WO2018216228A1

WO2018216228A1 - Method for regenerating urinary tract tissue using three-dimensional cell structure

Info

Publication number: WO2018216228A1
Application number: PCT/JP2017/020531
Authority: WO
Inventors: 哲也今村; 島村　満
Original assignee: 株式会社サイフューズ; 国立大学法人信州大学
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2018-11-29

Abstract

The purpose of the present invention is to establish, as one of bladder regenerative medical techniques, novel treatment which transplants a bone marrow-derived cell structure into a bladder and attempts to regenerate a functional bladder. The present invention provides: a low-compliance bladder or a contracted bladder including a three-dimensional structure which is constructed by laminating spheroids of bone marrow-derived cells on a spicule disposed on a substrate and has a thickness of at least 500 μm; or tissue regeneration, a function enhancer or the like for a low-compliance bladder.

Description

Method for regenerating urinary tract tissue using three-dimensional cell structure

The present invention relates to a low-compliance bladder that is difficult to treat in the urological field, or a three-dimensional cell structure produced by culturing cell aggregates (spheroids) from a cultured cell group and stacking them. Used to regenerate functional bladder tissue.

Normal urination is voluntarily performed with a sense of urine, because the bladder relaxes and the urethra contracts to urinate. On the other hand, urination is performed without applying abdominal pressure because the bladder contracts and the urethra relaxes. That is, normal urination is performed by the opposite movements of the bladder and urethra. However, urination disorder (lower urinary tract symptom) occurs when the contradictory functions of the bladder and urethra fail for some reason. For lower urinary tract symptoms, conservative treatment with drug treatment is the mainstream. However, it is difficult to treat with lower urinary tract symptoms when the urine accumulation function of collecting urine in the bladder causes a serious dysfunction.

Central nervous system disorders such as spina bifida, spinal cord injury, cerebrovascular disorder, pelvic nerve disorder with radical hysterectomy, neurogenic bladder resulting from peripheral nerve damage due to diabetes, side effects of anticancer drugs, etc. In diseases such as hemorrhagic cystitis caused by urinary bladder, interstitial cystitis of unknown cause, refractory overactive bladder, excessive bladder contraction (detrusor overactivity) and conflicting actions of bladder and urethra Dysfunction (dysfunction of urinary sphincter) occurs. If the bladder is exposed to a high-pressure environment for a long time due to detrusor overactivity or detrusor sphincter coordination failure, the original elasticity and expandability of the bladder disappears, the bladder cannot relax, and the urine cannot be collected sufficiently It becomes a low-compliance bladder, and in the late stage, the entire bladder is deformed and atrophied. Low-compliance bladders or atrophied bladders cause renal dysfunction from hydronephrosis due to vesicoureteral reflux, ureteral stenosis, etc., and urinary incontinence that significantly impairs social life.

Currently, conservative treatment using drugs such as anticholinergic drugs that suppress excessive detrusor contraction and enhance urine accumulation function is selected as a treatment for low-compliance bladder. However, resistance to conservative treatment, renal function and exacerbation of urinary incontinence are often unsuccessful, and bladder atrophy progresses. Surgical treatment is indicated if urinary incontinence is observed that severely impairs renal function or significantly reduces quality of life (QOL).

Surgical treatment for severely atrophied bladder includes surgery to form a low-pressure large-capacity bladder by forming a part of the intestine such as the ileum. The treatment by intestinal cystoplasty has been highly successful. However, intestinal cyst expansion is a very invasive operation because a part of the intestine is released and used. Furthermore, complications due to the use of the intestinal tract, such as bladder stones, intestinal stenosis, chronic diarrhea, and upper urinary tract stones may be serious. In addition, postoperative urination (normal urination voluntarily performed when feeling urinary) disappears, so intermittent urination throughout the life (periodically inserting a catheter from the urethra to drain urine out of the body) Is essential.

From these backgrounds, the present inventors have energetically researched a therapeutic method for regenerating bladder tissue that has been significantly damaged using bone marrow-derived cells to improve urination disorder. First, in order to examine whether or not the bladder tissue is regenerated by bone marrow-derived cells, a metal rod cooled with dry ice was applied to the mouse bladder to cause serious damage to the bladder smooth muscle, and the bladder contraction pressure decreased. A frozen injury bladder model was prepared (1). Subsequently, it was found that when a bone marrow-derived cell was directly injected and transplanted into a site where freezing injury was caused, a clear smooth muscle layer including a site constructed from smooth muscle cells differentiated from the transplanted bone marrow-derived cell was regenerated ( 1). Furthermore, it was reported that the urinary contraction pressure was improved in the cell transplanted group (1). Furthermore, the mechanism of functional bladder regeneration was also discussed.

The transplanted bone marrow-derived cells have the ability to differentiate into smooth muscle cells and the like. The bladder smooth muscle layer of the frozen injury bladder model had lost the smooth muscle layer and formed a space (scaffold) in which the transplanted bone marrow-derived cells could be engrafted (2). Furthermore, it was confirmed that expression of at least 19 types of cell growth factors was increased when freezing injury was given compared to intact bladder (2). In particular, cell growth factors whose expression has been increased include those known to be involved in smooth muscle cell regeneration (2). That is, bone marrow-derived cell transplantation in a frozen-injured bladder satisfied the three elements constituting cells, cells, cell scaffolds, and cell growth factors. From this, it was considered that functional bladder regeneration was established. At the same time, it was shown that bone marrow-derived cells can regenerate bladder tissue and improve urination function.

