Figure 14 shows CD31-stained in vivo subcutaneous grafts (20 μg / ml PLO) demonstrating vascularization of the scaffold at 1, 4, 8, and 12 weeks. 【0080】 The inventors also conducted experiments using Matrigel to compare the adhesion of NSC with that of scaffolds coated with PLO. The following steps were performed by the inventors: a) Remove the Matrigel from storage at -20°C and thaw it overnight in an ice bucket in a refrigerator at 4°C. b) The following day, the pipette tip, SFM, and culture dish were pre-cooled. c) Keep the Matrigel on ice at all times, spray with ethanol, and then bring the Matrigel onto the ice into a biological safety cabinet (BSC). Swirl the vial to mix thoroughly. d) 0.5 ml aliquots were prepared from a 10 ml bottle. e) Using a pre-cooled pipette and cold culture medium, Matrigel 1:25 was diluted in SFM (to prepare a dilute coating agent) (1:25 means 40 μL of Matrigel per 1 ml of SFM, 240 μL of Matrigel + 6 ml of SFM). f) Diluted Matrigel was then added to a pre-cooled culture dish, covering the entire surface. g) Next, an incubation step of 1 hour at room temperature was performed. h) Unbound Matrigel was aspirated and gently rinsed with SFM, and i) Matrigel was used immediately or kept at 4°C until about 1 hour before use. 【0081】 As can be seen in Figure 15, adhesion of NSC to Matrigel is verifiable but not optimal. Figure 15A shows adhesion to Matrigel 1:25, Figure 15B shows adhesion to uncoated scaffolding, and Figure 15C shows adhesion to scaffolding coated with PLO. 【0082】 2.1. Generation and transplantation of cellulose scaffolds in spinal cord injury in rodents Asparagus stalks were cut into cylindrical sections using a biopsy punch and decellularized to generate cellulose scaffolds (Figure 16A). The natural vascular bundles (VB) exceeded the length of the cellulose scaffold, and the scaffold had parenchyma with an average pore diameter of 39 ± 15 μm (Figure 16B). Aligned channels within the scaffold were characterized by scanning electron microscopy, and the Young's modulus was measured to be 128 ± 20 kPa parallel to the long axis (n=5). In animals with complete spinal cord transection at T8-T9, the scaffold was transplanted into the space between the spinal cord stumps with the long axis of VB parallel to the spinal cord (Figure 16D). No scaffold was transplanted in the control group (n=8), while the PLO group (n=11) received a cellulose scaffold coated with 100 μg / ml poly-L-ornithine (PLO) solution, and the ASP group (n=9) received an uncoated cellulose scaffold. Twelve weeks in vivo, spinal cord tissue was collected and hematoxylin and eosin staining was performed to evaluate the integration of the scaffold into the tissue (Figure 16E). The nuclei were observed migrating along the channels of the cellulose biomaterial, particularly within the vascular bundles (Figure 16C), completely extending beyond the lesion. The scaffold maintained its structure and diameter throughout the study, and no signs of a foreign body reaction were observed. Immunohistochemical staining for CD31 was performed to evaluate the vascularization of the biomaterial (Figures 16F and 16G). CD31-positive vessels were identified in the tissue surrounding the scaffold and within the vascular bundles in the biomaterial. 【0083】 Figure 16 shows plant-derived cellulose biomaterials in a rodent model of complete spinal cord transection. Figure 16A shows a decellularized plant-derived scaffold composed of vascular bundles and parenchyma (scale bar = 1 mm). Figure 16B shows a SEM of the microarchitecture of the cellulose scaffold (scale bar = 100 μm). Figure 16C shows hematoxylin and eosin staining of a cross-section of the cellulose scaffold after 12 weeks in vivo. As shown in the figure, host cells have infiltrated the vascular bundles of the scaffold (scale bar = 200 μm). Figure 16D shows the exposed complete spinal cord transection (left box) and the scaffold after transplantation (right box). Figure 16E shows hematoxylin and eosin staining of a sagittal section of spinal cord tissue with a cellulose scaffold (outlined) after 12 weeks in vivo (scale bar = 2 mm). Figure 16F shows immunohistochemical staining (brown) of CD31 in sagittal sections at the biomaterial tissue interface (representative image of the ASP group). CD31-positive blood vessels are indicated by arrows (scale bar = 200 μm). Figure 16G shows immunohistochemical staining (brown) of CD31 in sagittal sections at the biomaterial tissue interface (representative image of the PLO group). CD31-positive blood vessels are indicated by arrows (scale bar = 200 μm). 【0084】 2.2 Recovery of hindlimb locomotion after complete SCI 2.2.1. BBB Mobility Assessment Figure 17 shows the evaluation of locomotion after complete spinal cord transection. Figure 17A shows the mean BBB scores of each experimental group at 2 weeks post-SCI compared to 11 weeks post-SCI (two-way ANOVA, uncorrected Fisher's LSD method). **** P is less than 0.0001, * P=0.0158, mean ± sem, n=11 PLO animals, n=9 ASP animals, n=8 untreated animals). Figure 17B shows the mean KSAT swimming evaluation scores for each experimental group at 2 weeks post-SCI compared to 11 weeks post-SCI (2-way ANOVA, unadjusted Fisher's LSD method). ***P=0.0001, mean ± sem, n=11 PLO animals, n=9 ASP animals, n=8 untreated animals). Figure 17C shows the mean maximum angle achieved by each experimental group in the slope evaluation during the final week (mean ± sem, n=11 PLO animals, n=9 ASP animals, n=8 untreated animals). 【0085】 Hindlimb motor function was assessed weekly using the Basso, Beattie, and Bresnahan (BBB) ​​locomotion scales (Figure 17A). Two weeks after complete spinal cord transection, BBB assessments were performed to confirm complete hindlimb paralysis, and one animal was excluded based on its score greater than 5. There were no differences between the experimental groups in this initial postoperative BBB assessment. Over an 11-week recovery period, animals implanted with and without PLO-coated scaffolds showed significant functional recovery, while animals without scaffolds did not show significant changes in BBB scores. This was determined by comparing the mean score at week 2 to the mean score at week 11 for each experimental group, and by performing two-way ANOVA and unadjusted Fisher's LSD test. At the final week, the mean BBB score for rats treated with PLO-coated scaffolds was 5.6 ± 0.87 points, which corresponds to slight movement of two joints and extensive movement of the third. The group that received uncoated scaffolding had a mean score of 4.3 ± 0.96, indicating that all three joints of the hind limb were slightly mobile. In contrast, the control group that did not receive scaffolding had a mean score of 3.35 ± 1.0, reflecting that only two joints were mobile. Importantly, none of the animals in the control group achieved a score of 6 or higher, while several animals in the PLO and ASP groups achieved BBB scores of 7 (all three joints mobile extensively), 8 (foot contact without weight bearing), and 9 (foot contact with weight bearing, but only in posture). 【0086】 2.2.2. KSAT Swimming Evaluation During swimming, animals rely on buoyancy to support their weight, allowing for unloaded limb movements. Consequently, proprioceptive and cutaneous feedback from the hind limbs is significantly reduced compared to walking on land. Therefore, the Karolinska Institute's Swimming Assessment Tool (KSAT) was used to identify changes in hind limb motor ability after SCI, while minimizing the influence of afferent feedback. In this assessment, healthy animals achieve a maximum score of 19 based on the intensity and frequency of limb and tail movements. There were no differences between the experimental groups in this initial postoperative swimming assessment. After 11 weeks of recovery, rats with PLO-coated scaffolds showed a significant improvement in swimming performance compared to their initial postoperative assessment, while controls did not achieve the same improvement (Figure 17B). The mean KSAT score for rats with PLO-coated scaffolds was 3.79 ± 0.58, compared to 3.18 ± 0.64 for rats with uncoated scaffolds and 2.25 ± 0.68 for mice without scaffolds. Animals treated with scaffolding therapy showed improvements primarily in hind limb movement and torso stability. 