Method for producing peripheral nerve-like microtissue and its use

Abstract

The present invention relates to a method for producing a peripheral nerve-like microtissue and its use, which is a method for producing a peripheral nerve-like microtissue having a diameter of 100±20 μm, which is composed of 100 to 500 cells by isolating and culturing adult stem cells (PNSCs) derived from peripheral nerves, and forming cell-cell and cell-extracellular matrix bonds through suspension culture of the isolated and cultured PNSC cells. The microtissue produced by culturing in a suspension culture environment has a structural characteristic in which 100 to 500 cells are aggregated through cell-cell bonds by β-catenin, extracellular matrix (ECM) produced and secreted from PNSCs between the cells is accumulated, and the accumulated ECM is bound to the cells by β1-integrin. This is similar to the peripheral nerve structure and constituent cells that are regenerated after injury. The peripheral nerve-like microtissue can secrete neuroactive factors that act centrally on nerve regeneration to induce nerve tissue regeneration.

Description

[Technical Field]

[0001] The present invention relates to a method for producing peripheral nerve-mimicking microtissues and their uses. [Background technology]

[0002] Damage to nerve tissue is irreversible, leading to complications such as the permanent loss of motor, sensory, and autonomic nervous functions. As a result, nerve damage causes serious social, economic, and medical problems. Spinal cord injury, one of the most common nerve injury diseases, is estimated to affect approximately 200,000 patients in the United States, with approximately 10,000 new cases occurring each year. Depending on the severity, treatment costs can run into the billions of won per patient, with annual costs reaching approximately 200 million won, making it a representative intractable disease with high economic and social unmet needs. In South Korea, a survey found that there are approximately 70,000 patients with permanent spinal cord injury.

[0003] Nerve injury can be classified as primary or secondary. Primary injury occurs when nerve tissue and blood vessels are damaged by physical compression caused by trauma. Following primary injury, secondary injury occurs due to subsequent inflammatory responses, free oxygen, free radicals, ischemia and hypoxia, edema, and cell death. The only treatments available after injury include daily high-dose corticosteroids, surgical removal of damaged tissue, hematomas, and bones compressing nerve tissue, and spinal fusion. Currently, there are no therapeutic agents that can prevent or mitigate secondary injury and regenerate damaged nerves after primary injury.

[0004] Stem cell therapy is a biopharmaceutical that can be used for intractable diseases such as nerve injury that cannot be treated with conventional therapeutic agents. Stem cell therapy has been shown to alleviate and suppress secondary damage that occurs after nerve injury, and has been shown to mediate regenerative effects after nerve injury through indirect mechanisms of action, such as anti-inflammation, immune response regulation, anti-radical, cytoprotection, angiogenesis induction, and neuroactive secretion, as well as through direct mechanisms of action, such as differentiating into neurons or oligodendrocytes and promoting the growth and production of nerve axons and myelin.

[0005] Stem cell therapy for nerve regeneration has been proposed to improve regeneration by secreting neuroactive factors from transplanted cells. Representative neuroactive factors (NFs) that play a major role in nerve regeneration include brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). Neuroactive factors activate the endogenous tissue regeneration mechanism of surrounding cells after nerve injury. After transplantation, neuroactive factors secreted from the engrafted cell therapy bind to Trk receptors or p75 receptors around the injury. NTR It has been suggested that BDNF and NGF bind to receptor-positive cells to promote neuronal survival and axon formation, activating endogenous neuronal tissue homeostasis mechanisms and acting on regeneration. BDNF and NGF have been reported to primarily act on the cytoprotection and axon formation of rubospinal neurons and adrenergic / sensory neurons.

[0006] Stem cells used as therapeutic agents for nerve injury are mostly mesenchymal stem cells (MSCs) derived from bone marrow (BM), umbilical cord (UC), and adipose tissue (AT). Preclinical and clinical studies are currently being conducted on neural stem cells (NSCs) derived from the central nervous system, and NSCs or oligodendrocyte progenitor cells (OPCs) derived from embryonic stem cells or induced pluripotent stem cells (ISTS). Skin- or hair-follicle-derived neural crest stem cells (SCs) are stem cells present in peripheral nervous system tissues and can differentiate into Schwann cells, neurons, and mesenchymal cells. These cells share similar properties to neural crest-derived cells, which exist temporarily during development, and are therefore used as a cell source for regenerative therapies after nerve injury. However, to maximize the nerve regenerative function of stem cell therapeutic agents, techniques are needed to increase gene expression and protein secretion of neuroactive factors.

[0007] Although artificial gene transfer can increase neural activity gene expression and protein secretion in stem cells, safety concerns due to artificial manipulation have hindered clinical application. However, combining 3D culture technology with 3D culture technology to provide cells with a 3D environment can enhance endogenous gene function. 3D culture provides a 3D environment similar to that found in the human body, enabling cell-cell and cell-ECM (extracellular matrix) interactions, resulting in increased intracellular gene and protein expression and production. Porous scaffolds or hydrogels are widely used to create 3D environments. However, when 3D structures are constructed by seeding cells into such biomaterials, co-transplantation of such structures and cells can potentially raise regulatory issues. Therefore, a technology that allows cells to form 3D structures without the support of scaffolds or hydrogels is needed. Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to provide a method for producing peripheral nerve-like microtissues capable of inducing nerve regeneration by culturing adult peripheral nerve-derived stem cells (PNSCs) in a suspension culture environment, where approximately 100 to 500 cells form cell-cell and cell-ECM bonds via β-catenin and integrin-β1, accumulating peripheral nerve-specific ECM produced and secreted from PNSCs in the intercellular space, and secreting neuroactive factors produced and secreted from PNSCs, such as one or more neurotrophic family factors selected from the group consisting of BDNF, NGF, neutrophin-3, and neurotrophin-4; ephrin family factors; one or more GDNF family factors selected from the group consisting of GDNF and artemin; or one or more CNTF family factors selected from the group consisting of IL-6, CNTF, and LIF.