Previously, in the field of basic urology, low-compliance bladder and bladder atrophy model animals were not established, but radiation injury bladder similar to low-compliance bladder and bladder atrophy by irradiating rat bladder A model has been established (3). In addition, the frozen injury bladder model is reversible, and there remains a problem that the injury site recovers even by natural healing.
Low compliance bladder or bladder atrophy is irreversible. For this reason, when an irreversible injury is caused by irradiating the bladder, the smooth muscle cells and the number of nerve cells in the tissue of the radiation injury bladder model are remarkably reduced as in the case of the freezing injury (3). The difference from freezing injury is not the formation of a space due to the disappearance of the smooth muscle layer (cells), but the progression of fibrosis (3). Moreover, as a bladder function, the urination frequency which shows the reduction | decrease of a micturition interval and a single urination volume, and the high residual urine volume are shown (3).

As in the case of using the frozen injury bladder model, research has been conducted in which bone marrow-derived cells are directly injected and transplanted into a bladder that has been damaged by irradiation, and bladder regeneration by bone marrow-derived cells is evaluated. The histological analysis 4 weeks after transplantation revealed that the bladder smooth muscle layer was reconstructed in the cell transplant group, and the area of the reconstructed muscle layer was the control group in which the sham operation was performed without transplanting bone marrow-derived cells. (3). Similarly, neuronal cells were also significantly increased (3). Also in the bladder function, the control group shows irregular urination and high residual urine, whereas the cell transplant group regularly urinates about 1.5 ml in 5 or 6 minutes, which is similar to normal bladder. The residual urine amount was significantly reduced (3). These results suggest that bone marrow-derived cells can regenerate functional bladder even in irreversible disorders, similar to low-compliance bladder and bladder atrophy.

However, some problems remain in the conventional cell transplantation methods, that is, injection transplantation of bone marrow-derived cells directly into the injured part. For example, when preparing the cells to be transplanted, the cells immediately before the transplantation are damaged by adhering to the culture dish during the culture period, peeling the increased cells with an enzyme treatment such as collagenase, and performing centrifugation. . In general, in the case of single-cell injection transplantation, it is known that the engraftment rate in the recipient is very low. In the case of freezing injury, there was a gap structure in which the transplanted cells could enter, but in the case of radiation injury, such a gap structure was not formed. Therefore, the cell engraftment rate in the radiation-injured bladder is remarkably reduced as compared with the frozen injury bladder. The biggest problem in direct cell implantation transplantation is that there is a concern that the range of the injury site will be expanded and the degree of injury will be worsened from the method in which the needle is directly inserted into the affected area (injury site) and transplanted.

Therefore, we made a bone marrow-derived cell sheet by using a temperature-responsive culture dish, transplanted a patch of the bone marrow-derived cell sheet to a radiation-injured bladder, and examined whether a functional bladder was regenerated. It was. As a result, similar to direct injection transplantation, smooth muscle layer reconstruction, nerve cell regeneration, and improved bladder function were observed (4). The functional bladder regeneration mechanism was also discussed. In the group transplanted with the bone marrow-derived cell sheet, it was confirmed that the expression of 24 types of cell growth factors was significantly increased compared with the control group in which the sham operation using the cell-free sheet was performed (4). That is, it was thought that a functional bladder was regenerated by the paracrine effect by the bone marrow-derived cells constituting the cell sheet.

On the other hand, the major difference from direct injection transplantation was that bone marrow-derived cells constituting the cell sheet were confirmed to be attached correctly at the transplantation site, but the cells constituting the bladder, smooth muscle cells This is the point where differentiation into nerve cells was not observed. That is, the cells themselves did not differentiate and participate in regeneration. In addition, a single-layer cell sheet has no strength and requires a scaffold to which cells adhere.

1. Imamura, Kinebuchi, Ishizuka, Seki, Igawa, Nishizawa.
Implemented mouse bone marrow-derived cells reconstructed layered smooth muscle structures in injured urine blades. Cell Transplantation 17: 267-278, 2008
2. Imamura, Yamamoto, Ishizuka, Gotoh, Nishizawa.
The Microenvironment of freeze-injured mouse primary bladders enables successful tissue engineering.
Tissue Engineering 15: 3367-3375, 2009
3. Imamura, Ishizuka, Zhang, Hida, Gautam, Kato, Nishizawa.
Bone marrow-delivered cells implied into radiation-injured urine blades restructuring blades tissues in rats.
Tissue Engineering Part A, 18: 1698-1709, 2012
4). Imamura, Ogawa, Minagawa, Yokoyama, Nakazawa, Nishizawa, Ishizuka.
Engineered bone marrow-derived cell sheets restore structure and function of radiation-invented rat-bladder blades.
Tissue Engineering Part A.1. 21: 9-10, 1600-10, 2015

Currently, as a treatment for low-compliance bladder or atrophic bladder, conservative treatment with drugs or intestinal cystoplasty is performed, but no effective effect can be obtained despite significant patient burden. . In addition, improvement in the quality of life of patients cannot be expected. Therefore, a novel treatment is established as one of the regenerative medicine of the bladder by transplanting the bone marrow-derived cell structure into the bladder and attempting to regenerate a functional bladder.