【0087】 2.2.3. Slope Test Prior to spinal cord injury, all animals were trained to perform the incline test. The animals were placed on an incline, and the incline was adjusted to determine the maximum angle at which the animals could maintain their position without falling. The incline test was performed every two weeks after spinal cord injury to assess sensorimotor recovery. At the final week, no significant differences in recovery were observed among the experimental groups (Figure 17C). 【0088】 2.2.4. 5HT staining Figure 18 shows 5-HT immunohistochemical staining of sagittal sections of spinal cord injury at T8-T9. Figure 18A shows 5-HT staining of spinal cord transplanted with an ASP scaffold (outlined). IHC was performed using DAB as the chromophor (brown) (scale bar = 2 mm). Figure 17B shows 5-HT staining of spinal cord without transplantation. IHC was performed using DAB as the chromophor (brown) (scale bar = 2 mm). 【0089】 Immunohistochemical staining with 5-HT revealed some sprouting of rostral serotonergic axons in response to injury (Figure 18). However, caudal tissue in response to complete transection was not stained with 5-HT due to the denaturation of serotonergic activity. 【0090】 2.3. Retrograde neural pathway tracing of ascending sensory nerve fibers reveals enhanced regeneration. Figure 19 shows retrograde nerve pathway tracing of the ascending sensory afferent pathway by CTb injection into the sciatic nerve. Figure 19A shows the axon traced with the most distal rostral CTb. Each point represents the distance (mm) between the injury site and the axon traced with the most distal rostral CTb in one animal. The bars represent the mean ± sem for each group (one-way ANOVA). * (P=0.0264, n=4 PLO animals, n=2 ASP animals, n=3 untreated animals). Figure 19B shows a Hoechst (blue) stained section of the T10 spinal cord (caudal to the injury), confirming the presence of CTb (red) in the vertebral column and lamina 4 (scale bar 300um). Figure 19C shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal without a scaffold. Axons (red) traced with CTb can be seen in the vertebral column (scale bar 500um). Figure 19D shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal with an uncoated scaffold. Axons (red) traced with CTb can be seen in the vertebral column (scale bar 500um). Figure 19E shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal with a PLO-coated cellulose scaffold transplanted. The axons (red) traced by CTb can be seen in the spinal column (scale bar 500um). 【0091】 After 11 weeks of recovery, neuroanatomical neural pathway tracing was performed in rats of each experimental group to identify sprouted and regenerated nerve fibers at the injury site. For retrograde tracing, cholera toxin subunit B (CTb), conjugated to Alexa Fluor 647, was injected into the sciatic nerve to label sensory axonal projections along the spinal column of the spinal cord. Confocal laser scanning microscopy imaging of T10 spinal cord tissue caudal to the injury revealed a clear CTb-positive signal in the spinal column (Figure 19B), which was not observed in T6 sections cranial to the injury. Sagittal sections of spinal cord tissue showed that CTb-labeled axons projected caudally toward the injury site (Figures 19C, D, and E). Compared to animals without a scaffold, animals with a PLO-coated scaffold had a significantly smaller, on average distance between the axon traced by the most distal rostral CTb and the site of injury (Figure 19A), which may indicate reduced axonal regression or enhanced regeneration. In animals treated with a PLO-coated scaffold (Figure 19E), the axon traced by the most distal rostral CTb was identified at an average distance of 3.2 mm from the site of injury. In contrast, control animals without a scaffold had axons traced by the CTb at an average distance of 4.8 mm from the site of injury (Figure 19C). 【0092】 2.4 Antegrade markers of the corticospinal tract Figure 20 shows anterograde marking of the corticospinal tract by injection of dextranamine into the hindlimb motor cortex. Figure 20A shows axons traced with dextranamine at the most distal caudal end. Each point represents the distance (mm) between the site of injury and the axon traced with dextranamine at the most distal caudal end in one animal. The bars represent the mean ± sem for each group (one-way ANOVA, P=0.0903, n=4 PLO animals, n=4 ASP animals, n=3 untreated animals). Figure 20B shows a Hoechst (blue) stained cross section of the T6 spinal cord (rostral to the injury), confirming the presence of dextranamine (green) in the corticospinal tract (scale bar 300um). Figure 20C shows a Hoechst (blue) stained sagittal section of the spinal cord of a scaffoldless SCI animal. Axons traced with dextranamine (green) can be seen in the corticospinal tract (scale bar 500um). Figure 20D shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal transplanted with an uncoated cellulose scaffold. Axons traced with DA (green) can be seen in the corticospinal tract (scale bar 500 μm). Figure 20E shows a Hoechst (blue) stained sagittal section of the spinal cord of an SCI animal transplanted with a PLO-coated cellulose scaffold. Axons traced with DA (green) can be seen in the corticospinal tract (scale bar 500 μm). 【0093】 In addition, the sprouting of the corticospinal tract (CST) at the spinal cord injury site was analyzed by stereotactic injection of dextranamine conjugated to Alexa Fluor 488 into the hindlimb motor cortex. Successful tracer uptake was confirmed by confocal laser scanning microscopy of a T6 spinal cord section cranial to the injury, which showed strong positive dextranamine labeling of axons in the corticospinal tract (Figure 20B). In sagittal sections, dextranamine-labeled axons were identified at the rostral interface between the biomaterial and the spinal cord (Figures 20D and 20E), but were not present in T10 spinal cord sections caudal to the injury. The most caudal dextranamine-labeled axons were identified at an average distance of 0.33 mm from the developmental site in animals with PLO-coated scaffolds (Figures 20A and 20E), at an average distance of 0.45 mm from the developmental site in animals with uncoated scaffolds (Figure 20D), and at an average distance of 1.6 mm from the developmental site in animals without scaffolds (Figure 20C). Axons traced with DA were observed to project caudally toward the spinal cord along the dorsal side of the PLO-coated cellulose biomaterial (Figure 20E). 【0094】 2.5. Infiltration of nerve cells and axonal sprouting within cellulose biomaterials Figure 21 shows that immunostaining for β-III tubulin and neurofilament-200 reveals that nerve cells are attached to the scaffold and migrating along the channels. Figure 21A shows β-III tubulin (red) and Hoechst (blue) staining of sagittal sections in PLO-coated biomaterial. Cell bodies (identified by arrows) can be seen inside the scaffold (scale bar 50 μm). Figure 21B shows β-III tubulin (red) and Hoechst (blue) staining of sagittal sections in PLO-coated biomaterial. Axons (identified by arrows) can be seen sprouting inside the scaffold (scale bar 50 μm). Figure 21C shows neurofilament-200 (green) and Hoechst (blue) staining of sagittal sections at the interface (dashed line) between the PLO-coated biomaterial and the spinal cord (scale bar 200 μm). Figure 21D shows neurofilament 200 (green) and Hoechst (blue) staining of sagittal sections at the interface (dashed line) between the PLO-coated biomaterial and the spinal cord. NF200-positive cells can be seen infiltrating the biomaterial (asterisk), and NF200 axonal projections extend from the dorsal to the ventral side of the biomaterial (arrow) (scale bar 50 μm). 