[0009] Another object of the present invention is to provide a peripheral nerve-like microtissue, which is a spherical cell structure formed by bonding 100 to 500 PNSCs together and cultured in suspension in a culture medium containing human serum albumin (HSA), dexamethasone (DEX), and N-acetylcysteine ​​(NAC), and has a diameter of 100±20 μm, and is composed of cell-cell junctions of PNSCs and junctions between PNSCs and extracellular matrix (ECM).

[0010] It is yet another object of the present invention to provide a pharmaceutical composition for treating nerve damage and diseases, which contains the peripheral nerve-like microtissue as an active ingredient.

[0011] A further object of the present invention is to provide a pharmaceutical composition for treating neuroinflammatory diseases, which comprises the peripheral nerve-like microtissue as an active ingredient. [Means for solving the problem]

[0012] To achieve the above objectives, the present invention provides a method for producing peripheral nerve-like microtissues, which includes: 1) culturing peripheral nerve-derived stem cells (PNSCs) in a monolayer; and 2) recovering the monolayer-cultured PNSCs and subjecting them to suspension culture in a culture medium containing human serum albumin (HSA), dexamethasone (DEX), and N-acetylcysteine ​​(NAC).

[0013] The present invention also provides a peripheral nerve-like microtissue consisting of 100 to 500 PNSCs bound together, which are cultured in suspension in a culture medium containing HSA, DEX, and NAC, and which has a diameter of 100±20 μm and is composed of cell-cell junctions of PNSCs and junctions between PNSCs and the extracellular matrix (ECM).

[0014] The present invention also provides a pharmaceutical composition for treating nerve damage, which comprises the peripheral nerve-like microtissue as an active ingredient.

[0015] The present invention also provides a pharmaceutical composition for treating neuroinflammatory diseases, which comprises the peripheral nerve-like microtissue as an active ingredient. [Effects of the Invention]

[0016] The present invention relates to a method for producing a peripheral nerve-like microtissue and its use, specifically a method for producing a peripheral nerve-like microtissue with a diameter of 100±20 μm, which is composed of approximately 100 to 500 cells, by isolating and culturing peripheral nerve-derived adult stem cells (PNSCs) and then forming cell-cell and cell-extracellular matrix junctions through suspension culture of the isolated and cultured PNSCs. The microtissue produced by culturing in a suspension culture environment has the structural characteristic of approximately 100 to 500 cells aggregating through cell-cell junctions mediated by β-catenin, accumulating extracellular matrix (ECM) produced and secreted by PNSCs between the cells, and binding the accumulated ECM to the cells via β1-integrin. The cells that make up the fibroblasts are composed of stem cells derived from immature peripheral nerves, as well as Schwann progenitor cells, repair Schwann cells, myelinating Schwann cells, and interstitial stromal cells. This is similar to the structure and cells of peripheral nerves that regenerate after injury. Functionally, the fibroblasts have a peripheral nerve-like microstructure and are able to induce nerve tissue regeneration by secreting neuroactive factors that act centrally on nerve regeneration. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a diagram showing a method for producing peripheral nerve-like microtissues for nerve regeneration using peripheral nerve-derived adult stem cells (PNSCs). [Figure 2] 1 is a diagram showing the correlation between the size and the number of PNSC cells that compose peripheral nerve-like microtissues. [Figure 3] 1 is a diagram showing a method for controlling the frequency and size of microtissue formation depending on the concentration of human serum albumin (HSA) added to a suspension culture medium. [Figure 4] 1 is a diagram showing a method for controlling the size of peripheral nerve-like microtissues by adjusting the seeding density of PNSC cells. [Figure 5]This is a graph showing that the accumulation of ROS (radical oxygen species) in micro-tissues increases proportionally with size. [Figure 6] 1 is a graph showing cell viability depending on the size of microtissues. [Figure 7] 1 is a graph showing the protective effect of NAC (N-acetyl cysteine) and DEX (dexamethasone) on cells constituting fine tissues. [Figure 8] 1 is a diagram showing the mechanism by which NAC and DEX protect cells constituting microtissues. [Figure 9] 1 is a diagram showing the structural characteristics of peripheral nerve-like microtissues produced in a culture environment for industrial-level production. [Figure 10] 1 is a diagram showing the effect of strengthening the Wnt signaling pathway in peripheral nerve-like microtissues. [Figure 11] 1 is a diagram showing the characteristics of cells constituting peripheral nerve-like microtissues produced in a culture environment for industrial-level production. [Figure 12] 1 is a diagram showing the neuroactive gene expression characteristics of peripheral nerve-like microtissues produced in a culture environment for industrial-level production. [Figure 13] 1 shows the results of evaluating cell damage caused by ROS in peripheral nerve-like microtissues. [Figure 14] 1 shows the results of comparing the expression rates of cell death regulators in peripheral nerve-like microtissues. [Figure 15] 1 shows the results of confirming significantly higher cell viability and lower annexin V expression in microtissues compared to PNSCs. [Figure 16] 1 shows the results of confirming that the neuroactive protein secretion ability of PNSCs is enhanced through the formation of microtissues. [Figure 17] 1 shows the results of comparative evaluation of neural activity, anti-inflammatory, and angiogenesis-inducing potency through cell-based analysis using conditioned media obtained from microtissues and PNSCs. [Figure 18]1 shows the results of comparative evaluation of neural activity, anti-inflammatory, and angiogenesis-inducing potency through cell-based analysis using conditioned media obtained from microtissues and PNSCs. [Figure 19] 1 shows the results of comparative evaluation of neural activity, anti-inflammatory, and angiogenesis-inducing potency through cell-based analysis using conditioned media obtained from microtissues and PNSCs. [Figure 20] 1 shows the results of comparative evaluation of neural activity, anti-inflammatory, and angiogenesis-inducing potency through cell-based analysis using conditioned media obtained from microtissues and PNSCs. [Figure 21] 1 shows the results of evaluating the survival rate and efficacy of peripheral nerve-like tissue after in vivo transplantation. [Figure 22] 1 shows the results of assessing the content of neuroactive factors in the spinal cord one week after administration of PNSCs or microtissues. [Figure 23] 1 shows the results of confirming myelination and axon growth through administration of PNSCs or microtissues compared to animals not administered. [Figure 24] 1 shows the results that myelination and axon growth are significantly higher when microtissues are administered compared to PNSCs. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method for producing peripheral nerve-like microtissues, which includes the steps of: 1) culturing peripheral nerve-derived stem cells (PNSCs) in a monolayer; and 2) recovering the monolayer-cultured PNSCs and culturing them in suspension in a culture medium containing human serum albumin (HSA), dexamethasone (DEX), and N-acetylcysteine ​​(NAC).

