EP1868659A1

EP1868659A1 - Method for production of a tissue transplant construct for reconstruction of a human or animal organ

Info

Publication number: EP1868659A1
Application number: EP06722790A
Authority: EP
Inventors: Gouya Ram-Liebig; Ursula Ravens; Manfred Wirth
Original assignee: Technische Universitaet Dresden
Current assignee: Urotiss GmbH
Priority date: 2005-04-13
Filing date: 2006-04-12
Publication date: 2007-12-26
Also published as: DE102005017000B4; US7851217B2; WO2006108408A1; US20110081721A1; DE102005017000A1; US20060233767A1

Abstract

The invention relates to a method for production of a tissue transplant construct for the reconstruction of a human or animal organ. The method comprises the steps: a) isolation and two-dimensional cultivation of organ-specific tissue cells, b) application of the organ-specific tissue cells to a biologically-compatible, collagen-containing membrane and c) cultivation of the organ-specific tissue cells on the membrane with biochemical and mechanical stimulation of the organ-specific tissue cells.

Description

description

A method of making a tissue graft construct for reconstructing a human or animal organ

The invention relates to a method for the production of a tissue transplant construct for the reconstruction of a human or animal organ, to a tissue graft construct of this kind and to the use of such a tissue transplant construct. More particularly, the invention relates to a tissue graft construct for reconstructing the bladder.

The urinary bladder consists of the mucosal layer and the muscular system. Mucosa cells are urothelial cells that cover the inside of the urinary bladder in multiple layers and protect it against urine. The musculature, called the Detrusor, consists of smooth muscle cells and interstitial cells in between, which are fibroblasts or fibroblastic cells. These cells contain a subpopulation of c-Kit positive cells. The muscle cells are for contraction, d. H. the bladder emptying responsible. The interstitial cells are responsible for the transmission of the electrical impulses, which originate from muscle cells, and thus for the function of the muscle cells as a unit.

Reconstruction of the bladder may be necessary due to congenital or acquired diseases such as meningomyelocele, bladder and congestive dystrophy, trauma, malignancy, neuropathic dysfunction, detrusor instability. Reconstruction of the bladder is clinically

M 05 1 534 5. doc the use of intestinal sections. This is associated with multiple complications such as infection, incontinence, malignant degeneration, diarrhea, electrolyte imbalance, malabsorption and increased morbidity. It has been proposed to carry out the reconstruction, ie the replacement of damaged or diseased areas of a bladder, by means of tissue transplant constructs intended to serve as a matrix for the regrowing original tissue. In this context, the cultivation of the patient's own bladder cells on the membrane and thus the in vitro generation of viable bladder tissue has been proposed several times [1, 2, 3]. However, this has a variety of problems.

Furthermore, tissue graft constructs for the reconstruction of a human urinary bladder are known which comprise a biological, membrane-containing membrane to which one or more layers of organ-specific urothelial cells are applied. The outer layer of the urothelial cells is a layer of terminally differentiated tissue cells [4].

However, not only urothelial cells as a barrier against urine are required for the reconstruction of a bladder with respect to their physiological function, but also smooth muscle cells to allow their contraction. However, it has not yet been possible to reconstruct bladder tissue with seeded membranes whose contractility corresponds to that of the native tissue, since complete generation of the muscle cells after implantation was not achieved. One reason for this is presumably that adult smooth muscle cells lose their more differentiated phenotype in vitro [5, 6]. It is also unknown whether smooth muscle

M 05 1 534 5 .doc completely redifferentiate cells after implantation [6]. On the other hand, intercellular communication between smooth muscle cells may be impaired by the lack of interstitial cells in the tissue graft construct, which may lead to deficient dysfunction after construct implantation [7, 8].

The object of the invention is to eliminate the disadvantages of the prior art. In particular, a method for producing a tissue transplant construct is to be specified which, with regard to its physiological contractile functionality, resembles more strongly than hitherto healthy natural tissue.

This object is solved by the features of claims 1, 20, 26 and 27. Advantageous embodiments of the invention will become apparent from the features of claims 2 to 19, 21 to 25.

In accordance with the invention, there is provided a method of making a tissue graft construct for reconstructing a human or animal organ comprising the steps

(a) isolating and two-dimensionally culturing organ-specific tissue cells;

(b) applying the organ-specific tissue cells to a biocompatible, collagen-containing membrane; and

M 05 1 534 5 .doc (c) culturing the organ-specific tissue cells on the membrane under biochemical and mechanical stimulation of the organ-specific tissue cells.

According to the invention, the organ-specific tissue cells are isolated before application to the membrane and cultured two-dimensionally. After the multiplication of the two-dimensionally cultured tissue cells, the tissue cells thus obtained are applied to the membrane and further cultured there under biochemical stimulation and with mechanical stimulation. Biochemical stimulation and mechanical stimulation are preferably performed simultaneously. The mechanical stimulation can take place continuously or at intervals.