【0095】 Figure 22 shows Luxor Fast Blue (LFB) staining of sagittal tissue sections for myelin evaluation at spinal cord injury sites. Figure 22A shows typical LFB staining at the scaffold-tissue interface in animals transplanted with PLO-coated cellulose scaffolds. The outline indicates the cellulose scaffold (scale bar = 200 μm). Figure 22B shows LFB staining at the scaffold-tissue interface in animals transplanted with uncoated cellulose scaffolds (outline shown) (scale bar = 200 μm). Figure 22C shows LFB staining at the cystic tissue interface in animals without scaffolds (scale bar = 200 μm). 【0096】 Figure 23 shows Luxor Fast Blue (LFB) staining of sagittal tissue sections for myelin evaluation at spinal cord injury sites. Figure 23A shows typical LFB staining at the scaffold-tissue interface in animals transplanted with PLO-coated cellulose scaffolds. The outline indicates the cellulose scaffold (scale bar = 200 μm). Figure 23B shows LFB staining at the scaffold-tissue interface in animals transplanted with uncoated cellulose scaffolds (outline shown) (scale bar = 200 μm). Figure 23C shows LFB staining at the cystic tissue interface in animals without scaffolds (scale bar = 200 μm). 【0097】 Figure 24 shows immunohistochemistry of glial fibrillary acidic protein (GFAP) in sagittal spinal cord sections after SCI. Figure 24A shows GFAP (green) stained spinal cord sections from animals transplanted with PLO-coated cellulose scaffolds. The nuclei were stained with DAPI (blue). The outlines show the cellulose scaffolds (scale bar = 200 μm). Figure 24B shows GFAP (green) stained spinal cord sections from animals transplanted with uncoated cellulose scaffolds (outlines shown). The nuclei were stained with DAPI (blue). (Scale bar = 200 μm). Figure 24C shows GFAP (green) stained spinal cord sections from animals without scaffolds. The nuclei were stained with DAPI (blue). (Scale bar = 200 μm). 【0098】 Histological analysis of spinal cord tissue revealed host cell infiltration into the scaffold from the rostral and caudal interfaces. Within the scaffold, both cell bodies and axonal projections expressing β-III tubulin, an early neuronal marker, were identified (Figures 21A and B). This finding suggests that endogenous adult neural stem cells infiltrated the biomaterial and initiated differentiation into the neural lineage. In addition, numerous NF200-positive axons were observed within and around the scaffold (Figure 21C). Interestingly, at the rostral and caudal interfaces of the biomaterial, we observed clusters of neurofilament-positive projections branching from the dorsal to ventral side of the spinal cord, perpendicular to the axis of the neural pathway (Figure 21D). Finally, tissue sections from each experimental group were stained with Luxor Fast Blue (LFB) to assess axonal myelination at the injury site (Figures 22, 23, and 24). As expected in traumatic spinal cord injury, significant myelin degeneration was observed in the tissue surrounding the lesion, which was consistent with Wallerian degeneration. However, in animals transplanted with PLO-coated scaffolds, dark blue LFB-stained bands were observed at the rostral and caudal interfaces of the PLO-coated scaffold and tissue, suggesting the possibility of poly-L-ornithine-induced remyelination (Figures 22A-22C, 23A-23C). Immunostaining of glial fibrillary acidic protein (GFAP) in sagittal spinal cord sections after SCI is shown in Figure 24. In Figure 24A, the GFAP-stained (green) spinal cord sections are from animals transplanted with PLO-coated cellulose scaffolds, and the nuclei are stained with DAPI (blue), indicating the cellulose scaffold (scale bar = 200 μm). In Figure 24B, the spinal cord section stained with GFAP (green) is from an animal that received an uncoated cellulose scaffold (with an outline drawn), the nucleus is stained with DAPI (blue), and the scale bar is 200 μm. In Figure 24C, the spinal cord section stained with GFAP (green) is from an animal without a scaffold, the nucleus is stained with DAPI (blue), and the scale bar is 200 μm. 【0099】 Consideration Complete spinal cord injury (SCI) results in loss of both sensorimotor and autonomic function distal to the injury site due to disruption of ascending and descending pathways. Axonal regrowth and restoration of appropriate synaptic connections after SCI remain major medical challenges. Various therapeutic strategies have attempted to induce corticospinal tract sprouting or establish relay neural circuits that can mediate functional recovery. Neural circuits within the injured spinal cord have been shown to exhibit flexibility after treadmill training or electrical epidural stimulation, which can lead to some degree of motor recovery. However, maladaptive synaptic connections after SCI can be maladaptive and can result in spasticity or neuropathic pain. In recent decades, biomaterial-based SCI therapeutic strategies have been investigated as means to support axonal growth and to modulate the inhibitory environment at the lesion site by locally delivering cells or pharmaceuticals. For example, some groups are creating bioactive scaffolds that utilize cell signaling molecules to enhance axonal regrowth, myelination, and functional recovery, while others have 3D-printed scaffolds with microarchitectures that mimic structures within the spinal cord to support the formation of neural relays. Furthermore, some scaffolds are being used as carriers to deliver various types of stem cells to damaged spinal cords to promote repair. 【0100】 The inventors have developed a 3D cell culture scaffold made of decellularized asparagus stalks that supports the attachment, proliferation, and differentiation of adult rat neural stem cells. After decellularization, primary neural stem cells isolated from the hippocampus of adult Fisher 344 rats were seeded onto the cellulose scaffold. Microscopic studies of the scaffold revealed a system of aligned channels of various diameters, which the inventors predict will enable efficient transport of nutrients and oxygen. 【0101】 During several hours of seeding, it was demonstrated that NSCs attached to the uncoated scaffold as both individual cells and neurospheres of varying sizes (Figure 2). Furthermore, the inventors noted the difference in the degree of cell spread between PLO-coated and uncoated scaffolds. It was also found that NSCs seeded on the PLO-coated scaffold migrated out of the attached neurospheres to form a basal cell monolayer. This behavior can potentially be explained by the fact that PLO enhances migration by promoting filopodia formation. 【0102】 Subsequent F-actin staining confirmed the attachment of neurospheres to the biomaterial and highlighted the migration of NSCs to the scaffold channels. Furthermore, NSCs were found to proliferate within the 3D culture system, as demonstrated by the Alamar Blue assay (in Figure 3). Taken together, these data suggest that the scaffold is biocompatible and possesses suitable physical characteristics to enable the proliferation of neural stem cells. 【0103】 The behavior of cells cultured in 3D systems, including their proliferation rate, often differs from that in 2D systems. Interestingly, the percentage of AlamarBlue reagent reduction is consistently lower in 3D compared to 2D monolayer culture systems, suggesting a slight inhibition of NSC proliferation on the scaffold. This difference may be partly due to increased heterogeneity in 3D culture systems where NSCs assemble into neurospheres and attach to the scaffold. The outer surface of these neurospheres consists of rapidly proliferating cells, while the inner layer of NSCs tends to quiescent or necrotize due to reduced access to oxygen, nutrients, and growth factors. Therefore, this difference in AlamarBlue reduction may be due to heterogeneity rather than purely to differences in proliferation rate. 【0104】 Finally, NSCs grown on the scaffold were cultured in differentiation medium, and their expression of the cytoplasmic markers GFAP and βIII-tubulin was assessed after 7 days. Notably, this plant-derived scaffold was found to enhance the differentiation of NSCs into neurons and astrocytes. The increase in GFAP+ and βIII-tubulin+ cells on the scaffold compared to 2D controls is likely due to the complex interplay of chemical and tissue distributional cues provided by the scaffold. 