[0019] Preferably, in the step of carrying out the suspension culture, the culture vessel cm 2 0.25 to 2.5 x 10 per area 5 PNSCs can be seeded, but are not limited to this.

[0020] Preferably, the culture medium may contain 0.01 to 1% HSA, 0.1 to 5 μM DEX, and 0.1 to 10 mM NAC, but is not limited thereto.

[0021] Preferably, the suspension culture can induce cell-cell junctions of the PNSCs.

[0022] Preferably, the peripheral nerve-like microtissue is a spherical cell structure in which 100 to 500 PNSCs are bound together, and has a diameter of 100±20 μm, but is not limited thereto.

[0023] The present invention provides a peripheral nerve-like microtissue consisting of 100 to 500 PNSCs bound together, which are cultured in suspension in a culture medium containing HSA, DEX, and NAC, and which has a diameter of 100±20 μm and is composed of cell-cell junctions between PNSCs and PNSCs-extracellular matrix (ECM) junctions.

[0024] Preferably, the peripheral nerve-like microtissue is produced and secreted by PNSCs, and has a structure in which collagen type-VI and laminin accumulate in the intercellular matrix, with cell-extracellular matrix binding mediated by CD29 and cell-cell binding mediated by β-catenin, but is not limited thereto.

[0025] Preferably, the peripheral nerve-like microtissue is composed of peripheral nerve-derived adult stem cells, Schwann progenitor cells, repair Schwann cells, myelin Schwann cells, and mesenchymal stromal cells, and more preferably, GFAP- / S100β- / Sox10+ undifferentiated neural crest cells, GFAP+ / S100β+ / Myelin+ myelin-positive Schwann cells, GFAP+ / GAP43+ / Myelin- myelin-negative Schwann progenitor cells, and GFAP- / CD140a+ stromal cells, but is not limited thereto.

[0026] Preferably, the peripheral nerve-like microtissue has an activated Wnt / β-catenin signaling pathway.

[0027] Preferably, the peripheral nerve-like microtissue comprises any one or more neuroactive factors selected from the group consisting of BDNF, EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNB1, EFNB2, EFNB3, CTNF, GDNF, LIF, NGFB, NTF3, NTF5, NRG1, NRG2, NRG3, NRG4, and ZFP91; any one or more growth factors selected from the group consisting of EGF, FGF1, FGF2, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF18, FGF19, FGF20, FGF23, IGF1, and GAS6; CLC, CTF1, CSF1, CSF2, CSF3, GH1, GH2, FLT3LG , IDO1, IL2, IL3, IL5, IL7, IL9, IL10, IL11, IL12A, IL12B, IL15, IL20, IL21, IL22, IL23A, IL24, IL26, IL28A, IL29, IFNA1, IFNB1, IFNW1, IFNK, IFNE1, IFNG, KITLG, LEP, PRL, TGFB, TPO, and TSLP; or increased expression of any one or more angiogenesis-inducing factors selected from the group consisting of ANGPT1, ANGPT2, ANGPT4, EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNB3, EPO, PDGFC, PDGFD, VEGFA, VEGFB, and VEGFC.

[0028] The present invention also provides a pharmaceutical composition for treating nerve damage, which comprises the peripheral nerve-like microtissue as an active ingredient.

[0029] Preferably, the pharmaceutical composition can promote the regeneration of nerve tissue, but is not limited thereto.

[0030] The present invention also provides a pharmaceutical composition for treating neuroinflammatory diseases, which comprises the peripheral nerve-like microtissue as an active ingredient.

[0031] More specifically, the peripheral nerve-like 3D microtissue of the present invention is characterized by the following composition, structure, and biological components, providing evidence that it can be used as a nerve regeneration therapeutic agent.

[0032] (1) The peripheral nerve-like microtissue is a spherical structure composed of approximately 100 to 500 PNSCs cells, and is a cell structure with a diameter of 100±20 μm.

[0033] (2) Peripheral nerve-like microtissues are composed of peripheral nerve-derived adult stem cells, Schwann progenitor cells, reparative Schwann cells, myelin Schwann cells, and mesenchymal stromal cells.

[0034] (3) The peripheral nerve-like microtissue is composed of peripheral nerve-specific extracellular matrices, laminin and collagen type-IV, which are produced and secreted from PNSCs within the interstitium, and exhibits structural properties in which structural stability is enhanced through cell-cell and cell-ECM junctions by β-catenin and integrin-β1.

[0035] (4) Peripheral nerve-like microtissues possess biological properties such as enhanced structural stability and increased secretion of neuroactive factors from their constituent cells. Peripheral nerve-like microtissues create a microenvironment that facilitates cell-cell and cell-extracellular matrix interactions, activating the Wnt / β-catenin and Integrin-β1 / FAK signaling pathways and inducing increased expression of downstream target genes. As a result, compared to PNSCs, peripheral nerve-like microtissues accumulate peripheral nerve-specific ECM and exhibit increased mRNA and protein secretion of neuroactive factors produced and secreted by PNSCs, including Artemin, BDNF, CNTF, GDNF, IGF, IL-6, NGF, and NT-3.