As a result of intensive studies to solve the above-mentioned problems, the inventor forms spheroids from single cells, laminates them on needles to create a thick three-dimensional structure, and transplants it to the bladder As a result, the present invention was completed.
That is, the present invention is as follows.
(1) Tissue regeneration for low-compliance bladder or atrophic bladder, or a low-compliance bladder comprising a three-dimensional structure having a thickness of at least 500 μm constructed by laminating spheroids of bone marrow-derived cells on acicular bodies arranged on a substrate Function improvement material.
(2) The material according to (1), wherein the low-compliance bladder is a bladder having a diseased tissue or a bladder having a dysuria.
(3) Bladder having pathological tissue caused by side effects of radiation therapy, bladder having pathological tissue caused by side effects of drug treatment or surgical treatment, and pathological conditions that have been congenital or acquired The material according to (2), which is at least one selected from the group consisting of bladders having tissues.
(4) Bladder with dysuria is a bladder with lower urinary tract symptoms caused by side effects of radiation therapy, bladder with lower urinary tract symptoms caused by side effects of drug treatment or surgical treatment, and congenital or acquired (2) The material according to (2), which is at least one selected from the group consisting of bladders having lower urinary tract symptoms.
(5) The material according to (4), wherein the lower urinary tract symptom is a urinary symptom, urination symptom, or post-urination symptom caused by impaired function of the bladder or urethra.
(6) The low-compliance bladder is composed of a lower urinary tract having pathological tissue, a lower urinary tract having lower urinary tract symptoms, an upper urinary tract having pathological tissue, and an upper urinary tract having symptoms that impair the function of the upper urinary tract The material according to (1), which is at least one selected from the group.
(7) The lower urinary tract having pathological tissue caused by side effects of radiotherapy, the lower urinary tract having pathological tissue caused by side effects of drug treatment or surgical treatment, and the congenital or The material according to (6), which is at least one selected from the group consisting of a lower urinary tract having a diseased tissue that has acquired onset.
(8) Lower urinary tract having lower urinary tract symptoms, lower urinary tract having lower urinary tract symptoms developed by side effects of radiation therapy, lower urinary tract having lower urinary tract symptoms, lower urinary tract symptoms developed by side effects of drug treatment or surgical treatment The material according to (6), which is at least one selected from the group consisting of a tract and a lower urinary tract having a congenital or acquired urinary tract symptom.
(9) The upper urinary tract having pathological tissue caused by side effects of radiation therapy, the upper urinary tract having pathological tissue caused by side effects of drug treatment or surgical treatment, and the congenital or The material according to (6), which is at least one selected from the group consisting of upper urinary tracts having acquired pathological tissues.
(10) The upper urinary tract having symptoms that impair the function of the upper urinary tract has developed due to side effects of the upper urinary tract having symptoms that impair the function of the upper urinary tract, which is caused by side effects of radiotherapy, drug treatment or surgical treatment It is at least one selected from the group consisting of the upper urinary tract having symptoms that impair the function of the upper urinary tract and the upper urinary tract having symptoms that impair the function of the upper urinary tract that is congenital or acquired (6) The material described in 1.
(11) The material according to (10), which is a symptom of impaired function of the upper urinary tract, vesicoureteral reflux or hydronephrosis.
(12) The material according to any one of (1) to (11), wherein cells constituting the bladder organ, lower urinary tract organ, or upper urinary tract organ are induced by the paracrine effect or differentiated into the cells.
(13) A tissue regeneration or function improving material for a tissue having sexual dysfunction, comprising a three-dimensional structure having a thickness of at least 500 μm constructed by laminating bone marrow cell-derived spheroids on acicular bodies arranged on a substrate.
(14) A tissue having sexual dysfunction is a sexually functional tissue having a diseased tissue caused by side effects of radiation therapy, a sexually functional tissue having a diseased tissue caused by side effects of drug treatment or surgical treatment, and congenital or acquired (13) The material according to (13), which is at least one selected from the group consisting of sexually functional tissues having pathologically developed pathological tissues.
(15) The material according to (13), wherein the sexual dysfunction is erectile dysfunction or ejaculation disorder, which is mainly male sexual dysfunction.
(16) The material according to any one of (13) to (15), wherein a cell constituting a sexually functional tissue is induced by a paracrine effect or differentiated into the cell.

The smooth muscle layer is reconstructed by transplanting a three-dimensional structure of bone marrow-derived cells into the radiation-injured bladder. In addition, cells differentiated from bone marrow-derived cells constituting the structure into smooth muscle cells are also included. Regarding bladder function, significant frequent urination is improved and residual urine volume is reduced. From these results, transplantation therapy using bone marrow-derived cell structures can be established as a novel treatment for low-compliance bladder or atrophic bladder.

