Use of cell preparation for treatment of underactive bladder and method for producing same

Abstract

To provide a medicinal use of a cell preparation for treating underactive bladder, and a method for producing the same.SOLUTION: Provided is a use of a cell preparation in the manufacture of a pharmaceutical product for a medical use related to underactive bladder, wherein the cell preparation comprises a cell cluster, and the cell cluster is formed by seeding a plurality of mesenchymal stem cells in a culture device having a plurality of culture wells and culturing the mesenchymal stem cells under a condition that a surface of the culture well has a low protein adhesion coating. Also provided is a method of producing a cell preparation for medical use on an underactive bladder, comprising: seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells, wherein a surface of the plurality of culture wells comprises a low protein adhesion coating; and culturing the mesenchymal stem cells to form a cell cluster.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell preparation, and in particular to the use of a cell preparation for the treatment of underactive bladder and a method for producing the same. [Background technology]

[0002] Detrusor underactivity (DU) is a common and complex urinary dysfunction, the etiology of which is associated with factors such as nerve damage, muscle degeneration, post-surgical complications, or metabolic diseases. The main clinical manifestations include difficulty in voiding the bladder, a weak urinary stream, or a state where patients have to rely on assistance via catheterization, which significantly impacts the quality of life of patients. As the aging society continues, the incidence of underactive bladder continues to rise, making this a critical issue that urgently needs to be resolved in the clinical field of urology.

[0003] Conventional clinical treatment methods include drug therapy, physical stimulation training, invasive surgery, etc., but all of them have significant drawbacks and limitations, such as:

[0004] 1. Drug therapy can stimulate bladder contractions using acetylcholine enhancers, parasympathomimetics, etc., but these generally only provide short-term relief and can cause side effects such as nausea, diarrhea, and arrhythmia. Drugs cannot repair degenerated or damaged muscle and nerve tissue, so their therapeutic effect is limited in moderate to severe cases.

[0005] 2. Physical therapy includes methods such as bladder training, pelvic floor exercises, and electrical stimulation therapy, but these methods require long-term implementation and have slow effects. These methods cannot directly restore bladder muscle function and are unlikely to be effective for those with severe atrophy or neurological deficits.

[0006] 3. Regarding invasive treatments, measures such as long-term catheterization, bladder augmentation, or implanted neuromodulation devices may temporarily improve urinary function, but they carry risks of infection, complications, and increased patient dependency, and require highly specialized procedures and are expensive, affecting long-term patient compliance.

[0007] With the advancement of regenerative medicine and stem cell therapy technologies, cell therapy has gradually become a viable strategy for treating underactive bladder. Traditional 2D culture, the most common cell preparation method currently available, cannot mimic the actual 3D environment in the body, limiting intercellular signaling and tissue structure formation, resulting in incomplete cell function expression and affecting both post-transplant survival and therapeutic efficacy. Furthermore, cells are susceptible to mechanical damage during manipulation and transplantation, further reducing therapeutic efficacy. These drawbacks are particularly pronounced when applied to the treatment of underactive bladder. Conventional techniques cannot effectively promote the repair and functional regeneration of bladder smooth muscle tissue, making it difficult for patients to regain normal urination.

[0008] Therefore, there is an urgent need for a new type of cell therapy preparation and its manufacturing method that can more accurately mimic the three-dimensional structure in the body, promote intercellular signaling, and increase survival rates after transplantation, thereby effectively improving the contractile function of bladder muscles and increasing the effectiveness of treating bladder contractile weakness. Summary of the Invention [Means for solving the problem]

[0009] In view of the above, the present invention provides a manufacturing use in which a cell preparation is used to prepare a pharmaceutical for treating underactive bladder, wherein the cell preparation comprises a cell cluster, and the cell cluster is formed by seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells and culturing the cells under conditions in which the surface of the culture well has a low-protein adhesion coating.