【0105】 Furthermore, since stiffness is one of the most important mechanical properties that direct cell fate, mechanical tests were performed to determine the elastic modulus of the scaffold. The Young's modulus of the scaffold was determined to be 430 ± 139 kPa, which is softer than polystyrene cell culture plates (E = 3,730,000 kPa) but stiffer than brain and spinal cord tissue. Previous studies have shown that softer growth substrates tend to be more favorable to neural differentiation. Therefore, the difference in stiffness between the scaffold and the cell culture plate may contribute to increased neural differentiation. Similarly, the anisotropy of the scaffold may also modulate differentiation towards more robust neurogenesis, as has been reported previously. 【0106】 In further research, the inventors investigated the ability of the natural microarchitecture found in plant-derived scaffolds to provide physical support for regenerating axons and to induce axonal extension across lesions within the spinal cord. The results showed that plant cellulose can become vascularized after transplantation into the spinal cord, and that endogenous neurons expressing β-III tubulin and NF200 readily migrate to channels in the scaffold that are linearly oriented along the rostral-caudal axis. In addition to directing axonal extension during regeneration, the biomaterial may contribute to motor recovery through its interaction with astrocytes, which are known to play a crucial role in SCI pathology. Post-SCI, activated microglia induce gene expression and morphological changes in astrocytes around the lesion. The process of reactive astrogliosis is regulated by complex signaling pathways, particularly NFκB or TGF-β signaling, ultimately leading to the formation of dense glial scars. By occupying space within the injury site, the cellulose scaffold may have reduced the number of reactive astrocytes migrating to the lesion and interfered with the signaling mechanisms involved in the propagation of reactive astrogliosis, thereby attenuating tissue damage and enhancing motility. In addition, previous reports have shown that modulation of the cytoskeleton within astrocytes can be induced by microgrooves, so the presence of channels on the cellulose scaffold surface may have influenced the alignment and morphology of astrocytes at the injury site. 【0107】 In further research, the inventors also tested the ability of plant-derived cellulose scaffolds to support repair when combined with a coating agent of poly-L-ornithine, a positively charged synthetic amino acid that adheres to cellulose through electrostatic interactions. PLO is commonly used in neural stem cell culture, and previous studies have demonstrated its ability to improve cell adhesion and migration. Specifically, PLO has been shown to promote filopodia formation in neural progenitor cells in vitro by increasing the expression of α-actin 4, a key effector of structural flexibility. Based on this, the inventors hypothesized that PLO may have a similar effect in vivo after SCI, and that increased sprouting of severed axons may assist in the establishment of neural relays across the scaffold, thereby mediating functional recovery. 【0108】 Animals transplanted with uncoated cellulose scaffolds showed slight recovery of motor function in BBB assessment, and this recovery was found to be enhanced by the addition of a poly-L-ornithine coating agent. To evaluate axonal repair after complete transection, the inventors performed retrograde nerve pathway tracing with CTb and injected CTb into the sciatic nerve. This nerve pathway tracing experiment revealed the sprouting and regeneration of sensory axons in animals transplanted with PLO-coated scaffolds. Importantly, the inventors found that in animals with PLO-coated scaffolds, vertebral axons extended over longer distances to the injury site compared to animals without scaffolds. Our findings are consistent with those of Schackel, T. et al., *Peptides and Astroglia Improve the Regenerative Capacity of Alginate Gels in the Injured Spinal Cord. Tissue Eng Part A 25, 522-537 (2019)*, which found that hydrogels coated with PLO / laminin, transplanted into animals with hemicervical transection, increased host cell migration and slightly increased neurite growth. The improved regeneration of sensory nerve fibers identified in PLO-treated animals may have contributed to the recovery of motor function in these animals, considering the importance of sensory feedback in motor function after SCI. 【0109】 In addition to sensory axon sprouting, the inventors found enhanced myelination at injury sites in animals with PLO-coated scaffolds, as evidenced by Luxor Fast Blue staining indicating myelin around the scaffold. Recent studies have demonstrated PLO's ability to enhance myelin repair in animal models of local demyelination. The inventors hypothesize that myelin repair may be a contributing mechanism underlying PLO-induced motor recovery in their SCI model. Furthermore, since myelin breakdown products are known to be inhibitors of neuroplasticity, the positive effect of PLO on myelination at injury sites may be twofold. Therefore, by enhancing remyelination, PLO may also create a more favorable environment for axon sprouting, which is consistent with the inventors' view. 【0110】 Environmental enrichment (EE) has also been included as part of a multifaceted neurorehabilitation strategy due to its ability to enhance neuroplasticity and mitigate neuropathic pain induced by SCI. Environmental enrichment is the manipulation of the housing environment, including new toys, tunnels, nesting materials, puzzles, and running wheels, as well as opportunities for socialization with congenital animals. By enhancing flexibility, a stimulating environment supports functional improvement in animal models of stroke and SCI. Studies have shown that EE promotes flexibility through various mechanisms, including increased production of neurotrophic factors and changes in dendritic spine density. In rodent SCI models, enrichment enhances the regenerative capacity of neurons through Creb-binding protein-mediated histone acetylation, which increases the expression of genes related to regeneration. Numerous past studies have reported that environmental enrichment substantially improves sensory and motor recovery after bruising SCI. Similarly, in animal models of stroke, exposure to a stimulating environment induces neuroanatomical changes, including dendritic cell remodeling, axonal sprouting, and the release of growth factors. Environmental enrichment has also been reported to promote white matter recovery after stroke by reducing microglial activation. Taken together, environmental enrichment is very promising as part of a multidisciplinary SCI treatment strategy. 【0111】 In short, the inventors demonstrated that cellulose scaffolds can support the proliferation and differentiation of neural stem cells in vitro. These findings suggest that plant-derived scaffolds can facilitate the production or proliferation of a large number of specifically differentiated cells necessary for NSC research or regenerative medicine. 【0112】 The inventors also demonstrated the potential of functionalizing plant-derived cellulose scaffolds with peptides for use in multidisciplinary SCI therapeutic strategies, including environmental enrichment. Specifically, they found that coating biomaterials with poly-L-ornithine supported hindlimb motor recovery and nerve tissue repair in a rat model with complete amputation. When endogenous neurons were infiltrated into the biomaterials, the cells migrated along the linearly oriented channels of the plant scaffolds. Furthermore, retrograde nerve tracing highlighted the regeneration of sensory pathways in the spinal cord after treatment with PLO-coated scaffolds. Overall, the inventors' results point to an attractive potential patient treatment strategy using plant-derived scaffolds in combination with other therapies. 