The invention is based on the surprising discovery that the mechanical stimulation of the tissue cells cultured on the membrane leads to transplant constructs whose membrane is densely interspersed with tissue cells, the tissue cells having their natural phenotype. The tissue cells are preferably muscle tissue cells.

The term "organ-specific tissue cells" should be understood to mean that tissue cells of the same or a similar organ to be reconstructed are applied to the membrane. If, for example, a bladder is to be reconstructed, then organ-specific tissue cells are to be understood as meaning urothelial cells and / or muscle tissue cells.

Organs are functional units of the body. A preferred example is the bladder.

M 05 1 534 5 .doc The terms "membrane" and "matrix" are used interchangeably herein unless otherwise specified. The membrane should contain the components of the extracellular matrix (ECM), in particular its growth factors. Preferably, the membrane is a biological membrane, more preferably a biological membrane, which is degraded in the body to non-toxic substances and replaced by an endogenous tissue structure.

A collagen-containing membrane is to be understood here as a membrane based mainly on collagen.

Reconstruction is the replacement of damaged or diseased areas of a human or animal organ, especially a bladder.

The tissue cells are preferably muscle tissue cells, more preferably bladder muscle tissue cells. The muscle tissue cells are particularly preferably smooth muscle cells and interstitial cells (i.e., fibroblasts or fibroblastic cells containing a subpopulation of c-kit positive cells) of the muscle layer of the bladder in their natural relationship. Bladder muscle tissue cells are also referred to below as detrusor tissue cells or detrusor cells.

Biochemical stimulation is understood to mean urothelial induction and serum induction. A preferred example of biochemical stimulation is the mitogenic stimulation of the urothelium and the serum.

M 05 1 534 5. doc In a preferred embodiment, the muscle tissue cells, after being applied to the membrane, are cultured under mitogenic stimulation. Preferably, the muscle tissue cells applied to the membrane are two-dimensionally cultured muscle tissue cells from passages 2 through 7, more preferably 3 through 6, most preferably passage 3.

Culturing the muscle tissue cells in step (c) should be done with urothelial induction and adapted serum concentration. Urothelial induction can be achieved by using a medium that has been conditioned by urothelium. The medium may be a medium conditioned with proliferating urothelium or a medium conditioned with terminally differentiated urothelium.

By "proliferating urothelium" or "proliferative urothelium" is meant urothelial cells that have proliferated on an acellular membrane in a culture medium with a constant FCS addition (for example, 5%). The urothelial cells were then incubated in DMEM and the medium harvested. This medium is referred to as a proliferating urothelium conditioned medium. This medium can then be supplemented with FCS.

By "terminally differentiated urothelium" is meant urothelial cells that have proliferated on an acellular membrane in a culture medium with a decreasing FCS addition (for example, from 5% to 1%) under fibroblastic induction. The urothelial cells were then incubated in DMEM and the medium harvested. This medium is considered to be terminal

M 05 1 534 5 .doc differentiated urothelium conditioned medium. This medium can then be supplemented with FCS.

The urothelium may originate from the mucosal layer of the urinary bladder, preferably the bladder, from whose muscle layer the muscle tissue cells have been taken. Fibroblasts used to induce terminally differentiated urothelial cells can be obtained from the submucosal layer of the same urinary bladder.

The culture of the muscle tissue cells in step (c) should be in a urothelium conditioned medium which further contains fetal bovine serum (FCS) or autologous serum. Preferably, the medium contains 5% FCS or autologous serum. In a preferred embodiment, the concentration of FCS or autologous serum is gradually reduced to 0% during culture of the muscle tissue cells on the membrane.

Two-dimensional cultivation is understood to mean culturing in a plane, for example on the bottom of a culture vessel. Three-dimensional cultivation is understood as culturing in a three-dimensional space, for example in such a way that the tissue cells cover the membrane surface and also pass through the membrane.

The mechanical stimulation in step (c) of the method according to the invention can be carried out by stretching the membrane to which the tissue cells are applied. The membrane can be stretched statically and / or cyclically. Thus, this can be mechanical stimulation

M 05 1 534 5 .doc (cl) stretching the membrane for a given period of time;

(c2) the relaxation of the membrane for a given period of time; and

(c3) repeating n steps of steps (cl) and (c2), where n is an integer greater than or equal to 1.

When mechanical stimulation is performed by stretching the membrane, the membrane is preferably stretched along its longitudinal axis.

In a preferred embodiment, the muscle tissue cells and the urothelium are from a urinary bladder, most preferably from the same urinary bladder.

The invention is particularly suitable for the production of a tissue graft construct for the reconstruction of the urinary bladder. A method of making a tissue transplant construct for reconstruction of the bladder

(a *) isolating and two-dimensionally cultivating muscle tissue cells of the bladder;

(b *) applying the muscle tissue cells to a biocompatible, collagen-containing membrane; and

(c *) cultivating the muscle tissue cells on the membrane under urothelial induction and adapted serum concentration as well as mechanically stimulating the muscle tissue cells.