[0010] In the above invention, the number of mesenchymal stem cells is 5.0 × 10 4 pieces / cm 2 ~1.0×10 7 pieces / cm 2 The culture time is 24 to 72 hours.

[0011] In the above invention, the diameter of the culture well of the culture device is 200 μm to 500 μm, and the depth of the culture well of the culture device is 100 μm to 200 μm.

[0012] In the above invention, the cell clumps contain a plurality of target cells, and the number of the target cells contained in each cell clump is 30 to 600.

[0013] In the above invention, after treating underactive bladder using the cell preparation, when the bladder tissue after treatment is compared with the bladder tissue before treatment, a statistically significant change is clearly observed in the expression level of at least one gene selected from the Inhba gene, Kif5c gene, ANGPTL7 gene, Mcpt1 gene, Mcpt2 gene, Pla2g2a gene, Prss35 gene, Rasl2-9 gene, Spp1 gene, Sult1c2a gene, and Wif1 gene in the bladder tissue.

[0014] The present invention further provides a method for producing a cell preparation used to treat underactive bladder, comprising seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells, the surface of which comprises a low-protein adhesion coating, and culturing the mesenchymal stem cells to form cell clusters.

[0015] In the above invention, the number of mesenchymal stem cells is 5.0 × 10 4 pieces / cm 2 ~1.0×10 7 pieces / cm 2 The culture time is 24 to 72 hours.

[0016] In the above invention, the diameter of the culture well of the culture device is 200 μm to 500 μm, and the depth of the culture well of the culture device is 100 μm to 200 μm.

[0017] In the above invention, the cell clumps contain a plurality of target cells, and the number of the target cells contained in each cell clump is 30 to 600.

[0018] In the above invention, the method for producing a cell preparation further includes a step of separating and recovering the formed cell clumps from the culture device.

[0019] In summary, the present invention provides a method for using a cell preparation for the treatment of underactive bladder and a method for producing the same. The cell preparation of the present invention forms a three-dimensional cell cluster, promoting cell-cell interactions and the expression of physiological functions, thereby improving the survival rate and functionality of cells after transplantation. The cell preparation particularly effectively promotes the repair of bladder muscle tissue and the recovery of contractile function, thereby improving clinical symptoms of reduced bladder contractility. By utilizing specific cell culture conditions and cell cluster preparation methods, the present invention overcomes the technical drawbacks of conventional two-dimensional culture methods, such as the inability to mimic the in vivo microenvironment, limited cell function, and reduced transplant efficiency. Furthermore, by providing a cell preparation that clearly demonstrates significant regulatory effects on the expression of multiple genes in bladder tissue, the present invention provides an innovative cell preparation and a method for producing the same that is safe and effective for the treatment of underactive bladder. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows the results of analyzing the expression levels of relevant genes in cell clumps after 24 to 48 hours of culture by quantitative polymerase chain reaction (qPCR). [Figure 2] FIG. 2 compares bladder size after treatment with cell clusters in an animal model of underactive bladder. [Figure 3]FIG. 3 shows the change in bladder pressure after treatment with cell clusters in an animal model of underactive bladder. [Figure 4] FIG. 4 shows statistical results of multiple bladder function indicators following treatment with cell clusters in an animal model of underactive bladder. [Figure 5] FIG. 5 is a flow diagram of the steps of a method used to prepare a cell preparation according to one specific embodiment of the present invention. [Figure 6] FIG. 6 is a flow chart illustrating steps of a method used to prepare a cell preparation in accordance with another exemplary embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] In order to make the advantages, spirit, and features of the present invention more easily and clearly understood, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that these specific embodiments are merely representative examples of the present invention, and the specific methods, devices, conditions, materials, etc. exemplified therein are in no way used to limit the present invention or the corresponding specific embodiments. Furthermore, the elements in the drawings are merely used to represent their relative positions and are not drawn to their actual scales. The step symbols of the present invention are merely used to distinguish different steps, and do not represent the order of the steps.