[0036] (5) The peripheral nerve-like microtissues can activate the nerve regeneration mechanism through the secretion of neuroactive factors, including the active ingredients Artemin, BDNF, CNTF, GDNF, IGF, NGF, and NT-3, and can induce mechanisms that promote nerve cell and axon regeneration and myelin production within the injured area.

[0037] (6) Compared with the final PNSCs, the peripheral nerve-like microtissues have enhanced neuroactive gene expression and protein secretion, which can enhance the neural regeneration biological mechanism of PNSCs and increase the neural regeneration effect compared with PNSCs.

[0038] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0039] Example 1 This example provides a method for controlling the size of microtissues by adjusting the number of PNSC cells that compose peripheral nerve-like microtissues. PNSCs harvested in a monolayer culture environment are suspended in suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1% human serum albumin (HSA, green cross), 1 μM dexamethasone, and 1 mM N-acetylcysteine ​​to DMEM / F12 culture medium. An ultra-low attachment (ULA) culture vessel is used to prevent the seeded PNSCs from attaching to the culture vessel and induce cell-cell junctions. PNSCs suspended in suspension culture medium were seeded at 100, 200, 500, 750, 1000, 2500, and 5000 cells per well of a 96-well ULA culture vessel (SPL Life Sciences, Seoul, Korea), and then centrifuged at 500 × g for 10 minutes to collect the cells in the center of the vessel. Photographs were taken on days 1, 2, and 3 of culture, and the diameter of the microtissues was measured using photoplanometry with ImageJ (NIH, Bethesda, MD).

[0040] As shown in Figure 2(A), PNSCs seeded in a ULA 96-well culture vessel did not adhere to the surface of the culture vessel. Cell-cell aggregation and junctions were formed within 2 hours of culture, and the morphology of the microtissue formed during the preculture period was maintained. It can be seen that the size of the peripheral nerve-like microtissue increases proportionally with the number of cells constituting it. As shown in Figure 2(B), the correlation coefficient (R) between the size of the microtissue and the number of PNSCs constituting it was 0.99, suggesting a method for controlling the size of peripheral nerve-like microtissues by adjusting the number of cells seeded. However, the size of the microtissues formed did not change with the culture period.

[0041] <Example 2> This example provides a method for controlling the formation, number, and size of peripheral nerve-like microtissues by adjusting the concentration of human serum albumin (HSA) added to the suspension culture medium. PNSCs collected in a monolayer culture environment are suspended in suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1 μM dexamethasone and 1 mM N-acetylcysteine ​​to DMEM / F12 culture medium. To prepare peripheral nerve-like microtissues, a ULA 6-well culture vessel (SPL Life Sciences) was used. The ULA 6-well culture vessel contained 1 cm 2 1.0 x 10 per area 5 PNSCs were seeded in a culture vessel and cultured in suspension for 24 hours in 3 mL of medium containing 0, 0.01, 0.1, or 1% HSA. The number and size of the peripheral nerve-like microtissues formed were measured by photoplanometry using ImageJ.

[0042] Figure 3(A) shows the difference in the frequency and size distribution of microtissue formation depending on the HSA concentration in the culture medium during suspension culture. The number and size of microtissues formed in cultures without HSA and cultures with 0.01%, 0.1%, and 1% HSA were measured using ImageJ. As shown in Figures 3(B)-(D), the frequency of microtissue formation was significantly increased by the addition of HSA compared to the HSA-free group. In the HSA-added group, no significant difference was observed in the frequency of microtissue formation depending on the HSA concentration. However, the size of peripheral nerve-like microtissues significantly increased proportionally with the HSA concentration, suggesting that the formation and size of microtissues can be controlled by adjusting the HSA addition and concentration in a suspension culture environment.

[0043] Example 3 This example provides a method for controlling the number and size of peripheral nerve-like microtissues by adjusting the density of PNSCs seeded per culture vessel area for industrial mass production of peripheral nerve-like microtissues. PNSCs collected in a monolayer culture environment are suspended in a suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1% human serum albumin (HSA, Green Cross), 1 μM dexamethasone, and 1 mM N-acetylcysteine ​​to DMEM / F12 culture medium. ULA 6-well culture vessels (SPL Life Sciences) were used to produce peripheral nerve-like microtissues. The culture vessel contained 1 cm 2 2.5, 5.0, 7.5, 10.0, 15.0 x 10 per area 4 PNSCs were seeded, 3 mL of suspension culture medium was added, and the cells were cultured for 24 hours. The number and size of microtissues formed were measured using ImageJ.

[0044] Figure 4 shows the results of controlling the size of peripheral nerve-like microtissues by adjusting the PNSC seeding density. Although there was no significant difference in the number of microtissues formed depending on the PNSC density seeded per unit area, i.e., the number of cells, a correlation was confirmed in which the size of the microtissues increased as the cell seeding density increased. To produce peripheral nerve-like microtissues with a diameter of 100 ± 20 μm, the cells were seeded in a 100 cm 2 1.0~1.5×10 5 The conditions for seeding cells at a density and culturing them in suspension can be confirmed.

[0045] Example 4 As the size of microspheres increases, simple diffusion restricts air and metabolite exchange and nutrient supply, resulting in the accumulation of ROS within the microtissue, which can lead to cell damage and death. This example demonstrates the effect of the size of peripheral nerve-like microtissues on ROS-mediated cell damage in constituent cells. PNSCs harvested in monolayer culture were suspended in suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium was prepared by adding 1% human serum albumin (HSA, green cross) to DMEM / F12 culture medium. Ultra-low attachment (ULA) culture vessels were used to prevent the seeded PNSCs from attaching to the culture vessel and to induce cell-cell junctions. PNSCs suspended in suspension culture medium were seeded at 100, 200, 500, 750, 1000, 2500, and 5000 cells per well of a 96-well ULA culture vessel (SPL Life Sciences, Seoul, Korea), centrifuged at 500 × g for 10 minutes to collect the cells in the center of the vessel, and cultured for 24 hours. After 24 hours of suspension culture, the level of ROS accumulation in the microtissue was assessed using a confocal scanning microscope with CM-H2DCFDA (Molecular Probe, Eugene, OR).