M 05 1 534 5 .doc The muscle tissue cells include smooth muscle cells and interstitial cells, preferably in their natural proportion.

Preferably, the muscle tissue cells of the bladder, after being applied to the membrane, are cultured under mitogenic stimulation. More preferably, the muscle tissue cells of the urinary bladder applied to the membrane are two-dimensionally cultured muscle tissue cells of passage 2 to 7, more preferably 3 to 6, most preferably passage 3. It should be noted that with increasing two-dimensional passenger count the cells reduce their multiplication potential.

The culture of the bladder muscle tissue cells in step (c *) should be performed in a medium that is a urothelium conditioned medium, preferably a proliferating urothelium conditioned medium. Preferably, the concentration of serum (FCS or autologous serum) is gradually reduced during culturing, for example in three steps, for example from 5% to 1% to 0%.

The muscle tissue cells used to construct a transplant construct for reconstruction of the bladder are smooth muscle cells and interstitial cells, preferably in their natural ratio as found in the muscle layer of a natural healthy bladder. From the muscle layer of a bladder, the smooth muscle cells and interstitial cells can be obtained by the muscle layer of urothelium, lamina propria and serosa

M 05 1 534 5 .doc is released. The treated muscle layer is then minced and treated with collagenase. The individual cells can then be recovered by centrifugation, cultured two-dimensionally and passaged at confluency.

By means of the method according to the invention, it is possible for the first time to obtain tissue transplant constructs which have biocompatible membranes penetrated by tissue cells with a native phenotype. Due to the high degree of penetration, the membranes have a three-dimensional network of tissue cells, ie. in the case of bladder muscle tissue cells, a three-dimensional network of smooth muscle cells and interstitial cells. The presence of the interstitial cells promotes smooth muscle cell intercellular communication and is therefore of importance for the physiological functionality, in particular the contractility, of the graft construct after its implantation.

In a preferred embodiment, the method according to the invention comprises the isolation of muscle tissue cells from a bladder biopsy; the two-dimensional proliferation of muscle tissue cells in the culture flask; applying the passage 3 proliferated muscle tissue cells (P3 cells) to the surface of a biocompatible membrane; and the activation of the P3 cells under

(i) urothelial induction using a medium conditioned with proliferating urothelium and

(ii) serum induction and

(iii) mechanical stimulation.

M 05 1 534 5. doc When urothelial induction is performed for 32 days, serum induction involves reducing the serum concentration from 5% (days 1 to 14) to 1% (days 15 to 28) and then further to 0% (days 29 to 32). Mechanical stimulation is maintained throughout the entire period of urothelial induction (32 days) with the membrane preferably being stretched and relaxed cyclically.

The membrane surface is preferably up to 40 cm ^{2 in} size.

According to the invention, a tissue transplant construct for the reconstruction of a human or animal organ is also provided, which

(a) a biocompatible, collagen-containing membrane; and

(b) organ-specific tissue cells cultured on the membrane under biochemical and mechanical stimulation.

The tissue graft construct according to the invention is thus a contractible tissue graft construct.

Such a tissue graft construct can be obtained by means of the method according to the invention. Preferably, the organ-specific tissue cells are muscle tissue cells of a bladder. The muscle tissue cells of the bladder are smooth muscle cells and interstitial cells, preferably in their natural ratio.

M 05 1 534 5 .doc The tissue graft constructs according to the invention can be used for the reconstruction of a human or animal organ, preferably for the reconstruction of a human or animal bladder.

The beneficial results are based on the following findings: After applying the tissue cells to the membrane, the dynamic process of cell migration to the membrane was the response to the migration signals provided by the matrix of the membrane. Obviously, there are receptive proteins on the cell surface that communicate these migration signals into the interior of the cell.

Among the mitogenic effects of FCS, cells of passage 3 (so-called P3 cells) were able to confluently cover membrane surfaces of up to 40 cm ² within a few days, while covering the membrane by P7 cells of similar biopsy size, ie cells after seven passages in the culture flask under conventional conditions) was not complete. These results suggest that in two-dimensional cultures under conventional conditions in the culture flask the cells increasingly lose their proliferative capacity with increasing number of passages.

A major function of SMC is contraction. By de-differentiation, two-dimensional cultured bladder SMCs lose their contractility response to various in vivo agonists under conventional conditions [5]. On the other hand, the potential to redifferentiate in vitro dedifferentiated cells is unknown [6]. Considering that cellular tissue engineering with bladder

M 05 1 534 5 .doc SMCs is intended for clinical applications, the tissue cells should have their corresponding phenotype for the function of the graft when implanted in the host. For this reason, already at passage 3, the tissue cells were sown on three-dimensional biomaterials to enable them to at least partially maintain their differentiated phenotype by proliferating early enough in three dimensions through interaction with the supporting extracellular matrix. As a result, the physiological environment affected cell behavior [9, 10, H]. Indeed, when tissue cells were cultured two-dimensionally under conventional conditions for seven passages before being applied to the membrane, only less than 5% of the tissue cells stained positive with desmin and SMA antibodies, while about 20% of the P3 cells desmin and SMA expressed when cultured under the same conditions.