[0022] According to the present invention, so-called "mesenchymal stem cells (MSCs)" are a group of stem cells with diverse origins and multipotency that can form various cell types, such as myoblasts, osteoblasts, chondroblasts, and adipocytes, through induced differentiation. The mesenchymal stem cells used in the present invention can be obtained from various sources, such as bone marrow, adipose tissue, umbilical cord, or other mesenchymal tissues. These cells have good proliferation ability and differentiation plasticity under in vitro conditions, making them suitable as a basis for cell therapy materials in the field of regenerative medicine.

[0023] As used herein, unless otherwise specified, the term "treating" should be interpreted broadly and includes, but is not limited to, reducing, improving, inhibiting, preventing, or reversing bladder weakness (or underactive bladder). Specifically, treating can refer to restoring normal urination by enhancing the contractile function of the smooth muscle of the bladder, as well as reducing the risk of associated complications caused by urinary dysfunction, thereby improving the quality of life of patients.

[0024] According to the present invention, the disclosed cell preparation can be prepared in a dosage form suitable for parenteral administration, which can be selected from injection dosage forms including sterile aqueous solutions, suspensions, or lyophilized powders, and can be prepared to an appropriate concentration and dosage based on clinical needs. The cell preparation can be administered by intraperitoneal injection, subcutaneous injection, intramuscular injection, intravenous injection, or local intralesional injection, and in particular, by local injection into the bladder wall, cell aggregates can be directly delivered to target tissues, thereby improving the efficiency of tissue repair and functional regeneration.

[0025] Furthermore, the cell preparations disclosed herein may contain one or more pharmaceutically acceptable carriers or excipients to maintain cell viability and stability. In this case, the carriers or excipients may include water, physiological saline, phosphate buffer, sugar-containing solutions, alcohol-containing aqueous solutions, etc., and stabilizers, antioxidants, gelling agents, suspending agents, or sustained-release agents may be added as appropriate to enhance the shelf life and physicochemical properties of the cell preparation during use. The selection and optimization of the technical details of such preparations can be adjusted based on the expertise and experience of those skilled in the art.

[0026] The present invention relates to the use of a cell preparation as a pharmaceutical for treating underactive bladder, wherein the cell preparation comprises cell clumps. The cell clumps are formed by seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells, and culturing the cells under conditions in which the surfaces of the culture wells have a low-protein adhesion coating. In this specific example, the number of seeded mesenchymal stem cells is 5.0 x 10 4 pieces / cm 2 ~1.0×10 7 pieces / cm 2 The culture time is 24 to 72 hours, which promotes the natural cell aggregation and formation of cell cluster structures. Cell clusters formed using this method are expected to more accurately mimic the 3D microenvironment in the body, thereby contributing to the expression of cell function and pharmaceutical efficacy. However, in practice, the number of mesenchymal stem cells and the culture time are by no means limited to the above ranges. Further appropriate adjustments can be made based on the type of cell used, the origin of the cells, the growth characteristics of the cells, or the needs of the treatment to achieve optimal cell cluster formation and functional expression.

[0027] To solve the problem of insufficient bladder smooth muscle contractile function in underactive bladders, the present inventors conducted a series of studies to apply cell clusters to underactive bladders and established a mouse animal model to test functionality. In the studies, cultured cell clusters were injected into the bladder wall to evaluate their effects on bladder tissue repair and recovery of contractile ability. Please refer to Figures 1 to 4 for relevant experimental data and analytical results.