[0046] Figure 5 shows the accumulation of ROS (oxygen free radicals, reactive oxygen species) as a function of microtissue size. It can be seen that the fluorescence intensity, which indicates the degree of ROS accumulation, increases proportionally with the size of the microtissue. In particular, it can be seen that ROS accumulation increases sharply when the number of cells constituting the peripheral nerve-like microtissue is 1,000 or more. It can also be seen that the stability of the microtissue can be ensured by keeping the diameter of the microtissue below 200 μm and the number of cells constituting the microtissue below 1,000.

[0047] <Example 5> As the size of microspheres increases, oxygen and nutrient supply is limited by simple diffusion, which can result in the death of cells within the peripheral nerve-like microtissue. This example demonstrates the effect of the size of peripheral nerve-like microtissues on cell death of the constituent cells. PNSCs harvested in a monolayer culture environment are suspended in a suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1% human serum albumin (HSA, green cross) to DMEM / F12 culture medium. Ultra-low attachment (ULA) culture vessels are used to prevent the seeded PNSCs from attaching to the culture vessel and to induce cell-cell junctions. PNSCs were suspended in suspension culture medium and seeded at 100, 200, 500, 750, 1000, 2500, or 5000 cells per well of a 96-well ULA culture vessel (SPL Life Sciences, Seoul, Korea). The cells were then centrifuged at 500 × g for 10 minutes to collect them in the center of the vessel and cultured for 24 hours. After 24 hours of suspension culture, cell death was assessed by counting the number of ethidium homodimer-1 (EthD-1, Molecular Probe)-positive cells. Microscopic images of peripheral nerve-like structures were taken using a confocal scanning microscope and cell death was assessed using ImageJ.

[0048] As shown in Figure 6, it can be seen that cell death increases as the size of the peripheral nerve-like microtissue increases. Similar to the ROS accumulation results, it can be seen that cell death increases sharply when the number of cells constituting the peripheral nerve-like microtissue is 1,000 or more and the size of the cells is 200 μm or more. This example confirms that controlling the number and size of cells constituting the microspheres is an important regulatory factor in the production of peripheral nerve-like microtissues.

[0049] Example 6 During the generation of peripheral nerve-like microtissues, the limited exchange of oxygen and nutrients results in the accumulation of intracellular ROS, leading to cell damage and death. This example aims to demonstrate a cell protection method using dexamethasone (DEX) and N-acetylcysteine ​​(NAC) to prevent cell damage and death. PNSCs harvested in a monolayer culture environment are suspended in a suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1% HSA to DMEM / F12 culture medium. The cells were cultured in a ULA T75 flask (SPL Life Sciences) culture vessel with 1000 cells / mL. 2 1.0 x 10 per culture vessel area 5 Cells were seeded in 15 mL of suspension culture medium containing 1 mM NAC, 1 μM DEX, or both NAC and DEX, and cultured for 3 days. Cell death was assessed by LIVE / DEAD staining, and cell viability was assessed by counting the total number of DAPI-stained cells and the number of ethidium homodimer-1 (EthD-1)-positive cells. Expression of cell death regulators p38 MAPK and cleaved caspase, as well as the expression of the anti-cell death regulator p-Akt, were assessed by immunofluorescence staining.

[0050] As shown in Figure 7, the protective effects of NAC and DEX on cells constituting peripheral nerve-like microtissues can be confirmed. Compared to NAC, the addition of DEX demonstrated a greater cytoprotective effect, and the presence or absence of NAC did not significantly enhance the cytoprotective effect of DEX.

[0051] As shown in Figure 8, NAC and DEX significantly suppressed the expression of Hif, p38 MAPK, and cleaved caspase, which are cell death proteins that cause cell damage and death. In particular, when DEX and NAC were added to the culture medium of suspension cultures, they demonstrated a greater ability to suppress the expression of Hif, p38 MAPK, and cleaved caspase than when administered alone. Furthermore, the expression of p-Akt, a cytoprotective protein, was enhanced by the combined administration of NAC and DEX. This example demonstrates a method for suppressing cell death and increasing cell viability by adding low concentrations of DEX and NAC to the culture medium used in suspension cultures.

[0052] Example 7 This example demonstrates a method for industrial mass production of peripheral nerve-like microtissues. PNSCs harvested from monolayer culture are suspended in suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1% HSA, 1 μM DEX, and 1 mM NAC to DMEM / F12 culture medium. Suspension culture is performed using a ULA T75 flask (SPL Life Sciences) culture vessel. Culture vessel cm 2 1.0-1.5 x 10 per culture vessel area 5 Seed cells at 7.5–11.1 × 10 in a ULA T75 culture vessel. 6 The cells were seeded, 15 mL of suspension culture medium was added, and the cells were cultured for 3 days.

[0053] The microtissues were prepared as paraffin tissue blocks using standard methods, then cut into 5 μm-thick paraffin sections. HE staining and immunofluorescence staining were performed to evaluate their structural characteristics. The accumulation of collagen type-IV and laminin, which are peripheral nerve-specific extracellular matrices within the microtissues, was evaluated using immunofluorescence staining. Cell-cell and cell-extracellular matrix junctions within the microtissues were evaluated through the expression of β-cantenin and integrin-β1.

[0054] To evaluate Wnt signaling pathway-related mRNA expression in microtissues, RNA was isolated from PNSCs before and after suspension culture, and cDNA was prepared using reverse transcriptase. Gene expression was assessed using a PCR microarray constructed with starter vectors capable of amplifying Wnt signaling pathway regulators. Activation of Wnt signaling was assessed by evaluating the expression of Wnt receptors, ligands, and target genes activated by the Wnt pathway. mRNA expression was expressed as a fold increase compared to PNSCs before microtissue formation.