In bladder regeneration, epithelial-stromal interactions are known to regulate SMC proliferation and differentiation [12, 13]. To find out how detrusor cells respond to epithelial stimuli on acellular membranes, tissue cells were cultured that were cultured with media conditioned by urothelium (that is, urinary bladder epithelial cells). On the other hand, proliferative and terminally differentiated urothelium were used for this purpose. In fact, proliferative urothelium had a stronger proliferative effect on the cell cultures, while only a smaller effect was achieved by terminally differentiated urothelium. In any case, the urothelium-conditioned medium increased with proliferative urothelial cells and gradually reduced concentration and later

M 05 1 534 5 .doc the absence of serum [14], the desmin and SMA protein expression, although this could not fully induce desmin and SMA protein expression (60%).

It is known that mechanical stress affects the SMC in different ways, i. H. Proliferation, differentiation, formation of extracellular matrix and growth factors induce [15-19]. Interestingly, some of these effects are characteristic of a more differentiated phenotype, while others are more proliferating on a more synthetic, ie. H. indicate less differentiated condition. With the present invention, a more overgrown tissue with increased expression of desmin and SMA was achieved by mechanical stimulation. In addition, the tissue cells showed a different motility pattern than under conditions without mechanical stimulation. In the latter case, more persistent cells mostly moved in two directions across the membrane surface. Under mechanical stretching and relaxation, less persistent cells penetrated in several directions into the interior of the membrane by overcoming the matrix resistance by degrading their three-dimensional structure. The use of mechanical stimulation in urothelium-conditioned cultures allowed the SMCs to show their differentiated phenotype when the mitogenic effect of FCS was reduced and subsequently eliminated. In this case, about 80% of the tissue cells were positive for desmin and SMA. Desmin and SMA are markers for the differentiated phenotype of SMC.

The insoluble signals of the three-dimensional extracellular matrix, which interact with the soluble factors of the urothelium-conditioned medium, and mechanical forces in the

M 05 1 534 5 .doc Gradually adjusted serum concentrations promoted the migration, division and differentiation of SMC. The induction of SMC differentiation in response to the urothelium-conditioned medium is characteristic of an endogenous regulatory mechanism. In addition, the results of the present invention demonstrate that SMC registers the stress that also induces cell differentiation. These results indicate that the effects of the mechanical environment point to ways other than growth factor / receptor binding. Based on these findings, it is possible to develop effective cellular strategies for more proliferative or differentiated activity of tissue cells to improve tissue development.

The detrusor is known to have a large network of fibroblasts. In the examples described below, approximately 20% of the isolated detrusor cell population was interstitial cells that were positive for vimentin but negative for desmin and SMA, confirming results [8] that suggest it from immunoreactivity and ultrastructural analyzes, that these cells are fibroblasts. The c-kit immunoreactivity was additionally demonstrated in a subpopulation of interstitial cells. The interstitial cells showed only weak immunoreactivity against biochemical and mechanical stimuli, which induced the differentiation of the SMC. Since it is believed that the propagation of activating impulses in the bladder is mediated by interstitial cells, the presence of these cells in our model system may improve the features of graft functionality after implantation.

M 05 1 534 5 .doc The invention will be explained in more detail by means of examples with reference to the drawings. Show it

1 shows photographs for the characterization of bladder tetrorrystal cells on an acellular tissue membrane (FIG.

IA: staining with BCECF, 40X magnification; FIG.

IB: staining of cellular DNA with DAPI, 100 fold

Magnification);

Fig. 2 shows immunohistochemical staining of passage tetrusor cell cultures of passage 3 on acellular membranes using antibody for alpha smooth muscle actin (Fig. 2A: cultivation without urothelial induction and mechanical stimulation; Fig. 2B: cultivation under urothelial induction with reduction of serum concentration; 2C: Cultivation under urothelial induction and mechanical stimulation (10% of the membrane surface) with serum reduction, day 32, FIGS. 2D to 2F: penetrating SMC in the membrane on culture day 21 below

Maintaining its differentiated phenotype (positive reaction with alpha-smooth muscle actin antibody);

Fig. 3 Hematatoxylin and eosin staining with blast detrusor cells cultured under urothelial induction and without mechanical stimulation on day 18 on the membrane (Fig. 3A: Cultivation of bladder tetrusor cells under induction with terminally differentiated urothelial medium Fig. 3B: Cultivation of bladder tetrusor cells under induction with proliferating urothelium-conditioned medium);

M 05 1 534 5 .doc Fig. 4 hematatoxylin and eosin staining of urothelial induction and mechanical stimulation