FIG. 1 is a diagram showing a method for producing a rat radiation bladder irradiated bladder model.
FIG. 2 is a conceptual diagram for producing a three-dimensional structure of bone marrow-derived cells.
FIG. 3 is a diagram showing a method for transplanting a bone marrow-derived cell structure.
FIG. 4 is a diagram showing the measurement results of bladder function two weeks after transplantation of the bone marrow-derived cell structure.
FIG. 5 is a diagram showing various bladder function parameters two weeks after transplantation of the bone marrow-derived cell structure.
FIG. 6 is a diagram showing immunohistochemical staining of a bone marrow-derived cell structure that has arrived two weeks after transplanting the bone marrow-derived cell structure.
FIG. 7 is a diagram showing differentiation of bone marrow-derived cells constituting a three-dimensional structure into smooth muscle cells (whole).
FIG. 8 is a diagram showing differentiation of bone marrow-derived cells constituting the three-dimensional structure into smooth muscle cells (local).
FIG. 9 is a diagram showing a protocol of the present invention.
FIG. 10 is a diagram showing the results of measuring the bladder function 2 weeks (upper) and 4 weeks (lower) after transplanting the bone marrow-derived cell structure.
FIG. 11 is a diagram showing various bladder function parameters 4 weeks after transplantation of bone marrow-derived cell structures.
FIG. 12 is a diagram showing differentiation of bone marrow-derived cells into smooth muscle cells within the transplanted three-dimensional structure.
FIG. 13 is a diagram showing differentiation of bone marrow-derived cells into smooth muscle cells around the vascular structure ( ^* ) developed from the recipient.

1. Outline The present inventor has made a three-dimensional structure having a thickness by forming a spheroid from a single cell and laminating it on a needle-like body with respect to a transplantation method that should overcome the problems of each transplantation method from previous studies. I thought about making it and transplanting it to the bladder. The present invention succeeded in reconstructing a functional bladder by reconstructing a bladder tissue by transplanting a bone marrow-derived cell structure into a low-compliance bladder or a radiation injury bladder model that mimics a deflated bladder. Is.
The present invention establishes a novel therapeutic method as one of the regenerative medicine of the bladder from the research results that the bone marrow-derived cell structure is transplanted into the radiation-damaged bladder model to reconstruct the bladder tissue and improve dysuria. Is.

Bone marrow-derived cells that have developed and proliferated are obtained through primary and subculture in a collagen-coated culture dish or flask using bone marrow cells collected from the femur. Bone marrow-derived cells that have been cultured are seeded on 96-well U bottom plates to form spheroids. The formed spheroid is laminated on the needle-like body to construct a thick three-dimensional structure, and circulation culture is performed. After circulating culture is finished, the three-dimensional structure is extracted from the needle-like body and used as a bone marrow-derived cell structure for transplantation.

On the other hand, a radiation injury bladder model that mimics a low-compliance bladder or an atrophy bladder is prepared. The prepared bone marrow-derived cell structure is transplanted to the front wall of the radiation-injured bladder. Following transplantation, histological analysis assesses bladder tissue remodeling, and bladder function recovery assesses bladder function recovery.

The conventional direct injection transplantation method, which is a conventional transplantation method, has problems such as concern about the damage of the transplanted cells, a low cell engraftment rate in the recipient, and concern about expansion and deterioration of the damaged site. In cell sheet transplantation, a single-layer cell sheet is thin and brittle and requires a structure to be used as a scaffold, and there are problems such as restriction on differentiation of target organs and tissues into constituent cells. As a means of overcoming these problems, the present inventors succeeded in overcoming the problems by producing and using a three-dimensional structure from a single cell.

In the present invention, cells collected from the bone marrow of a subject are aggregated to produce spheroids (cell aggregates), which are stacked by piercing needles placed on a substrate to produce a three-dimensional cell structure. . By transplanting this structure into the subject's bladder, regeneration or function improvement of the bladder tissue or bladder organ is performed. In the present invention, for example, cells constituting the bladder organ are induced by a paracrine effect. The “paracrine effect” is one of signal transduction between cells. Substances secreted from cells (physiologically active substances such as cell growth factors and cytokines) pass through tissue fluids, not blood, around the cells. It means to act locally.

The bladder to be transplanted is a bladder having a pathological tissue that has developed due to a side effect of radiation therapy. On the other hand, cells constituting the bladder organ are induced by the paracrine effect. In addition, the tissue of the bladder organ is regenerated from the cells including the differentiation of the cells themselves constituting the structure.
In another aspect, bladder function is improved for bladders that have dysuria such as frequent urination and residual urine that have developed due to side effects of radiation therapy.

In still another embodiment, the cells constituting the bladder organ are induced by the paracrine effect on the bladder having a diseased tissue caused by the side effects of drug treatment and surgical treatment. In addition, the tissue of the bladder organ is regenerated from the cells including the differentiation of the cells themselves constituting the structure.
In still another aspect, the function of the bladder is improved with respect to a bladder having dysuria such as frequent urination and residual urine caused by side effects of drug treatment and surgical treatment.
In still another embodiment, cells that constitute the bladder organ are induced by a paracrine effect on a bladder having a pathological tissue that has been congenitally or acquired. In addition, the tissue of the bladder organ is regenerated from the cells including the differentiation of the cells themselves constituting the structure.

In yet another aspect, bladder function is improved for bladders with dysuria such as frequent urination and residual urine that are congenital or acquired.
In yet another embodiment, the cells constituting the lower urinary tract organs are induced by the paracrine effect on the lower urinary tract having pathological tissue caused by side effects of radiotherapy. In addition, the tissue of the lower urinary tract organs is regenerated from the cells including the differentiation of the cells themselves constituting the structure.