【0113】 Method explanation Preparation of biomaterial: Asparagus scaffolds were prepared as described by Modulevsky et al. Asparagus sections were cut using a 4 mm biopsy punch and placed in 50 ml Falcon tubes containing 0.1% sodium dodecyl sulfate (SDS) (Sigma-Aldrich). The samples were shaken at 180 RPM for 72 hours at room temperature. The resulting cellulose scaffolds were then transferred to new sterile microcentrifuge tubes, washed, and incubated in phosphate-buffered saline (PBS) for 12 hours. After the PBS washing step, the asparagus was then incubated in 100 mM CaCl2 at room temperature for 24 hours and washed three times with dH2O. The samples were then sterilized overnight in 70% ethanol. Finally, they were then washed twelve times with sterile 1×PBS. 【0114】 Cellulose scaffolds were prepared by cutting asparagus sections using a 4 mm diameter biopsy punch, and then placed in a 50 ml Falcon tube containing 0.1% sodium dodecyl sulfate (SDS) (Sigma-Aldrich). The samples were shaken at 180 RPM for 72 hours at room temperature. The resulting cellulose scaffolds were then transferred to a new sterile microcentrifuge tube, washed, and incubated in phosphate-buffered saline (PBS) for 12 hours. After the PBS washing step, the asparagus was incubated in 100 mM CaCl2 at room temperature for 24 hours and washed three times with dH2O. The samples were then sterilized overnight in 70% ethanol. Finally, the scaffolds were washed 12 times in sterile saline solution and then incubated overnight in 100 μg / ml poly-L-ornithine solution (30-70 kDa, Sigma-Aldrich, P3655). 【0115】 Scanning electron microscopy: Scanning electron microscopy was performed at 2 weeks. The scaffold on which NSCs were seeded was fixed with 4% PFA and dehydrated via a continuous gradient of ethanol (50%, 70%, 95%, and 100%). The sample was dried in a critical point dryer (SAMDRI-PVT-3D) and then coated with gold using a Hitachi E-1010 ion sputtering device at a current of 15 mA for 3 minutes. SEM imaging was performed using a JSM-7500F Field Emission SEM (JEOL) at voltages in the range of 2.00 to 10.0 kV. 【0116】 Mechanical Inspection: Tensile tests were performed on scaffolds (4 mm in diameter x 1.2 mm in height) placed on a CellScale UniVert (CellScale) compression platform. Each scaffold (n=15) was mechanically compressed to a maximum strain of 10% at a compression speed of 50 μm / s. The modulus of elasticity was determined from the slope of the linear region of the obtained stress-strain curve. 【0117】 Cell culture and seeding onto scaffolds: The obtained cellulose scaffolds were incubated overnight at room temperature in poly-L-ornithine (Sigma, 20 μg / ml in dH2O). The PLO-coated scaffolds were rinsed twice with sterile water and then transferred to 96-well plates. Adult rat hippocampal neural stem cells (Sigma, SCR022) were cultured in serum-free medium (1× knockout D-MEM / F-12 containing 2% StemPro Neural Supplement (ThermoFisher, A1050801), 20 ng / mL bFGF, 20 ng / mL EGF, and 2 mM GlutaMAX-I). The culture vessels were also coated with 20 μg / ml poly-L-ornithine and 10 μg / ml laminin as described above. 80 μL droplets containing 200,000 cells were placed on each scaffold and then incubated at 37°C and 5% CO2. After incubation for 4 hours, 2 mL of StemPro NSC SFM complete medium was added to each scaffold, followed by incubation for 48 hours. The scaffolds were then transferred to a new 96-well plate. The medium was changed once daily for 2–4 weeks. 【0118】 Staining and confocal microscopy: Cell adhesion and morphology were described in detail by phase-contrast microscopy on days 3, 7, and 14 after seeding. Staining and confocal microscopy were performed at 72 hours or 14 days of culture. Scaffolds seeded with NSCs were fixed in warm 4% paraformaldehyde for 10 minutes, then incubated in warm permeabilization buffer (0.5% Triton-X, 20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 0.05% sodium azide) for 3 minutes. The samples were then incubated in fluorescein phalloidin (1:100, ThermoFisher, F432) for 15 minutes to stain for F-actin. The samples were rinsed with PBS and incubated in Hoechst (1:200, ThermoFisher) for 10 minutes to label the nuclei. The scaffold was incubated in 0.2% Congo Red (Sigma) for 15 minutes, followed by a final rinse with PBS. The sample was placed on a Vectashield (Vector Labs) and confocal imaging was performed. 【0119】 Alamar Blue Cell Proliferation Assay: To measure cell proliferation, the Alamar Blue assay was performed according to the manufacturer's protocol. Briefly, PLO-coated asparagus scaffolds were placed in the wells of a 96-well plate, and 100,000 adult rat hippocampal neural stem cells were seeded into each scaffold. After 1, 2, or 5 days, the seeded scaffolds were transferred to new wells, and 200 μL of fresh medium containing 10% Alamar Blue (catalog BUF012A, Bio-Rad) was added to each scaffold. After incubation at 37°C for 4 hours, 100 μL of medium was removed from each well and placed in an empty well, and the absorbance at 570 nm and 600 nm was then read using a spectrophotometer (Epoch 2, BioTek). Each time point included biological triplicates for a cell-seeded scaffold (n=3), a 2D control (NSCs grown in PLO-coated wells, n=3), and a blank (culture medium only, n=3). Absorbance was corrected by subtracting the mean absorbance of the blank at 570 nm and 600 nm. 【0120】 NSC differentiation: For the differentiation of neurons and astrocytes, cells were cultured for 7 days in 1× knockout DMEM / F-12 supplemented with 2% B27 (catalog 17504044, ThermoFisher) and 2 mM GlutaMAX-I (ThermoFisher). 【0121】 Immunostaining for GFAP and β-tubulin: Cells or cell-seeded scaffolds were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Samples were incubated for 5 minutes in permeabilization buffer (0.5% Triton-X, 20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 0.05% sodium azide). After blocking for 10 minutes in 6% normal goat serum in 1×PBS, samples were incubated overnight at 4°C in rabbit anti-GFAP antibody (catalog AB5804, Sigma, 1:1000 in 1×PBS) or mouse anti-β-III tubulin antibody (catalog MAB1195, R&D systems, 10 μg / ml in 1×PBS). The following day, the samples were washed twice in 1×PBS (5 minutes, RT), and then incubated for 2 hours with either a secondary antibody: goat anti-rabbit IgG Alexa Fluor 488 (catalog A11008, Invitrogen, 1:500 in 1×PBS) or goat anti-mouse IgG Alexa Fluor 594 (catalog A11005, Invitrogen, 1:200 in 1×PBS). The samples were then washed in 1×PBS and counterstained with Hoechst 33342 (1:2000 in 1×PBS) for 10 minutes. 【0122】 Image analysis: For βIII tubulin staining, analysis was performed on 13 images at 40x magnification (2D and 3D) for each condition. The total number of cells was determined by counting Hoechst-labeled nuclei using FIJI. Cells were considered βIII tubulin-positive if signals from red and blue channels co-localized. For GFAP staining, analysis was performed on 6 images at 40x magnification (2D and 3D) for each condition. Cells were considered GFAP-positive if signals from green and blue channels co-localized. 