(10% of the membrane surface) cultured bladder detrusor cells on day 18 on the membrane (Figures 4A and 4B show the movement of the cells within the membrane)

Membrane);

Fig. 5 Hematatoxylin and eosin stains of the bladder detrusor cells cultured under urothelial induction and mechanical stimulation (10% of the membrane surface) on the membrane (Fig. 5A: cell layers on the membrane Fig. 5B: penetration of the cells within the membrane in Fig different directions; Fig. 5C: fully penetrated membrane on day 32); and

FIG. 6 shows immunohistochemical SMA staining of the bladder trastorus cells on the membrane, which were cultured under urothelial induction and mechanical stimulation (10% of the membrane surface). Compared to SMC, the fibroblastic cells did not stain with alpha-smooth muscle actin antibody and did not show the corresponding brown color.

Examples

The following examples illustrate the process of the invention for the preparation of urinary bladder tissue transplant constructs.

M 05 1 534 5 .doc Unless otherwise stated, the abbreviations used have the following meanings:

BCECF 2 ', 7' bis (2-carboxyethyl) -5 / 6-carboxyfluorescein DAPI 4 '-β-diamidino-2-phenylindole

DMEM Dulbecco's Modified Eagle's Medium,

ECM extracellular matrix,

FCS fetal bovine serum

PI propidium iodide SIS small intestinal submucosa

SMA alpha-smooth muscle actin

SMC smooth muscle cells

Materials and methods

Preparation of Acellular Tissue Membranes: Acellular tissue membranes (also referred to herein as acellular membranes) were prepared from porcine bladder, aorta, small intestine and skin samples. They were obtained in Triton X 1% (Sigma-Aldrich, Taufkirchen, Germany) for 24 to 48 h in the presence of 0.1% sodium azide with stirring in a water bath at 37 ⁰ C acellular. Extraction of all cellular elements was confirmed histologically. In addition, lyophilized small intestinal submucosa (SIS) was provided by Cook Biotech (Mönchengladbach, Germany).

Cell Isolation, Culture and Characterization: The muscle layer of porcine (n = 8) and human (n = 4) urinary bladder samples (0.5 x 0.5 cm) was dissected from the urinary epithelium, lamina propria and serosa, minced and collagenase B 0 5% 2 h (Worthingto Biochemical, Lakewood, NJ, USA). Single cells were collected by centrifugation.

M 05 1 534 5 .doc melt, and the pellets were transferred to culture flasks containing DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 5% FCS. The cells were cultured to confluency. P3 and P7 cells were cultured on the surfaces of acellular membranes (up to 40 cm ² ). The cultured cells were histologically and immunohistochemically stained using hematoxylin and eosin stain (H & E), SMA, vimentin and desmin antibodies (all Dako, Glastrup, Denmark) and c-kit antibodies (Sigma-Aldrich; Germany). The apoptosis index was determined with 4'-6-diamino-2-phenylindole (DAPI), the vitality of the cells with 2 ', 7' bis-carboxyethyl-5 and 6-carboxyfluorescein (BCECF) and propidium iodide (PI) (both MoBiTec , Göttingen, Germany).

Urothelium-conditioned media. To produce urothelium-conditioned media, urothelial cells (obtained from the mucosal layer of samples from the same urinary bladder were cultured on the surface of acellular membranes.) As in [4], urothelial cells were maintained in two distinct groups, either in a proliferative phenotype in the presence of 5% FCS or terminally differentiated by reducing the FCS concentration to 1% with fibroblastic induction (from the lamina propria of the same urinary bladder). Urothelial cells were then incubated in DMEM for 24 h and the conditioned media were harvested Scaffolds were each fed with medium conditioned with proliferating or terminally differentiated urothelium at the FCS levels discussed below.

FCS-conditioned media. FCS became urothelium-conditioned media, i. H. either with proliferative

M 05 1 534 5 .doc or supplemented with terminally differentiated urothelium conditioned medium, at a constant concentration of 5% from day 1 to day 32. Alternatively, FCS was reduced in three steps: 5% from day 1 to 14, 1% to day 28 and then serum-free for 4 days. In the control groups, cells were cultured with media containing the same serum concentrations but lacking urothelial conditioning. The culture media were changed every three days, except in serum-free conditions, where they were changed on a daily basis to ensure the availability of sufficient nutrients to the cells.

Mechanical stimulation. A special bioreactor was used to detect the effects of mechanical stimulation on the membrane-cell composites. Cell-sutured membranes were stretched by 5, 10, and 20%, respectively, of the surface, causing passive stretching of the cells. The device consisted of a motor driven system that allowed simultaneous mechanical stretching of four rectangular shaped membranes (Figure 1). The membrane-cell composites, d. H. the membrane to which the tissue cells have been applied was placed in parallel and in sterile chambers. The media were injected separately through a roller pump into the different chambers at 1 ml / h and collected on the opposite side in a sterile bottle. The cultures were supplied with 95% air and 5% CO2 at 37 ° C. The membranes were uniformly stretched in the horizontal direction and attached to each side between two plates. The effects of mechanical stretching were studied under continuous elongation and under cyclic elongation and relaxation (20 sec.