[0055] As shown in Figure 9, after 3 days of culture, 2,350 ± 451 microtissues measuring 86.5 ± 27.3 μm were produced in a T75 flask. The microtissues had a structure in which PNSCs were bound together. Immunofluorescence staining revealed the accumulation of peripheral nerve-specific ECM, collagen type-VI and laminin, within the microtissues. β-catenin and CD29 (integrin-β1), which represent cell-cell and cell-ECM binding, were uniformly expressed within the microtissues. This confirms that the microtissues produced through the culture process achieved structural stability by the accumulation of peripheral nerve ECM between cells, which acts as a substrate for PNSC binding and binds to cells. Furthermore, the densely packed cells were bound together by β-catenin, strengthening cell-cell junctions, suggesting that a structure similar to that of human tissues could be produced, rather than simply by cell aggregation.

[0056] Figure 10 demonstrates activation of the Wnt / β-catenin signaling pathway through the formation of peripheral nerve-like microtissues. It was confirmed that both canonical and non-canonical Wnt signaling pathways induce several- to several-thousand-fold increases in mRNA expression through the formation of microtissues. In particular, the addition of NAC and DEX during microtissue preparation protected against cell death and induced increases in mRNA expression. It was also confirmed that the formation of microtissues increased the mRNA expression of APC, CTNB1, and GSK3B, which are central regulatory genes in the canonical signaling pathway of PNSCs, by several to several tens of times (Tables 1 and 2). Table 1 shows the canonical Wnt pathway, and Table 2 shows the non-canonical Wnt pathway.

[0057] The Wnt signaling pathway is activated through the formation of microtissues, and in tests investigating the expression of water-soluble compounds, ligands, and subordinate target genes that affect Wnt signaling, gene expression increased several to several tens of times compared to PNSCs before microtissue formation (Tables 3, 4, and 5). Table 3 lists Wnt receptors, Table 4 lists Wnt ligands, and Table 5 lists Wnt targets. It was confirmed that the expression of functional genes that regulate cell migration and differentiation significantly increased for Wnt target genes.

[0058] This example provides a method for adjusting the density of PNSC cells seeded in an industrialized mass production system, protecting cells within the microtissue by adding NAC and DEX to provide structural stability, and simultaneously activating the Wnt signaling pathway of PNSCs through the formation of microtissues to enhance the biological function of PNSCs.

[0059] [Table 1]

[0060] [Table 2]

[0061] [Table 3]

[0062] [Table 4]

[0063] [Table 5]

[0064] Example 8 This example demonstrates the structural and biological properties of peripheral nerve-like microtissues mass-produced at an industrial scale. PNSCs harvested in a monolayer culture environment are suspended in a suspension culture medium at a density of 1.0E+06 / mL. The suspension culture medium is prepared by adding 1% HSA, 1 μM DEX, and 1 mM NAC to DMEM / F12 culture medium. The suspension culture is performed using a ULA T75 flask (SPL Life Sciences) culture vessel. The culture vessel cm 2 1.5 x 10 per culture vessel area 5 Seed the cells at 1.1 x 10 in a ULA T75 culture vessel. 7 After seeding the cells, 15 mL of suspension culture medium was added and cultured for 3 days. The resulting microtissues were prepared into paraffin tissue blocks using standard methods, and then cut into 5 μm-thick paraffin sections. To evaluate the constituent cells, immunofluorescent staining was performed using markers for neurons, neural crest cells, glial cells, Schwann cells, and myelin.

[0065] To evaluate the expression of neuroactive mRNA in microtissues, RNA was isolated from peripheral nerve-like microtissues before and after suspension culture, and cDNA was prepared using reverse transcriptase. Gene expression was evaluated using a PCR microarray consisting of a set of primers to amplify neuroactive mRNA. The mRNA expression of anti-inflammatory and angiogenic factors in microtissues was also evaluated using PCR microarray. Because the secretion titer of neuroactive proteins in microtissues plays an important role in nerve regeneration, the protein content of representative neuroactive proteins, BDNF, GDNF, IGF-1, IL-6, NGF, and NT-3, in the culture medium was analyzed using ELISA after microtissue preparation.

[0066] Figure 11 shows the characteristics of the peripheral nerve-like cells in the microtissue produced by the mass production method. The cells in the microtissue express the neural crest-derived cell markers CD105, nestin, and p75. NTR The expression of these cells was maintained, resembling the characteristics of peripheral nerve-derived stem cells. However, after 3 days of culture, the expression of GFAP, GAP43, and S100β, which were not expressed in the pre-culture state, was detected in the PNSCs within the microtissue. This suggests that cells that had differentiated into Schwann cells were present, and in particular, cells with the characteristics of Schwann progenitor cells, which co-expressed Sox2 and Sox10, were present. It was confirmed that the PNSCs were composed of myelinating Schwann cells expressing MBP and intraneuronal interstitial cells expressing CD140b, confirming that the PNSCs were composed of cells similar to those found in peripheral nerves that regenerate after injury. This example provides a method for producing peripheral nerve-like microtissues that resemble the cells that make up peripheral nerves during the process of regeneration after injury, and are composed of GFAP- / S100β- / Sox10+ undifferentiated neural crest cells, GFAP+ / S100β+ / Myelin+ myelin-positive Schwann cells, GFAP+ / GAP43+ / Myelin- myelin-negative Schwann progenitor cells, and GFAP- / CD140a+ interstitial cells.

[0067] Figure 12 shows the expression profile of neuroactive genes in the mass-produced peripheral nerve-like microtissues. Expression of neuroactive genes in the mass-produced peripheral nerve-like microtissues was evaluated using PCR microarray analysis. Compared to the expression of neuroactive mRNAs in PNSCs before microsphere formation, the expression of these genes significantly increased after microtissue formation. In particular, the addition of NAC and DEX during microtissue formation significantly increased gene expression. Peripheral nerve-like microtissues showed several- to several-tens-fold increases in expression of neurotrophic family mRNAs, including BDNF, NGF, Neurotrophin-3, and Neurotrophin-4; ephrin family mRNAs; GDNF family mRNAs, including GDNF and Artemin; and CNTF family mRNAs, including IL-6, CNTF, and LIF, all of which play key roles in nerve regeneration. This demonstrates the significant application of peripheral nerve-like microtissue formation to nerve regeneration (Table 6).