[0028] Figure 1 shows the results of quantitative polymerase chain reaction (qPCR) analysis of the expression levels of related genes in cell clusters after 24 to 48 hours of culture. In the figure, the related genes are hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). The inventors of the present invention used qPCR technology to measure the expression levels of the above-mentioned genes in the cell clusters. As clearly shown in Figure 1, the expression of HGF and VEGF genes in the 3D cell clusters (3D-24 hours and 3D-48 hours) was significantly elevated compared to conventional 2D cultured cells. HGF and VEGF are key growth factors that promote tissue repair and angiogenesis, and their elevated expression indicates that the cell clusters have the potential to enhance tissue repair. Please note that the statistical symbols shown in the figures indicate different significance levels, with P≦0.05 (*) representing a statistically significant difference, P≦0.01 (**) representing a highly significant difference, and P≦0.001 (***) representing a very highly significant difference.

[0029] Figure 2 shows a comparison of bladder size after treatment with cell clusters in an animal model of underactive bladder. Figure 3 shows the change in bladder pressure after treatment with cell clusters in an animal model of underactive bladder. In the figure, the normal group is an animal that has not received any treatment, while the control group and the treatment group both have symptoms of underactive bladder induced by pharmacological methods, and the treatment group also receives injection treatment with cell clusters. As shown in Figure 2, the external size of the bladder in the cell cluster treatment group is significantly smaller than that of the untreated control group, clearly demonstrating that cell clusters can effectively improve the distension caused by insufficient bladder contraction force and promote the structural recovery of bladder muscle tissue. Furthermore, referring to the measurement results in Figure 3, the intravesical pressure reached in the cell clump treatment group during urination was significantly higher than that of the untreated control group, demonstrating that the cell clumps not only promoted the repair of bladder structure, but also effectively enhanced bladder contractile function, providing concrete technical support for the treatment of reduced bladder contractile force.

[0030] Figure 4 shows the statistical results of multiple bladder function indexes after treatment with cell clusters in an animal model of underactive bladder. As shown in Figure 4, the multiple bladder function indexes include four parameters: voided volume, bladder capacity, infusion volume, and voided time. In the underactive bladder control group, the voided volume, bladder capacity, and infusion volume were all significantly higher than those in the normal group, clearly indicating a decrease in bladder contractility and emptying ability, resulting in excessive distension and difficulty in urination. In addition, the time required for urination was significantly prolonged, which typically reflects underactive bladder. However, in the cell cluster treatment group, all of the above parameters showed a tendency to recovery, and the bladder capacity, infusion volume and urination volume were significantly lower than those of the untreated control group, clearly indicating that the bladder contractile force had improved and the urinary retention and excretion ability had recovered, and the urination time had also been significantly shortened and approached the level of the normal group, clearly indicating that the urination efficiency had improved. Overall, cell cluster treatment can effectively alleviate the pathological phenomenon of excessive bladder distension and delayed urination, and promote the regeneration of bladder smooth muscle contractile function, clearly demonstrating the clinical potential for treating decreased bladder contractile force.

[0031] Taking the above experimental results together, the cell aggregates of the present invention have a remarkable effect on repairing bladder muscle tissue and restoring contractile function, clearly demonstrating their applicability for improving conditions caused by decreased bladder contractile force, and supporting the feasibility of a novel cell therapy strategy that is urgently needed in clinical practice.

[0032] The present invention also provides a method for preparing a cell preparation, see Figure 5. Figure 5 is a flow chart illustrating steps of a method for preparing a cell preparation according to a specific embodiment of the present invention. As shown in Figure 5, one of the specific embodiments is a method applicable to preparing a cell preparation for medical use related to underactive bladder, which includes step S1 of seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells, the surfaces of which include a low-protein adhesion coating, and step S2 of culturing the mesenchymal stem cells to form cell clusters.

[0033] In another specific embodiment, the method may further include one separate step, see Figure 6. Figure 6 is a flow chart illustrating steps of a method used to prepare a cell preparation according to another specific embodiment of the present invention. As shown in Figure 6, the method used to prepare the cell preparation in this specific embodiment further includes step S3 of separating and recovering the formed cell clumps from the culture device, which is advantageous for subsequent application as a manufacturing process for a cell preparation provided for medical use.