M 05 1 534 5 .doc and 10 s relaxation or 10 s stretch and 10 s relaxation).

The mechanical stretching can be carried out by means of a bioreactor. Details of the bioreactor are described in DE 101 51 822.

Results

The detrusor cells freshly isolated from the porcine or human urinary bladder had a spindle-shaped morphology, with approximately 80% of the cells staining positive with antibodies to desmin and SMA, and about 20% were vimentin-positive cells showing no desmin and SMA expression , 15 to 20% of these cells stained positive with c-kit antibody. The fraction of desmin and SMA positive cells decreased with the increasing number of passages under 2D conditions and was less than 60% at passage 7, with a variability between different animals and patients was detected.

There were no large differences between cell cultures on the matrices from different sources. Fluorescence microscopic analyzes (Figure 1) showed that the cells adhered to the scaffold for up to 32 days and remained viable (> 80%). DAPI staining of cultured cells on the membranes revealed nuclei with an apoptosis rate of less than 20%.

Fig. 1 shows images for characterizing the tissue cells on the membrane. Figure IA shows, at 40X magnification, an assay for determining the viability of tissue cells on the membrane. The dye used was BCECF (green color). The fluorescent dye BCECF is specific for living cells and showed a high cell vitality of the tissue

M 05 1 534 5 .doc cells 32 days after the application of tissue cells to the membrane. Fig. IB shows in 10X magnification staining of the cellular DNA with DAPI. Recognizable are several dividing cell nuclei on the membrane 14 days after application of the tissue cells.

Histological analysis showed that P3 cells were evenly distributed on the surface of the membrane five days after application, while P7 cells obtained from similarly sized biopsies show only partial and less uniformly distributed growth. At day 32, the cell populations on the membranes were nearly 10-fold higher with P3 cells than with P7 cells. However, the percentage of cells staining positive for desmin and SMA decreased substantially to about 20% in cultures with P3 cells and to 5% in cultures with P7 cells when the cells on the surface of larger membranes (> 10 cm ² ) were cultivated.

Although the removal of the serum resulted in the suppression of cell proliferation and migration, it did not effectively induce differentiation of SMC on the membranes (20 to 30% expression of desmin and SMA in P3 cells, 5 to 10% in P7 cells by day 32).

FIGS. 2A to 2F show recordings of immunohistochemical analyzes of membrane-cell composites using SMA antibodies, each in 20-fold magnification. Passage 3 tissue cells were transferred to the membrane. Fig. 2A shows cultured tissue cells at rest, i. H. Tissue cells without urothelial induction and without mechanical

Stimulation were cultured. These tissue cells showed about 20% positive staining with SMA antibody after 32 days

(Brown colour) . Fig. 2B shows that about 60% of the tissue cells have a differentiated (mature) phenotype on the membrane surface.

M 05 1 534 5 .doc area on day 32 after serum removal, when the tissue cells were cultured under medium conditioned with proliferative urothelium. Figures 2C to 2F show cells cultured on the membrane surface under urothelial induction and mechanical stimulation. More than 80% of the cells show positive responses with SMA antibody on day 32 after serum removal (Figure 2C). In serum-depleted medium (1% FCS), on day 21, urothelial and mechanical stimulation led tissue cells to the interior of the membrane, with the tissue cells retaining their differentiated phenotype (Figures 2D-2F).

Cell proliferation and migration was induced by a medium that had been conditioned with proliferating urothelium. In fact, as early as day 18, the PS cells cultured in this medium formed 3 to 5 layers on the surface of the membrane, whereas cultures fed with medium conditioned with terminally differentiated urothelium formed only 1 until 2 layers of cells formed (Figure 3).

Figures 3A and 3B show images of H & E staining of cultured membrane-cell composites on day 18 after application of the P3 tissue cells to the membrane at 20x magnification. In the culture system, conditioned with terminally differentiated urothelium without mechanical stimulation, the tissue cells formed 1 to 2 layers and then penetrated the membrane (Figure 3A). Compared to this system, the medium conditioned with proliferating urothelium induces a higher proliferation of PS tissue cells and penetration of the membrane (Figure 3B). In fact, 3 to 5 cell layers on the surface of the

M 05 1 534 5 .doc Membrane can be identified, which is also interspersed with more strongly pene- trated cells (Fig.3B).