[0068] As peripheral nerve-like microtissues were formed, we examined the mRNA expression of growth factors, immune response regulators, and angiogenesis inducers. As shown in Tables 7 to 9, we observed significant increases in growth factor, immune response regulator, and angiogenesis inducer mRNA levels during microtissue formation compared to PNSCs before formation. Expression of EGF, FGF, and IGF-1, along with neuroactive factors, increased significantly (Table 7). Expression of IL10 mRNA, a key cytokine that regulates excessive inflammatory responses, increased by more than 50-fold (Table 8). Expression of angiogenesis inducers ANGPT, EPNA, EPO, PDGF, and VEGF mRNA also increased significantly, confirming that microtissue formation can enhance and activate the biological functions of PNSCs (Table 9).

[0069] [Table 6]

[0070] [Table 7]

[0071] [Table 8]

[0072] [Table 9]

[0073] Example 9 In this example, we evaluated ROS-induced cell damage in mass-produced peripheral nerve-like microtissues. Microtissues composed of the same number of cells (1.0E+07 cells) and PNSCs were incubated with 100 nM sodium arsenite for 1 hour to induce ROS cell damage. After sodium arsenite-mediated cell damage, PNSCs and microtissues were treated with RIPA buffer to obtain cell lysates. Cell lysates were electrophoresed via PAGE and transferred to a PVDF membrane. Expression of cell death-regulating proteins pc-Jun, p-p38MAPK, p-MAPKAPK-2, p-JNK, and cleaved caspase 3 was assessed. To semiquantitatively analyze expression rates, band density was measured using an image analyzer (Image J) and expression rates were compared. Furthermore, the viability of PNSCs and microtissues after sodium arsenite treatment was analyzed by LIVE / DEAD staining and annexin V expression.

[0074] As shown in Figure 13, sodium arsenite induced cell death in PNSCs. Sodium arsenite treatment significantly increased the expression of pc-Jun, p-p38MAPK, p-MAPKAPK-2, p-JNK, and cleaved caspase 3, which mediate cell death. In contrast, the expression of pc-Jun, p-p38MAPK, p-MAPKAPK-2, p-JNK, and cleaved caspase 3, which were significantly lower in the microtissues than in PNSCs, was significantly reduced. Densitometric comparison of the expression of cell death regulators (Figure 14) again confirmed that the microtissues had significantly lower expression of cell death regulators than PNSCs (p<0.01). After inducing cell death through sodium arsenite treatment, cell viability was quantitatively assessed by LIVE / DEAD staining and annexin V expression. Similar to the expression of cell death regulators, significantly higher cell viability and lower annexin V expression were observed in the microtissues compared to PNSCs (Figure 15, p<0.01).

[0075] These results provide a method to improve resistance to ROS compared to PNSCs through microstructural construction.

[0076] Example 10 In this example, the efficacy of mass-produced peripheral nerve-like microtissues was evaluated. Stem cell therapeutics are known to exert their effects through an indirect mechanism of action, relying on substances secreted from administered stem cells. Regeneration of damaged nerve tissue requires the recruitment, growth, and axonal growth of neural stem cells. Furthermore, the therapeutic agent must be able to suppress excessive inflammatory responses induced after injury, and must also be able to promote regeneration through revascularization within the damaged tissue.

[0077] Neuroactive proteins secreted from PNSCs and microtissues were evaluated by measuring the neuroactive proteins in the conditioned medium collected during PNSC culture and microtissue preparation. The contents of representative neuroactive proteins, BDNF, GDNF, IGF-1, IL-6, NGF, and NT3, in the conditioned medium were measured by ELISA. To compare and evaluate the neuroactive protein secretion capacity, bone marrow-derived mesenchymal stem cells (BMSCs) were used as a control group. The neuroactive effects of substances secreted from PNSCs and microtissues were assessed on a cell-by-cell basis using SH-SY5Y, a neural crest-derived neural stem cell line.

[0078] The ability to induce neural stem cell growth was assessed by analyzing cell growth after the addition of conditioned medium and comparing the percentage of increased dsDNA content with the dsDNA content before culture. The neural regeneration efficacy was assessed by adding conditioned medium, differentiating SH-SY5Y cells into neurons, and measuring the length of the resulting axonal processes using image analysis.

[0079] The anti-inflammatory efficacy of PNSCs and microtissues was evaluated using RAW264.7 cells. RAW264.7 cells were depleted with 100 μg LPS, and after 6 hours, the TNF-α and IL-1β levels secreted by RAW264.7 cells in the culture medium were analyzed by ELISA. Conditioned medium obtained from PNSCs or microtissues was added at the time of LPS depletion, and the degree to which the conditioned medium suppressed the secretion of inflammatory cytokines from RAW264.7 cells was assessed for anti-inflammatory efficacy.

[0080] The angiogenesis-inducing ability of PNSCs and microtissues was evaluated based on HUVEC cells. Conditioned medium obtained from PNSCs or microtissues was added, and the ability to induce HUVEC cell growth and inhibit sodium arsenite-mediated cell death was evaluated to compare the angiogenesis-inducing ability.

[0081] Similar to the trend in neuroactive mRNA expression, we confirmed that the neuroactive protein secretion ability of PNSCs was enhanced through microtissue formation (Figure 16). Compared to bone marrow-derived mesenchymal stem cells (BMSCs), PNSCs had significantly higher secretion abilities of neuroactive proteins, including BDNF, GDNF, IGF-1, IL-6, NGF, and NT-3 (p<0.01). We also confirmed that the neuroactive protein secretion ability of PNSCs was significantly enhanced through microtissue formation. Secretion of all neuroactive proteins tested was significantly higher in microtissues than in PNSCs (p<0.01).