[0034] In this specific embodiment, the diameter of the culture well of the culture device used is 200 μm to 500 μm, and the depth of the culture well of the culture device is 100 μm to 200 μm. Controlling the dimensions of the culture well effectively promotes the formation of stable and uniform cell clusters within the local microspace of mesenchymal stem cells, thereby improving the homogeneity of the cell culture and its adaptability during subsequent transplantation. However, in practice, the diameter and depth of the culture well can be adjusted based on the requirements of the cell type, cell size, or the size of the cell clusters expected to be formed, and are by no means limited to the specific ranges mentioned above.

[0035] In this specific example, the formed cell clumps contain multiple target cells, with the number of target cells contained in each cell clump ranging from 30 to 600. By appropriately controlling the number of cells, it is possible to prevent necrosis in the central region of the cells while taking into consideration both the structural stability of the cell clumps and nutrient permeability, and to increase the survival rate and functional expression of the cell clumps after transplantation. However, in practice, the number of cells contained in each cell clump can be adjusted depending on the cell type, culture conditions, or pharmaceutical application to achieve optimal physiological activity and efficacy during use.

[0036] In this example, the cell preparation of the present invention was applied to a mouse animal model with underactive bladder for treatment, and then bladder tissue samples were collected and analyzed, and the changes in gene expression before and after treatment were compared. This gene level evaluation confirmed the effect of the cell preparation on bladder tissue repair and functional recovery. As a result, the following genes were found in the bladder tissue: Inhba (inhibin beta A chain), Kif5c (kinesin family member 5C), ANGPTL7 (angiopoietin-related protein 7-like), Mcpt1 (mast cell protease 1), Mcpt2 (mast cell protease 2), Pla2g2a (phospholipase A2 group IIA), Prss35 (protease, serine 35), Rasl2-9 (RAS-like, family 2, locus 9), Spp1 (secreted phosphoprotein 1), and the bladder tissue: It was clearly demonstrated that the expression level of at least one of the following genes was statistically significantly changed: the Sult1c2a (Sulfotransferase Family 1C Member 2A) gene, the Sult1c2a (Sulfotransferase Family 1C Member 2A) gene, and the Wif1 (Wnt Inhibitory Factor 1) gene, demonstrating that the cell preparation of the present invention can effectively promote the repair and functional recovery of bladder tissue.

[0037] In addition, gene analysis was performed to further compare the differences in gene expression levels between the cell preparation of the present invention and mesenchymal stem cells in the single-cell format of conventional two-dimensional (2D) culture. As a result, the cell preparation of the present invention contains a variety of proteins, including vascular endothelial growth factor (VEGF), fibroblast growth factor 10 (FGF10), fibroblast growth factor 14 (FGF14), platelet-derived growth factor D (PDGFD), bone morphogenetic protein 2 (BMP2), colony stimulating factor 3 (CSF3), CXC chemokine receptor type 4 (CXCR4), stanniocalcin 1 (STC1), matrix metalloproteinase-2 (MMP2), elastin (ELN), insulin-like growth factor 1 (IGF), and other proteins. The expression levels of genes such as IGF1, MMP10 (Matrix Metalloproteinase-10), AREG (Amphiregulin), and IL-1b (Interleukin-1 beta) genes were significantly elevated compared to those of common single-cell mesenchymal stem cells, revealing special and advantageous differences in expression. The cell cluster structure prepared by the present invention effectively promotes the elevation of the above-mentioned key genes, thereby enhancing the angiogenesis, tissue repair, and anti-fibrotic functions of cells, as well as enhancing intercellular signaling and physiological activity.In addition, compared with conventional 2D-cultured single-cell mesenchymal stem cells, the cell preparation of the present invention has a significantly longer residence time in the animal body, resulting in better survival rate and tissue retention ability after transplantation, clearly demonstrating its enhanced practicality and potential for clinical application as a cell therapy. In summary, the cell preparation of the present invention is superior to mesenchymal stem cells obtained by conventional 2D culture methods in terms of mimicking the microenvironment, expressing functional mechanisms, existing in the body, and efficacy during use.