The percentage of desmin and SMA positive cells in the second group (ie cells cultured with proliferative urothelial medium) was about 60% at day 32 (Figure 4B) compared to 40% in the first group (ie cells , which were cultured with terminally differentiated urothelial medium), when the serum was depleted in the three steps. Under the second conditions (with proliferating urothelium conditioned medium), the additional mechanical stretching not only induced cell penetration and migration in different directions in the presence of serum (day 1 to 28, Figures 4, 5) but also increased the level of desmin and SMA-positive cells (Figs. 2C to 2F). In this culture system, over 80% of the PS cells stained positive for desmin and SMA on day 32 when the serum is reduced in the three steps (Figure 2C) (10% extension of the membrane surface). The remaining 20% of the cell population (interstitial cells) stained positive with the veinin antibody, while they stained negative for SMA and showed only weak sensitivity to the mechanical deformations and biochemical stimuli (Figure 6). A subpopulation (15-20%) of these cells stained positively with C-Kit antibody. Thus, the presence of C-kit positive

Cells in the constructs confirmed. The proportion of desmin and SMA-positive cells under the same conditions

(urothelial induction and three-step FCS reduction) was 66% and 69%, respectively, in the mechanical elongation of the constructs by 5% and 20% of the membrane surface, respectively.

M 05 1 534 5 .doc In cultures with constant serum concentration (5% FCS, day 1 to 32), P3 cells migrated into the tensioned membrane when fed with urothelium-conditioned medium but showed only partial desmin and SMA expression (40%). Desmin and SMA positive cells in proliferating urothelium conditioned medium and 30% desmin and SMA positive cells in medium differentiated with terminally differentiated urothelium).

Figures 4A and 4B show images of H & E staining of the P3 cells on the acellular membrane at 20x magnification. The spatial cell movement in the interior of the three-dimensional structure of the acellular membrane is visible under urothelial induction and mechanical stimulation on day 18 after application of the cells to the membrane (FIGS. 4A, 4B). The cells abolish cell-cell adhesion during migration. The degradation of the extracellular matrix by the migrating cells could be detected. Of particular note is the elongated shape of the migrating cells under extension.

Figures 5A to 5C show further images of H & E staining of the membrane-cell composites in 20X magnification. Visible is the penetration of the membrane through the P3 tissue cells under urothelial induction and mechanical stimulation (10% of the membrane surface). The highly mobile cells moved on the entire surface of the membrane and formed many layers thereon (Figure 5A). They strongly penetrated the interior of the membrane in different directions and initially maintained cell-cell adhesion (Figure 5A), degrading the extracellular matrix components and overcoming their spatial barrier

M 05 1 534 5 .doc (Figs. 5A to 5C). The membrane was fully penetrated by tissue cells at day 32 after serum removal (Figure 5C).

6 shows a picture of an SMA staining of a membrane-cell composite. Evident are the cells that have penetrated into the membrane. The interstitial cells did not stain positively with SMA antibody under urothelial induction and mechanical stimulation. In comparison, the smooth muscle cells showed a positive reaction with the antibody (brown color) under the same conditions. The interstitial cells are indicated by arrows.

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M 05 1 534 5 .doc 6 Kropp BP, Zhang Y, Tomasek JJ, Cowan R, Furness PD 3rd, Vaughan MB, Parizi M, Cheng EY. Characterization of cultured bladder smooth muscle cells: assessment of in vitro contrac- tility. J Urol, 162: 1779, 1999

7 Hashitani H, Yanai Y, Suzuki H. Roei of interstitial cells and gap junctions in the transmission of spontaneous Ca2 + signals in detrusor smooth muscles of the guinea-pig urinary bladder. J Physiol, 559: 567, 2004

8 Drake MJ, Hedlund P, Andersson KE, Brading AF, Hussain I, Fowler C. Morphology, phenotype and ultrastructure of fibroblastic cells from normal and neuropathic human detrusor: absence of myofibroblast characteristics. J Urol, 169: 1573, 2003

9 Bottger BA, Hedin U, Johansson S, Thyberg J. Integrin-type fibronectin receptors of rat arterial smooth muscle cells: isolation, partial characterization and role in cytoskeletal organization and control of differentiated properties. Differentiation, 41: 158, 1989

10 Hedin U, Bottger BA, Luthman J, Johansson S, Thyberg J. A Substrate of the cell attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype , Dev Biol, 133: 489, 1989

11 Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Various effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol _. 107: 307, 1988

M 05 1 534 5 .doc 12 Baskin LS, Sutherland RS, Thomson AA, Hayward SW, Cunha GR. Growth factors and receptors in bladder development and obstruction. Lab Invest, 75: 157, 1996

13 Liu W, Li Y, Cunha S, Hayward G, Baskin L. Diffusible growth factors induce bladder smooth muscle differentiation. In Vitro Cell Dev Biol Anim, 36: 476, 2000

14 Camoretti-Mercado B, Liu HW, Halayko AJ, Forsythe SM, KyIe JW, LiB. Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor. J Biol Chem, 275: 30387, 2000