[0082] We compared the neural activity, anti-inflammatory, and angiogenesis-inducing potential of conditioned medium obtained from microtissues and PNSCs through cell-based assays. As shown in Figure 17, the addition of conditioned medium obtained from PNSCs or microtissues increased neural stem cell axon formation, and axon length was dependent on the concentration of the added conditioned medium. Quantitative analysis confirmed that the addition of conditioned medium from PNSCs and microtissues significantly increased neural stem cell axon length (Figure 18, p<0.01). We also evaluated the ability of the conditioned medium to induce neural stem cell growth. Both PNSCs and microtissue-conditioned medium were able to induce neural stem cell growth (Figure 18). In particular, conditioned medium obtained from microtissues demonstrated significantly greater neural stem cell and axon growth than conditioned medium from PNSCs (p<0.01).

[0083] The anti-inflammatory effects of PNSCs and microtissues were confirmed in Figure 19. TNF-α and IL-1β secretion was significantly increased in RAW264.7 cells cultured with LPS, and the conditioned medium obtained from PNSCs and microtissues was able to suppress the secretion of inflammatory cytokines from the cultured cells, demonstrating its anti-inflammatory effects. In particular, the conditioned medium derived from microtissues showed a significantly higher ability to suppress inflammatory cytokine secretion (p<0.01) compared to PNSCs, confirming that the formation of microtissues can provide a high level of anti-inflammatory efficacy.

[0084] The angiogenesis-inducing ability of PNSCs and microtissues was evaluated using a HUVEC cell-based assay. The addition of conditioned medium derived from PNSCs and microtissues induced significant growth of vascular endothelial cells, confirming their significant protective ability against ROS-mediated HUVEC cell formation (Figure 20). In particular, the addition of conditioned medium derived from microtissues compared to PNSCs induced significant cell growth of HUVECs, a type of vascular endothelial cell, (p<0.01), demonstrating significant cytoprotective effect against ROS-mediated cell damage (p<0.01).

[0085] These results confirmed the neuroactivating, anti-inflammatory, and angiogenesis-inducing effects of PNSCs, and provided a method for enhancing the neuroactivating, anti-inflammatory, and angiogenesis-inducing effects of PNSCs that affect nerve regeneration by forming microtissues from PNSCs.

[0086] Example 11 In this example, the survival rate and efficacy of mass-produced peripheral nerve-like structures were evaluated after in vivo transplantation. Nude mice were injured by compressing the spinal cord between thoracic vertebrae 7 and 8. Three days after injury, 1.0E+05 PNSCs or microtissues composed of the same cells were injected intraspinally. The survival rate in the spinal cord after PNSC or microtissue administration was assessed using qPCR targeting the human-specific Alu gene. The levels of human-specific neuroactive proteins BDNF, GDNF, IGF-1, NGF, and NT-3 in the spinal cord after PNSC or microtissue administration were analyzed using ELISA. The nerve regeneration efficacy of the injected PNSCs or microtissues was assessed by morphometric analysis of myelin regeneration and axon growth within the lesion epicenter.

[0087] As shown in Figure 21, the intraspinal administration of PNSCs and microtissues showed a proportional decrease over time. Compared to PNSCs, the microtissues showed a significantly higher cell survival rate throughout the study period (p<0.01). After two weeks of administration, only 5% of PNSCs remained, and after four weeks, no cells remained. In contrast, after administration of microtissues, 12.4% remained after two weeks, and 3.4% remained after four weeks. These results suggest that microtissues are significantly more resistant to ROS than PNSCs, and that this resistance to cell damage is also effective in vivo, resulting in a higher survival rate.

[0088] It is widely known that the number of remaining cells correlates with the potency of those cells. The neuroregenerative effects of PNSCs or microtissues are expected to be primarily mediated by indirect effects mediated by active factors secreted by the cells, rather than by a direct mechanism of action. Figure 22 shows the results of assessing the content of neuroactive factors in the spinal cord one week after administration of PNSCs or microtissues. Similar to the survival rate, the levels of all neuroactive proteins analyzed in the spinal cord after administration of microtissues were significantly higher (p<0.01). Compared to PNSCs, the levels of BDNF, GDNF, IGF-1, IL-6, NGF, and NT-3 were all higher when microtissues were administered, confirming the evidence for the neuroregenerative effects of these factors.

[0089] After spinal cord injury, nerve regeneration is expected to reestablish a neural network through myelination and axonal growth at the epicenter of the injury, resulting in the recovery of motor and sensory functions of the spinal cord. In this example, myelination and axonal growth were observed in animals administered with PNSCs or microtissues compared to untreated animals, indicating that nerve regeneration can be improved through administered cells (Figure 23). In particular, myelination and axonal growth were significantly higher when administered with microtissues compared to PNSCs (Figure 24, p<0.01), confirming the high nerve regeneration efficacy of microtissues.

[0090] These results suggest that structural stability and functional potency can be enhanced through microtissues, thereby improving survival rate and potency after in vivo transplantation, and thus increasing nerve regeneration efficacy.

Claims

1. 1) culturing human peripheral nerve-derived stem cells (PNSCs) in a monolayer; 2) recovering the monolayer-cultured PNSCs and culturing them in suspension in a culture medium containing 0.01-1% human serum albumin (HSA), 1 μM dexamethasone (DEX), and 1 mM N-acetylcysteine ​​(NAC); A method for producing a peripheral nerve-like microtissue, comprising:

2. In the step of carrying out the suspension culture, the culture vessel cm ２ 0.25 to 2.5 x 10 per area ５ The method for producing a peripheral nerve-like microtissue according to claim 1, characterized in that PNSCs are seeded.

3. The method for producing a peripheral nerve-like microtissue according to claim 1, wherein the suspension culture induces cell-cell junctions of the PNSCs.

4. The method for producing a peripheral nerve-like microtissue according to claim 1, characterized in that the peripheral nerve-like microtissue is a spherical cell structure to which 100 to 500 PNSCs are bound, and has a diameter of 100±20 μm.

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