In yet another aspect, the lower urinary tract function is improved with respect to the lower urinary tract having lower urinary tract symptoms caused by side effects of radiation therapy.
In yet another embodiment, the cells constituting the lower urinary tract organs are induced by the paracrine effect on the lower urinary tract having pathological tissues that have developed due to side effects of drug treatment and surgical treatment. In addition, the tissue of the lower urinary tract organs is regenerated from the cells including the differentiation of the cells themselves constituting the structure.
In still another embodiment, the lower urinary tract function is improved with respect to the lower urinary tract having lower urinary tract symptoms caused by side effects of drug treatment and surgical treatment.
In yet another embodiment, the cells constituting the lower urinary tract organs are induced by the paracrine effect on the lower urinary tract having a pathological tissue that has been congenitally or acquired. In addition, the tissue of the lower urinary tract organs is regenerated from the cells including the differentiation of the cells themselves constituting the structure.

In yet another aspect, lower urinary tract function is improved relative to the lower urinary tract having a lower urinary tract symptom congenitally or acquired.
In yet another embodiment, the cells constituting the upper urinary tract organs are induced by the paracrine effect on the upper urinary tract having pathological tissue caused by side effects of radiation therapy. In addition, the tissue of the upper urinary tract organ is regenerated from the cells including the differentiation of the cells themselves constituting the structure.
In yet another embodiment, the upper urinary tract function is improved with respect to the upper urinary tract having vesicoureteral reflux or hydronephrosis caused by side effects of radiation therapy.
In yet another embodiment, the cells constituting the upper urinary tract organs are induced by the paracrine effect on the upper urinary tract having pathological tissue that has developed due to side effects of drug treatment or surgical treatment. In addition, the tissue of the upper urinary tract organ is regenerated from the cells including the differentiation of the cells themselves constituting the structure.

In still another embodiment, the upper urinary tract function is improved with respect to the upper urinary tract having vesicoureteral reflux or hydronephrosis caused by side effects of drug treatment or surgical treatment.
In yet another embodiment, the cells constituting the upper urinary tract organs are induced by the paracrine effect on the upper urinary tract having pathological tissue that has been congenitally or acquired. In addition, the tissue of the upper urinary tract organ is regenerated from the cells including the differentiation of the cells themselves constituting the structure.

In yet another aspect, the upper urinary tract function is improved with respect to the upper urinary tract having congenital or acquired cystoureteral reflux or hydronephrosis.
In yet another embodiment, cells constituting a sexually functional tissue organ are induced by a paracrine effect on a sexually functional tissue having a diseased tissue caused by side effects of radiation therapy. In addition, the tissue of the sexually functional tissue organ is regenerated from the cell including the differentiation of the cell itself constituting the structure.
In yet another aspect, sexual function is improved for sexually functional tissues having erectile dysfunction and the like caused by side effects of radiation therapy.
In yet another embodiment, cells constituting a sexually functional tissue organ are induced by a paracrine effect on a sexually functional tissue having a diseased tissue caused by side effects of drug treatment or surgical treatment. In addition, the tissue of the sexually functional tissue organ is regenerated from the cell including the differentiation of the cell itself constituting the structure.

In yet another embodiment, sexual function is improved for sexually functional tissues having erectile dysfunction caused by side effects of drug treatment and surgical treatment.
In yet another embodiment, cells constituting a sexually functional tissue organ are induced by a paracrine effect on a sexually functional tissue having a pathological tissue that has been congenitally or acquired. In addition, the tissue of the sexually functional tissue organ is regenerated from the cell including the differentiation of the cell itself constituting the structure.
In yet another embodiment, sexual function is improved with respect to sexually functional tissues having an erectile dysfunction that is congenital or acquired.

2. Preparation of three-dimensional structure (1) Bone marrow-derived cell The cell used for preparation of the three-dimensional structure is a bone marrow-derived cell. Bone marrow-derived cells refer to cells that have undergone primary culture in collagen-coated culture dishes collected from bone marrow, have grown and adhered to the culture dishes, and are mainly mesenchymal cells including stem cells. It may be a mixture of types of cells, or may be separated by a cell sorter using a plurality of cell markers.

The bone marrow-derived cells are cultured or maintained in a medium suitable for each cell, and are prepared as necessary. In addition, various antibiotics, fetal bovine serum, etc. can be added to a culture medium as needed.
When the culture is continued in this manner, bone marrow-derived cells aggregate to form cell aggregates, that is, spheroids. The ability to form spheroids can be examined, for example, by morphological inspection using an optical microscope.

(2) Method for producing a three-dimensional structure A method for producing a three-dimensional structure of a cell by arranging cells in an arbitrary three-dimensional space is known (WO2008 / 123614). In this method, a needle-like body is arranged in a sword mountain shape on a substrate, and the needle-like body is placed by piercing a cell mass.
In the present invention, a three-dimensional structure (three-dimensional structure) is produced by laminating spheroids using the above method. Since an automatic stacking robot for realizing the above method is already known (Bio 3D printer “Regenova” (registered trademark), Cyfuse Co., Ltd.), the three-dimensional structure is preferably manufactured using this robot.