【0123】 Animal Care and Environmental Enrichment: All procedures described were approved and followed standards set forth by the Ethics Review Board of the Animal Care and Veterinary Services at the University of Ottawa. Young female Sprague Dolly rats were purchased from Charles River. As a habituation technique to improve welfare, the young rats were tickled. The experimenter trained the rats for four days following Punksep's rat tickling method, which consisted of a total of two minutes of tickling, with a 15-second rest followed by 15 seconds of dorsal contact and restraint. After the tickling training was completed, all rats were tickled before every procedure. To improve coordination in all behavioral assays and to generate positive emotions with the experimenter, the ticklers were trained with young rats. Briefly, the rats were encouraged to stand on a plastic platform, and if successful, they were rewarded with a given food along with a tickle. The ticklers were trained for five days (four minutes daily). The rats were trained to perform a slope test, which involved placing them on a slope and adjusting the incline to determine the maximum angle at which the animals could maintain their position without falling. All rats were housed in pairs throughout the study and played together with other rats of the same species for 20 minutes each day. During group play, up to 10 rats were placed in a 1-meter diameter area equipped with climbing structures, tunnels, foraging toys, foraging enrichments, and nesting materials. Enrichments, including wooden blocks, nylon bones (Bio-Serv, K3580), bunny blocks (Bio-Serv, F05274), and metal swings, were supplied to all rats' home cages.In addition to the standard rat diet, all rats were given a daily feeding enrichment consisting of mini yogurt drops (Cedarlane Bio-Serve, F7577), banana chips (Cedarlane Bio-Serve, F7161), fruit-flavored pellets (Cedarlane Bio-Serve, F6038), ABC fruit blend (Cedarlane Bio-Serve, F7228), mealworms (Cedarlane Bio-Serve, 9264), vegetable-flavored pellets (Cedarlane Bio-Serve, F5158), and pumpkin (ED Smith). 【0124】 Surgical procedure for spinal cord transection: When rats reached a body weight of 250-300g, they were anesthetized with isoflurane USP-PPC and subcutaneously injected with normal saline (Baxter) and enrofloxacin (Baytril). A laminectomy was performed at the T8-T9 level to expose the spinal cord, the dura mater was removed, and the entire spinal cord was gently lifted with a hook and then transected with microscissors. Hemostasis was established using Surgifoam 1972 (Ethicon), and then 10 minutes later, the gap was measured to select a cellulose graft of an appropriate size. Prior to surgery, the animals were divided into four groups: sham group (laminectomy only), untreated group (complete transection only), ASP-treated group (cellulose graft only), and PLO-treated group (cellulose graft coated with PLO). PLO-coated scaffolds were rinsed twice with sterile water before transplantation. During scaffolding implantation, ARTISS fibrin sealant was applied to the space. Muscle and adipose tissue were brought together again with 3-0 Vicryl sutures (Johnson & Johnson), and the skin was closed with Michel clips (Fine Science Tools). Postoperative care was provided to the rats, including manual urination four times daily, pain monitoring and management with buprenorphine HCl as needed, and tracking of weight loss and dehydration. 【0125】 BBB mobility assessment: Hindlimb functional recovery was assessed weekly using a BBB open field assessment. Each rat was placed in a 1-meter diameter area covered with a non-slip floor and recorded by five cameras. Then, for each animal at each time point, a 4-minute video was scored by three blinded observers, and the average score of the three observers was calculated. Spasticity and movement occurring simultaneously with urination were ignored and confirmed by repeatedly reviewing the videos. Two weeks after spinal cord injury, one animal was excluded based on its BBB score exceeding 5. 【0126】 KSAT Swimming Assessment: Prior to SCI, all rats were acclimated to the pool once a day (maximum 5 minutes) for 5 days. During this pre-training, each rat was placed in a clear acrylic swimming tank (20 cm deep, 150 cm long) filled with water (27-30°C) and encouraged to swim through training by a tickler. Before SCI surgery, animals with poor swimming ability were excluded from the study. After the rats underwent SCI, swimming assessments were conducted every two weeks to evaluate functional recovery. Each animal swam across the swimming tank three times, with a 20-second rest between swims. Swimming was recorded by four cameras, and the videos were scored by three blinded observers using the Karolinska Institute Swimming Assessment Tool (KSAT). 【0127】 Retrograde marker of ascending sensory afferent pathways: Animals were also injected bilaterally with 2 μL of cholera toxin subunit B (CTb, ThermoFisher, catalog C34778, 1% solution in sterile PBS) conjugated to Alexa Fluor 647 (4 μL total per rat). Rats were anesthetized with 3% isoflurane, a linear incision was made along the femur, and the gluteus maximus was separated from the gluteus medius to expose the sciatic nerve. Using a surgical microscope, a wound was made in the proximal portion of the exposed nerve. The CTb solution was loaded into a Hamilton syringe, and the needle was inserted 5 mm into the sciatic nerve. CTb was injected in 0.5 μL increments, with a 30-second wait, and the needle withdrawn 1 mm after each injection. After injection was complete, all CTb discharged from the nerve was removed using a sterile Q-tip, and the incision was sutured with 4-0 Prolene sutures. Postoperatively, 2% bupivacaine was applied topically, and buprenorphine (0.05 mg / kg subcutaneously) and gappentin (Chiron, 50 mg / kg subcutaneously) were administered to the animals for analgesia. Pain was assessed using the TID, and additional buprenorphine was administered as needed. To prevent infection, enrofloxacin (Bayer, 10 mg / kg subcutaneously) was administered once daily for three days before and after surgery. 【0128】 Anterior markers of the corticospinal tract: Eleven weeks after surgery, each group of rats was injected with a neural tracer to visualize axons within and around the injury site. Animals were anesthetized with 3% isoflurane and placed in a stereotactic frame. After surgical handwashing, a sagittal incision was made to expose the apex of the skull, and the periosteum was scraped away. Using a Zeiss surgical microscope, the skull was leveled by measuring the dorsal-ventral axes at four random points, ensuring that these were within 0.05 mm of each other. Bregma was located, and its coordinates were used to calculate the target position for each injection. Eight holes were drilled into the skull using a surgical drill (Micro Drill, Harvard Apparatus) mounted on the stereotactic frame. Stereotactic injections were administered to the right and left motor cortex at the following coordinates: Injection 1: Anterior-posterior (AP) -0.5 mm, Medial-lateral (ML) ±2 mm, Dorsal-ventral (DV) 1.5 mm. Injection 2: Anterior-posterior (AP) -1 mm, Medial-lateral (ML) ±2.5 mm, Dorsal-ventral (DV) 1.5 mm. Injection 3: Anterior-posterior (AP) -1.5 mm, Medial-lateral (ML) ±2 mm, Dorsal-ventral (DV) 1.5 mm. Injection 4: Anterior-posterior (AP) -2 mm, Medial-lateral (ML) ±2.5 mm, Dorsal-ventral (DV) 1.5 mm. Injections were administered using a 5 μL Hamilton syringe. Two minutes later, 0.5 μL of 10% dextranamine Alexa Fluor 488 (ThermoFisher, catalog D22910, 10000 MW) was injected at each site at a rate of 250 nl / min, for a total injection volume of 4 μL per rat. A two-minute wait was allowed after each injection to ensure diffusion into the tissue. Once all eight injections were completed, the scalp was sutured with 4-0 Prolene sutures, and 2% transdermal bupivacaine was applied to the incision. The animals were given a two-week recovery period before euthanasia to transport the nerve tracers. For tissue retrieval, the animals were deeply anesthetized with isoflurane USP-PPC and euthanized by cardiac perfusion with 500 ml of 1×PBS followed by 500 ml of 4% paraformaldehyde.The brain and spinal cord were dissected and removed, fixed overnight with 4% paraformaldehyde at 4°C, and then stored in 70% ethanol at 4°C until embedding and sectioning. 