[0038] In summary, the present invention provides a medical use of a cell preparation for underactive bladder and a preparation method thereof. The cell preparation of the present invention can form a three-dimensional cell cluster, promoting cell-cell interactions and the expression of physiological functions, thereby improving the survival rate and functionality of cells after transplantation. In particular, it can effectively promote the repair of bladder muscle tissue and the recovery of contractile function, thereby improving symptoms of reduced bladder contractility. By utilizing specific cell culture conditions and cell cluster preparation methods, the present invention overcomes the technical drawbacks of conventional two-dimensional culture methods, such as the inability to mimic the in vivo microenvironment, limited cell function, and reduced transplant efficiency, and has clearly demonstrated a significant regulatory effect on the expression of multiple genes in bladder tissue, thereby providing a valid technical basis for the safe and effective application of cell preparations to underactive bladder.

[0039] It is hoped that the above detailed description of preferred specific embodiments will more clearly illustrate the features and spirit of the present invention, but the scope of the present invention is not limited by the preferred specific embodiments presented above. On the contrary, the purpose of doing so is to cover various modifications and equivalent variations within the scope of the patent claimed by the present invention. Therefore, the scope of the patent claimed by the present invention should be interpreted in the broadest possible manner based on the above description so as to cover all possible modifications and equivalent variations. [Explanation of symbols]

[0040] S1 Step S2 Step S3 Step

Claims

1. A use in which a cell preparation is used in the manufacture of a pharmaceutical product for medical use related to underactive bladder, wherein the cell preparation comprises cell clumps, and the cell clumps are formed by seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells and culturing the cells under conditions in which the surfaces of the culture wells have a low-protein adhesion coating.

2. The number of mesenchymal stem cells was 5.0 × 10 ４ pieces / cm ２ ~1.0 x 10 ７ pieces / cm ２ The use according to claim 1, wherein the culture time is 24 to 72 hours.

3. 2. The use according to claim 1, wherein the diameter of the culture well of the culture device is 200 μm to 500 μm, and the depth of the culture well of the culture device is 100 μm to 200 μm.

4. The use according to claim 1, wherein the cell clumps contain a plurality of target cells, and the number of the target cells contained in each cell clump is between 30 and 600.

5. The use according to claim 1, wherein, after the cell preparation is used for medicinal purposes for underactive bladder, when the bladder tissue after treatment is compared with the bladder tissue before treatment, a statistically significant change is clearly observed in the expression level of at least one gene selected from the Inhba gene, Kif5c gene, ANGPTL7 gene, Mcpt1 gene, Mcpt2 gene, Pla2g2a gene, Prss35 gene, Rasl2-9 gene, Spp1 gene, Sult1c2a gene, and Wif1 gene in the bladder tissue.

6. seeding a plurality of mesenchymal stem cells into a culture device having a plurality of culture wells, the surfaces of which comprise a low-protein adhesion coating; Culturing the mesenchymal stem cells to form cell clusters. A method for producing a cell preparation for medical use against underactive bladder, comprising:

7. The number of mesenchymal stem cells was 5.0 × 10 ４ pieces / cm ２ ~1.0 x 10 ７ pieces / cm ２ and the culture time is 24 to 72 hours.

8. 7. The manufacturing method according to claim 6, wherein the diameter of the culture well of the culture device is 200 μm to 500 μm, and the depth of the culture well of the culture device is 100 μm to 200 μm.

9. The manufacturing method according to claim 6, wherein the cell clumps contain a plurality of target cells, and the number of the target cells contained in each cell clump is 30 to 600.

10. The method of claim 6, further comprising the step of separating and recovering the formed cell clumps from the culture device.

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