The number and arrangement shape of the spheroids are not particularly limited and are arbitrary.
Further, the three-dimensional structure to be manufactured is at least 500 μm in thickness. The thickness of the obtained structure is, for example, 500 μm to 1000 μm, 500 μm to 1500 μm, 600 μm to 1200 μm, or 600 μm to 1800 μm. By setting this thickness, in addition to the paracrine effect when bone marrow cells are transplanted, blood vessels are induced in the transplanted tissue to reduce fibrotic lesions, and the transplanted bone marrow cells further to the tissue constituting the bladder tissue Differentiate directly. It is expected that improvement of fibrotic lesions and reconstruction of bladder tissue will proceed in unison.

3. Transplantation of three-dimensional structure The three-dimensional structure formed as described above is transplanted into the bladder of a subject (test animal). The transplantation method is not particularly limited, and is arbitrary.

4). Application When the three-dimensional structure is transplanted as described above, the smooth muscle layer is reconstructed. In addition, cells differentiated from bone marrow-derived cells constituting the structure into smooth muscle cells are also included.
The present invention can be applied to low-compliance or atrophic bladder, or low-compliance bladder. Sexual dysfunction is also applicable. That is, by transplanting the above three-dimensional structure, low-compliance bladder or atrophic bladder, or tissue regeneration or function improvement for low-compliance bladder, or low-compliance bladder or atrophic bladder, or low-compliance bladder can be treated.
In addition, by transplanting the above three-dimensional structure, tissue regeneration or function improvement for a tissue having sexual dysfunction can be performed, and further sexual dysfunction can be treated.

“Low-compliance bladder” means a state in which the original elasticity or expandability of the bladder is lost, the bladder cannot be relaxed, and the urine cannot be sufficiently collected. Is mentioned. “Atrophic bladder” means a state in which the entire bladder is deformed and atrophied. In addition, “bladder with pathological tissue” means that the bladder is exposed to a high-pressure environment for a long time due to excessive contraction of the bladder (detrusor overactivity) and failure of the opposite functions of the bladder and urethra (dysfunction of the sphincter). As a result, it means a bladder that cannot exhibit its original urine storage function and urination function due to irreversible functional disorders such as bladder smooth muscle, nerves, and urothelium.

“Lower urinary tract” means the urinary tract system that mainly refers to the bladder and urethra. “Lower urinary tract symptoms” is a general term for urinary storage symptoms, micturition symptoms, and post-urination symptoms caused by impaired function of the lower urinary tract. “Upper urinary tract” generally refers to the kidney and ureter. There are various causes of vesicoureteral reflux and hydronephrosis.

“Sexual dysfunction” refers to a general term for symptoms that are impaired and do not work well in a series of processes such as sexual stimulation, sexual desire, penile erection, intercourse, ejaculation, orgasm, and penile relaxation. It mainly refers to male sexual dysfunction. Male sexual dysfunction is a disorder that occurs in at least one of the above-described series of processes, such as erectile dysfunction and ejaculation disorder.
Causes of the low-compliance bladder or atrophic bladder, or low-compliance bladder, or sexual dysfunction include side effects of radiation therapy, side effects of drug treatment or surgical treatment, and those that have been congenital or acquired. The present invention is applicable to any bladder disease caused by the above causes.
After transplantation, it is confirmed whether or not it has a predetermined bladder function. This confirmation can be performed, for example, by urinary fluid examination (urodynamics) in the case of humans and by measurement of intravesical pressure in the case of animals. In addition, the evaluation of bladder function is performed by, for example, basal bladder pressure, urination threshold pressure, maximum urinary contraction pressure, single urination interval time, single urination volume, residual urine volume, bladder capacity, etc., alone or in combination as appropriate. Just do it. Histological examination by immunohistological staining can also be performed as appropriate.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) Preparation of rat radiation-damaged bladder model A 10-week-old female SD rat was fixed to a table based on a previously reported method and irradiated with a radiation-protected metal plate hollowed out near the bladder. MBR-1520R-4 (Hitachi Power Solutions Co., Ltd.) was used as a radiation irradiation device, and irradiation was performed 5 times in total at a frequency of 2 Gy / week, and then the animals were reared normally for 2 weeks. After two weeks of normal rearing, the structure of bone marrow-derived cells was transplanted by incising and fixing the subcutaneous bladder. After transplantation, it was observed for 2 weeks and analyzed by bladder function and dissection (FIG. 1).

(2) Production of cell structure A thick bone marrow-derived cell structure for transplantation was produced as follows.
Bone marrow-derived cells were isolated from GFP-expressing rats, and cells attached to Type I collagen dish were grown and cultured in 15% FBS DMEM HG medium. Next, the grown bone marrow cells are peeled off by trypsin treatment, turbid in 10% FBS DMEM LG medium, and seeded at a rate of 2 to 5 × 10 ⁴ cells / well on Prime Surface 96 well plate (U bottom) of Sumitomo Bakelite. It was. Three days later, the formed cell aggregates were laminated in a position / shape of 9 (vertical) × 9 (horizontal) × 3 layers (height) using a bio-3D printer / regenova. A 9 × 9 Kenzan with testicular spheroids was placed in a Perfusion chamber, and cultured in a circulating manner with 150 ml of 10% FBS DMEM LG at a rate of 2 to 3 ml / min. One week later, the structure (thickness 1500 μm) fused with spheroids was removed from the grid plate, stored in 10% FBS DMEM LG medium at 20 ° C., transported to the transplant destination, and used for transplantation. There are many other designs for creating patch-like structures. It is also possible to create a patch with one or two layers depending on a method of reducing cell density by stacking and fusing spheroids in a lattice shape, or by composition of spheroid and medium. A conceptual diagram summarizing several designs is shown in FIG.