【0129】 Quantitative analysis of neuroanatomical neural pathway tracing: Cross-sections of spinal cord tissue above and below the injury site were imaged using a confocal laser scanning microscope to confirm successful tracer uptake. In retrograde tracing, axons traced on CTb were observed in the vertebral column in the T10 section. In anterograde tracing, dextranamine-labeled axons were identified within the corticospinal tract in the T6 section. In each animal, three sagittal sections from the center of the spinal cord were imaged using a confocal laser scanning microscope, and the distance between the tracer and the injury site was measured using FIJI. In retrograde tracing on CTb (n=4 PLO animals, n=2 ASP animals, n=3 untreated animals), the distance (mm) between the axon traced on the most distal rostral CTb and the injury site was measured. In anterograde tracing with dextranamine (n=4 PLO animals, n=4 ASP animals, n=3 untreated animals), the distance (mm) between the axon traced with the most distal caudal dextranamine and the site of injury was measured. The mean was calculated for each group, and a one-way ANOVA was performed. 【0130】 GFAP immunostaining: Paraformaldehyde-fixed, paraffin-embedded tissue sections (5 μm thick) were deparaffinized and pre-treated using heat-mediated antigen retrieval in sodium citrate buffer (pH 6.0). The slides were then rehydrated in 1× TBST buffer and blocked on Rodent Block R (Biocare, RBR962H) for 30 minutes. The sections were then incubated with rabbit GFAP (1:3000, Sigma, AB5804) at room temperature for 1.5 hours. The sections were washed with 1× TBST and incubated with goat anti-rabbit-488 antibody for 2 hours in the dark at room temperature. These were then incubated with a quencher (Vector TrueView Autofluorescence Quenching Kit #SP-8400, Vector Labs) to reduce autofluorescence. The sections were then washed, incubated with 5 ug / ml DAPI (Thermo Scientific, #62248), and then covered with coverslips. 【0131】 Immunostaining of β-III tubulin: Paraformaldehyde-fixed, paraffin-embedded tissue sections (5 μm thick) were deparaffinized, and the tissues were permeabilized at room temperature with the following buffers: 0.5% Triton-X, 20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, and 0.05% sodium azide (for 5 minutes). The sections were incubated in block buffer (6% normal goat serum in 1 × PBS) for 10 minutes. The sections were rinsed twice in 1 × PBS and then incubated overnight at 4°C with mouse anti-β-III tubulin antibody (10 ug / ml, MAB1195, R&D Systems). The following day, after washing twice with 1×PBS, the sections were incubated at room temperature in goat anti-mouse alexa Fluor 594 polyclonal antibody (1:200, A11005, Thermofisher) for 2.5 hours, and then counterstained with Hoechst (1:2000). 【0132】 Immunostaining of Neurofilament 200: Paraformaldehyde-fixed, paraffin-embedded tissue sections (5 μm thick) were deparaffinized, and the tissue was permeabilized at room temperature in 1 × TBST (3 × 5 minutes). The sections were incubated in block buffer (5% normal goat serum in 1 × TBST) for 30 minutes, and then washed three times in 1 × TBST. The tissue was incubated overnight at 4°C in rabbit anti-NF200 (1:3000 dilution in 1 × PBS, catalog N4142, Sigma). The following day, after washing twice in 1 × PBS, the sections were incubated at room temperature in goat anti-rabbit alexa fluor 488 (catalog A11008, Thermofisher) for 2 hours, and then counterstained with Hoechst (1:2000). 【0133】 Statistical analysis: P-values ​​were calculated using a two-tailed Student's t-test. A P-value less than 0.01 was considered statistically significant. Numerical data are expressed as mean ± standard deviation. Experimental data were analyzed using GraphPad Prism. Differences between two groups were compared using t-tests, while differences between multiple groups were compared using ANOVA. 【0134】 As those skilled in the art will understand, only one or more exemplary embodiments have been described. 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Claims

[Claim 1] Cellulose scaffolds based on vascular plants for in vitro production of neural stem cells. [Claim 2] The scaffold according to claim 1, obtained by decellularizing a vascular plant or a part thereof. [Claim 3] The scaffold according to claim 2, which supports the proliferation of neural stem cells. [Claim 4] The scaffold according to claim 2 or 3, wherein the vascular plant is asparagus or celery. [Claim 5] The scaffold according to claim 1, which is coated with a biomolecule, synthetic biomolecule, ligand, protein, amino acid, antibody, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-l-lysine, low molecular weight, hydrogel, or Matrigel. [Claim 6] The scaffold according to claim 1, which is coated with synthetic biomolecules. [Claim 7] The scaffolding according to claim 1, which is coated with poly-L-ornithine (PLO). [Claim 8] The scaffold according to claim 1, having a porous structure comprising multiple vascular bundles of various diameters and multiple parenchyma cells. [Claim 9] The scaffold according to claim 8, wherein vascular bundles are scattered among parenchyma cells. [Claim 10] The scaffolding according to claim 8, wherein the vascular bundles are unevenly spaced and extend across the length of the scaffolding. [Claim 11] The scaffold according to claim 8, wherein parenchyma cells form the surface of the scaffold. [Claim 12] The scaffold according to claim 8, wherein the porous structure of the scaffold promotes the attachment, proliferation, or differentiation of neural stem cells. [Claim 13] The scaffold according to claim 8, wherein the porous structure of the scaffold allows neural stem cells to maintain their differentiation ability. [Claim 14] The scaffold according to claim 8, wherein the porous structure of the scaffold promotes the differentiation of neural stem cells into neurons, dopaminergic neurons, motor neurons, astrocytes, oligodendrocytes, or combinations thereof. [Claim 15] The scaffold according to claim 8, wherein the porous structure of the scaffold promotes the differentiation of neural stem cells into neurons and astrocytes. [Claim 16] The scaffold according to claim 7, wherein the poly-L-ornithine coating increases the production of neural stem cells by promoting the formation of filopodia. [Claim 17] The scaffold according to claim 8, wherein the porous structure of the scaffold promotes increased production of neural stem cells by enabling nutrient exchange, oxygen exchange, and removal of waste within the scaffold. [Claim 18] The scaffolding according to claim 1, wherein the porosity of the scaffolding is in the range of 10% to 95%. [Claim 19] The scaffolding according to claim 1, having an elastic modulus in the range of 1 kPa to 1000 kPa. [Claim 20] The scaffold according to claim 1, which is biocompatible. [Claim 21] The scaffolding according to claim 1, which is biodegradable. [Claim 22] The scaffolding according to claim 1, wherein the surface of the scaffolding is modified by a chemical or physical treatment. [Claim 23] The scaffold according to claim 22, wherein the modification enhances the attachment of neural stem cells to the surface of the scaffold. [Claim 24] The scaffolding according to claim 22, wherein the surface of the scaffolding is treated by surface functionalization, gamma radiation irradiation, radical treatment, oxidation treatment, or a combination thereof. [Claim 25] The scaffold according to claim 24, wherein the functionalization is achieved by providing a functional group that generates an electric charge on the surface of the scaffold. [Claim 26] The scaffold according to claim 25, wherein the functional group is a primary amine, a tertiary amine, a quaternary compound, an alcohol group, a carboxylic acid group, an aldehyde group, a sulfonyl group, or a combination thereof. [Claim 27] The scaffold according to claim 1, which promotes the three-dimensional proliferation of neural stem cells as neurospheres on the scaffold. [Claim 28] The scaffold according to claim 1, which promotes the growth of neurite-like projections by cells. [Claim 29] The scaffold according to claim 1, which promotes the growth of multiple neural stem cell layers. [Claim 30] The scaffold according to claim 1, which promotes the production of nerve cell proteins. [Claim 31] A scaffold according to any one of claims 1 to 30, comprising a plurality of linearly oriented channels. [Claim 32] The scaffold according to claim 31, which promotes the proliferation of neurons in a