15 Stegemann J.P., Nerem R.M. Effect of mechanical stimulation on smooth muscle cell proliferation and phenotype. BED,

Vol.50, 2001 Bioengineering Conference. ASME 2001

16 Galvin DJ, Watson RW, Gillespie JI, Brady H, Fitzpatrick JM. Mechanical stretch regulates cell survival in human bladder smooth muscle cells in vitro. At J Physiol Renal Physiol, 283: F1192, 2002

17 Albinsson S, Nordstrom I, Hellstrand P. Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization. J Biol Chem, 279: 34849, 2004

18 Zeidan A, Nordstrom I, Albinsson S, Malmqvist U, Sward K, Hellstrand P. Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors. Am J Physiol Cell Physiol, 284: C1387, 2003

19 Upadhyay J, Aitken KJ, Damdar C, Bolduc S, Bagli DJ. Ingrins expressed with bladder extracellular matrix after stretch injury in vivo mediate bladder smooth muscle cell growth in vitro. J Urol, 169: 750, 2003

M 05 1 5345 .doc 20 Sjuve R, Haase H, Ekblad E, Malmqvist U, Morano I, Amer A. Increased expression of non-muscle myosin heavy chain B in connective tissue cells of hypertrophic rat urinary bladder. Cell Tissue Res, 304: 271, 2001

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Claims

claims

A method of making a tissue graft construct for reconstructing a human or animal organ comprising the steps

(a) isolating and two-dimensionally cultivating organ-specific tissue cells;

(c) culturing the organ-specific tissue cells on the membrane under biochemical and mechanical stimulation of the organ-specific tissue cells.

2. The method according to claim 1, characterized in that the tissue cells are muscle tissue cells.

3. The method according to claim 1 or claim 2, characterized in that the muscle tissue cells are muscle tissue cells of the bladder.

4. The method according to claim 2 or claim 3, characterized in that the muscle tissue cells, after they are applied to the membrane to be cultured under mitogenic stimulation.

5. The method according to claim 4, characterized in that the muscle tissue cells applied to the membrane

M 05 1 534 5 .doc be, under mitogenic stimulation cultured muscle tissue cells of the passage 2 to 7 are.

6. The method according to claim 4, characterized in that the muscle tissue cells are cultured muscle tissue cells of the passages 3 to 6.

7. The method according to any one of claims 2 to 6, characterized in that the culturing of the muscle tissue cells in step (c) takes place under urothelial induction.

8. The method according to claim 7, characterized in that for urothelial induction a conditioned with proliferating urothelium medium is used.

9. The method according to claim 7, characterized in that for urothelial induction using a terminally differentiated urothelium conditioned medium is used.

10. The method according to any one of claims 7 to 9, characterized in that the urothelium comes from the mucosal layer of the bladder.

11. The method according to any one of claims 7 to 10, characterized in that the culturing of the muscle tissue cells in step (c) takes place in a medium conditioned by urothelium, which further contains fetal bovine serum (FCS) or autologous serum.

12. The method according to claim 11, characterized in that the culture medium contains 5% FCS or autologous serum.

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13. The method according to claim 12, characterized in that the concentration of FCS or autologous serum during the cultivation of muscle tissue cells on the membrane is gradually reduced to 0%.

14. The method according to any one of the preceding claims, characterized in that the mechanical stimulation in step (c) comprises stretching the membrane to which the tissue cells are applied.

15. The method according to claim 14, characterized in that the mechanical stimulation

(cl) stretching the membrane for a given period of time;

(c2) the relaxation of the membrane for a given period of time; and

16. The method according to claim 14 or claim 15, characterized in that the membrane is stretched along its longitudinal axis.

17. The method according to any one of claims 7 to 16, characterized in that the muscle tissue cells and the urothelium originate from a bladder.

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18. The method according to claim 17, characterized in that the muscle tissue cells and the urothelium originate from the same urinary bladder.

19. The method according to any one of claims 2 to 18, characterized in that the muscle tissue cells are smooth muscle cells and interstitial cells.

20. A tissue graft construct for reconstructing a human or animal organ, comprising

(a) a biocompatible, collagen-containing membrane; and

(b) organ-specific tissue cells cultured on the membrane under biochemical and mechanical stimulation and biochemically and mechanically stimulated.

The tissue graft construct of claim 20, comprising c-kit positive cells.

22. A tissue transplant construct according to claim 20 or 21, obtained by the method according to one of claims 1 to 19.

23. A tissue transplant construct according to any one of claims 20 or claim 22 for the reconstruction of a human or animal bladder, characterized in that the organ-specific tissue cells are muscle tissue cells of a bladder.

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24. A tissue transplant construct according to claim 23, characterized in that the muscle tissue cells of the bladder are smooth muscle cells and interstitial cells.

25. A tissue transplant construct according to claim 23 or claim 24, obtained by the method according to any one of claims 2 to 19.

26. Use of a tissue transplant construct according to any one of claims 20 to 22 for the reconstruction of a human or animal organ.

27. Use of a tissue transplant construct according to one of claims 23 to 25 for the reconstruction of a human or animal bladder.

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