(3) Transplantation In the transplantation method, the anterior bladder wall of a radiation-injured rat was incised, and the bone marrow-derived cell structure was fixed with a surgical suture and a tissue adhesive (surge cell) (FIG. 3).
(4) Measurement of bladder function A chart of measuring bladder function 2 weeks after transplantation is shown (FIG. 4). The peak sometimes seen in the chart is the timing at which urinary bladder stays and urinates. In the Sham operation group, as shown in the chart, the frequency of peaks is high, and noise is occasionally seen. In contrast, in the group transplanted with bone marrow-derived cells, the peak period during which the bladder pressure increased was prolonged. This indicates that frequent urination has improved and more urine has accumulated in the bladder.

(5) Evaluation of bladder function Furthermore, the result of evaluating the bladder function 2 weeks after transplantation is shown (FIG. 5). Bladder basal pressure (cmH2O) (sham group vs BMC transplantation group = 7.56 vs 8.44), urination threshold pressure (cmH2O) (sham group vs BMC transplantation group = 21.44 vs 27.5) Maximum contraction pressure during urination (cmH2O) (sham group) vsBMC transplantation group = 37.01 vs 44.05), single urination interval time (min) (sham group vs BMC transplantation group = 6.52 vs 6.55), single urination volume (ml) (sham group vs BMC transplantation group = 1.04 vs1) .18), residual urine volume (ml) (sham group vs BMC transplant group = 0.08 vs 0.02), bladder capacity (ml) (sham group vs BMC transplant group = 1.12 vs 1.20). Residual urine was significantly reduced in the BMC transplant group.

(6) Immunohistological staining Rats were dissected two weeks after transplantation, and the results of immunostaining after sectioning the bladder tissue transplanted with bone marrow-derived cell structures were shown. The cells of the transplanted structure expressed GFP and were detected in green. It was shown that bone marrow-derived cells migrated from the transplanted interface to the lesion site.
It was also shown that the constituent cells of the bone marrow-derived cell structure were differentiated into smooth muscle cells (FIG. 7).
Furthermore, among the transplanted bone marrow-derived cells, it was confirmed that the cells on the engraftment surface were being differentiated into cells expressing a smooth muscle marker (FIG. 8).

In order to evaluate changes over a longer period of time than tissue and bladder function evaluation over a long period of time, functional evaluation and tissue evaluation were performed 4 weeks after transplantation.

(1) Method A 10-week-old female SD rat was irradiated with a radiation-protected metal plate hollowed out near the bladder. MBR-1520R-4 (Hitachi Power Solutions Co., Ltd.) was used as the radiation irradiation device, and irradiation was performed 5 times in total at a frequency of 2 Gy / week, and then the animals were reared normally for 4 weeks. After normal breeding for 4 weeks, the structure of bone marrow-derived cells was transplanted by incising and fixing the subcutaneous bladder (n = 4). After transplantation, the cells were observed for 4 weeks and analyzed by bladder function and dissection (FIG. 9).

(2) Results Four weeks after transplantation (upper), in the control group in which the structure was not transplanted, urination intervals were short (frequent urination) or longer (decreased urination efficiency) (upper figure). . In addition, non-voiding contraction of the bladder is observed. On the other hand, regular urination is observed in the bone marrow-derived cell structure transplantation group (see the figure below). In the control group 4 weeks after transplantation (bottom), a clear frequent urination pattern (short urination interval, decreased single urination volume) was observed (upper figure). In the bone marrow-derived cell structure transplantation group, there is a regular urination pattern, and recovery of bladder function is observed (lower figure) (FIG. 10).

FIG. 11 is a diagram showing the results of evaluating various bladder functions. There were no significant changes in the basal bladder pressure, micturition threshold pressure, and micturition contraction pressure in both groups at 2 weeks and 4 weeks after transplantation. In addition, no difference was observed in the comparison between groups. On the other hand, in the control group, once the micturition interval time after implantation 4 weeks ^(** P <0.01), single voided volume ^(* P <0.05), bladder capacity of Sham-operated group ^(* P <0 .05) was significantly reduced compared to 2 weeks after transplantation. However, those parameters in the bone marrow-derived cell structure transplantation group were not significantly different from those after 2 weeks of transplantation. It is interpreted that the symptom of bladder capacity atrophy was alleviated by the transplanted structure being differentiated into the smooth muscle tissue of the bladder. Furthermore, after 4 weeks of transplantation, the interval of single urination († P <0.05) and the amount of single urination († P <0.05) significantly increased compared to the control group. It was high, and the worsening of atrophic bladder symptoms was alleviated. Regarding the amount of residual urine, the structure transplantation group did not change after 2 weeks and 4 weeks after transplantation, and was significantly lower than the control group († P <0.05). Thus, in the state where the atrophic bladder symptom greatly deteriorated 4 weeks after the transplantation, it is suggested that the transplantation therapy of the bone marrow-derived cell structure partially compensates by regenerating the bladder tissue.