plurality of linearly oriented channels, preferably the proliferation of endogenous neurons. [Claim 33] The scaffold according to claim 30, which promotes the growth and / or regeneration of neuronal sensory pathways. [Claim 34] A scaffold based on a vascular plant according to any one of claims 1 to 33, for use in regenerative medicine or the production of biopharmaceuticals. [Claim 35] A scaffold based on a vascular plant according to any one of claims 1 to 33, for use in neurotissue engineering. [Claim 36] A scaffold based on a vascular plant according to any one of claims 1 to 33, for use in the treatment of nerve disorders, abnormalities, or injuries. [Claim 37] A vascular plant-based scaffold according to any one of claims 1 to 33, for use in in vitro drug production or in vitro drug testing. [Claim 38] A vascular plant-based scaffold according to any one of claims 1 to 33, for use in the in vitro production or in vitro collection of neuronal proteins. [Claim 39] A vascular plant-based scaffold according to any one of claims 1 to 33, for use in the production of nerve grafts or nerve cell banks. [Claim 40] A vascular plant-based scaffold according to any one of claims 1 to 33, for use in the production of neural constructs for disease modeling in vitro. [Claim 41] A scaffold based on a vascular plant according to any one of claims 1 to 33, preferably for use in the treatment of spinal cord injury, in promoting motor recovery of the hind limbs and / or repair of nerve tissue in a subject having a spinal cord injury. [Claim 42] a) Seeding a culture medium containing neural stem cells onto a cellulose scaffold based on a vascular plant according to any one of claims 1 to 33 in a container; and b) Allowing the neural stem cells to attach to and proliferate on the cellulose scaffold for at least 48 hours; A method for in vitro production of neural stem cells, including the method described above. [Claim 43] The method according to claim 42, wherein nutrients and growth factors are injected into the culture medium to facilitate or increase the attachment and proliferation of neural stem cells to the scaffold. [Claim 44] The method according to claim 42, wherein the culture medium is DMEM MEM / F12, Aplha MEM, knockout DMEM / F12, FBS, FCS, goat serum, or horse serum. [Claim 45] The method according to claim 42, wherein the culture medium is A: 1× knockout DMEM / F-12 supplemented with 2% B27 (catalog 17504044, ThermoFisher), 2 mM GlutaMAX-I (ThermoFisher), and B: serum-free medium (1× knockout D-MEM / F-12) containing 2% StemPro Neural Supplement (ThermoFisher, A1050801), 20 ng / mL bFGF, 20 ng / mL EGF, or 2 mM GlutaMAX-I. [Claim 46] The method according to claim 42, wherein the method is carried out at a temperature in the range of 32°C to 42°C. [Claim 47] The method according to claim 42, wherein the procedure is carried out at a pH in the range of 6.8 to 7.

2. [Claim 48] The method according to claim 42, wherein the container is a flask, a culture vessel, a bioreactor, a petri dish, a multiwell plate, or a glass chamber. [Claim 49] The method according to claim 42, further comprising the step of coating the container with a natural biomolecule, synthetic biomolecule, ligand, protein, amino acid, antibody, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-l-lysine, low molecular weight, hydrogel, or Matrigel before step a). [Claim 50] The method according to claim 49, wherein the container is coated with natural biomolecules and synthetic biomolecules. [Claim 51] The method according to claim 49, wherein the container is coated with poly-L-ornithine and laminin. [Claim 52] Step a) further includes a pre-processing step, The scaffolding Sterilize the scaffold with 70% ethanol. Wash the scaffold with physiological saline solution, surfactant solution, physiological buffer, or salt solution, or The surface of the scaffolding is treated in advance. The method according to claim 42, wherein the method is pre-treated by [Claim 53] The method according to claim 52, wherein the washing step includes an incubation step of incubating the scaffold overnight in a salt solution. [Claim 54] The method according to claim 52, wherein the surface is pre-treated by chemical treatment, physical treatment, functionalization, succinylation, or a combination thereof. [Claim 55] The method according to claim 54, wherein the surface is pre-treated by treatment with a biomolecule, synthetic biomolecule, ligand, protein, amino acid, antibody, extracellular matrix, fibrin, laminin, collagen, fibronectin, poly-l-lysine, low molecular weight, hydrogel, Matrigel, or pharmaceutically acceptable compound. [Claim 56] The method according to claim 54, wherein the synthetic biomolecule for chemical treatment is poly-L-ornithine. [Claim 57] The method according to claim 54, wherein the pretreatment enhances cell production by modifying the surface of the scaffold and increasing adhesion to the surface of the scaffold. [Claim 58] The method according to claim 54, wherein the surface is functionalized by providing functional groups that generate an electric charge on the surface of the scaffold. [Claim 59] The scaffold according to claim 58, wherein the functional group is a primary amine, a tertiary amine, a quaternary compound, an alcohol group, a carboxylic acid, an aldehyde, a sulfonyl, or a combination thereof. [Claim 60] The method according to claim 42, further comprising a sterilization step prior to step a) wherein the scaffold is sterilized over a predetermined period of time. [Claim 61] The method according to claim 60, wherein the sterilization step is carried out by autoclaving, treatment with 70% ethanol, gamma radiation, or treatment with ethylene oxide. [Claim 62] The method according to claim 60, wherein the sterilization step is performed for 20 minutes to several days. [Claim 63] The method according to claim 42, wherein the production of neural stem cells is increased by modulating the rigidity of the scaffold. [Claim 64] The method according to claim 42, wherein the production of neural stem cells is increased by modulating the anisotropy of the scaffold. [Claim 65] The method according to claim 52, further comprising a quantitative step for quantifying the production of neural stem cells. [Claim 66] The method according to claim 65, wherein the quantification step is performed by a biomolecular quantification assay, a cell quantification assay, a cell viability assay, image analysis, immunohistochemistry, or electrophysiology. [Claim 67] The method according to claim 66, wherein, after the quantitative step, an optimization step is performed to optimize the growth conditions in the container, to optimize the amount of scaffold, to optimize the size of the scaffold, to optimize the structure of the scaffold, or a combination thereof. [Claim 68] The method according to claim 42, further comprising an incubation step of incubating the scaffold and neural stem cells at a temperature in the range of 30°C to 45°C. [Claim 69] The scaffold and neural stem cells are CO ２ The method according to claim 68, wherein the body is incubated with the body. [Claim 70] The scaffold and neural stem cells are in 5% CO2 at 37°C. ２ The method according to claim 69, wherein the body is incubated with the body. [Claim 71] The method according to claim 42, wherein neural stem cells, after attaching to a scaffold, differentiate into neurons, astrocytes, oligodendrocytes, or a combination thereof. [Claim 72] The method according to claim 42, wherein neural stem cells differentiate into neurons and astrocytes after attaching to a scaffold. [Claim 73] The method according to claims 71 and 72, wherein the differentiation of neural stem cells is determined by immunohistochemical assay, cell imaging, confocal microscopy, flow cytometry, or electrophysiology. [Claim 74] The method according to claim 56, wherein the poly-L-ornithine coating increases the production of neural stem cells by promoting the formation of filopodia.

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