FIG. 12 shows the result of individual dissection after evaluation of bladder function, preparation of a pathological section of the bladder and immunostaining, showing differentiation into smooth muscle cells within the transplanted structure. Four weeks after transplantation, the decrease in bladder capacity accompanying the worsening of the atrophy symptom gradually decreased. At that time, the group of cells differentiated into smooth muscle cells formed clusters and formed a smooth muscle layer. On the other hand, a large cluster of smooth muscle layers was not visible 2 weeks after transplantation.
In conclusion, (i) improvement of bladder function is observed, (ii) transplanted cells have differentiated into smooth muscle layers, and (iii) decrease in bladder capacity (volume) is prevented. Since it was confirmed at the same time, it was shown that “the transplanted cells themselves differentiated into bladder tissue to compensate for the missing tissue”.

Following the results shown in FIG. 12, differentiation of smooth muscle cells in the vicinity of the vascular structure was confirmed (FIG. 13). Four weeks after transplantation, a large blood vessel (* indicated portion) having a smooth muscle layer around it was formed. This blood vessel was thought to have been transmitted not from bone marrow-derived cells but from other tissues of the living body.
In thick cell structures, there was a concern that nutrients would be supplied to the entire transplanted cell, but as a result, living blood vessels infiltrated the fiber layer and structure, and the cells in the structure themselves were firmly And was alive. It was shown that the recipient animal differentiated into regenerative tissue while decreasing the bladder fiber layer.

Claims

Low-compliance bladder or atrophic bladder, or a tissue regeneration or function-improving material for low-compliance bladder, comprising a three-dimensional structure having a thickness of at least 500 μm constructed by laminating bone marrow-derived cell spheroids on acicular bodies arranged on a substrate .
The material according to claim 1, wherein the low-compliance bladder is a bladder having a diseased tissue or a bladder having dysuria.
Bladder having pathological tissue has bladder having pathological tissue caused by side effects of radiotherapy, bladder having pathological tissue caused by side effects of drug treatment or surgical treatment, and pathological tissue that has been congenitally or acquired The material according to claim 2, which is at least one selected from the group consisting of bladders.
Bladder with dysuria has bladder with dysuria caused by side effects of radiation therapy, bladder with dysuria caused by side effects of medication or surgical treatment, and dysuria that has been congenital or acquired The material according to claim 2, which is at least one selected from the group consisting of bladders.
The material according to claim 4, wherein the dysuria is frequent urination or residual urine.
Low compliance bladder selected from the group consisting of lower urinary tract with pathological tissue, lower urinary tract with lower urinary tract symptoms, upper urinary tract with pathological tissue, and upper urinary tract with symptoms that impair the function of upper urinary tract The material according to claim 1, wherein the material is at least one.
Lower urinary tract having pathological tissue caused by side effects of radiotherapy, lower urinary tract having pathological tissues caused by side effects of drug treatment or surgical treatment, and congenital or acquired The material according to claim 6, which is at least one selected from the group consisting of a lower urinary tract having a diseased tissue that has developed.
A lower urinary tract having a lower urinary tract symptom, a lower urinary tract having a lower urinary tract symptom caused by a side effect of drug treatment or a surgical treatment, The material according to claim 6, which is at least one selected from the group consisting of a lower urinary tract having a lower urinary tract symptom congenitally or acquired.
Upper urinary tract having pathological tissue caused by side effects of radiation therapy, upper urinary tract having pathological tissues caused by side effects of drug treatment or surgical treatment, and congenital or acquired The material according to claim 6, wherein the material is at least one selected from the group consisting of upper urinary tract having diseased tissue.
Upper urinary tract with symptoms of impaired function, upper urinary tract with symptoms of impaired upper urinary tract caused by side effects of radiation therapy, symptoms of impaired upper urinary tract caused by side effects of drug treatment or surgical treatment The material according to claim 6, which is at least one selected from the group consisting of an upper urinary tract having a symptom, and an upper urinary tract having a symptom of impaired function of the upper urinary tract that is congenital or acquired.
The material according to claim 10, wherein the symptom of impaired function of the upper urinary tract is a symptom of vesicoureteral reflux or hydronephrosis.
The material according to any one of claims 1 to 11, wherein cells constituting the bladder organ, the lower urinary tract organ or the upper urinary tract organ are induced by the paralacrine effect or differentiated into the cells.
A tissue regeneration or function improving material for a tissue having sexual dysfunction, comprising a three-dimensional structure having a thickness of at least 500 μm constructed by laminating bone marrow cell-derived spheroids on acicular bodies arranged on a substrate.
Tissues with sexual dysfunction are sexually functional tissues with pathological tissues that have developed due to side effects of radiation therapy, sexually functional tissues with pathological tissues that have developed due to side effects of drug treatment or surgical treatment, and are congenital or acquired The material according to claim 13, which is at least one selected from the group consisting of sexually functional tissues having a diseased tissue.
The material according to claim 13, wherein the sexual dysfunction is an erectile dysfunction or ejaculation disorder which is mainly male sexual dysfunction.
The material according to any one of claims 13 to 15, which induces or differentiates into cells constituting a sexually functional tissue by a paracrine effect.