IMPLANTABLE CELL DRESSING FOR TREATMENT OF DISEASE

Abstract

The present invention provides a multilayer matrix comprising at least two layers in which the first layer comprises a bioresorbable material and the second layer comprises a bioadhesive material, wherein the second layer contains cells. A method for producing a multilayer matrix and the use of a multilayer matrix are also provided.

Claims

1. A multilayer matrix comprising at least two layers in which the first layer comprises a bioresorbable material and the second layer comprises a bioadhesive material, wherein the second layer contains cells.

2. The multilayer matrix according to claim 1, wherein the percentage of cells exhibiting positive to CD105, CD73 and CD90 in the cells is 50% or more, and the percentage of cells exhibiting positive to CD45 and CD34 in the cells is 10% or less.

3. The multilayer matrix according to claim 1, wherein the percentage of cells exhibiting positive to CD142 in the cells is 50% or more, and the percentage of cells exhibiting positive to CD106 in the cells is 10% or less.

4. The multilayer matrix according to claim 1, wherein the cells are mesenchymal stem cells.

5. The multilayer matrix according to claim 1, wherein the mesenchymal stem cells are derived from fetal appendage.

6. The multilayer matrix according to claim 1, wherein a cell density comprised in the second layer is 0.310.sup.6 cells/cm.sup.2 or more.

7. The multilayer matrix according to claim 1, wherein the administrating area of the multilayer matrix per body weight is 0.1 cm.sup.2/kg or more and 6.0 cm.sup.2/kg or less.

8. The multilayer matrix according to claim 1, wherein the second layer comprises at least two bioadhesive materials.

9. The multilayer matrix according to claim 1, wherein the bioadhesive material is at least one which is selected from a group consisting of fibrinogen, thrombin, gelatine, laminin, integrin, fibronectin and vitronectin

10. The multilayer matrix according to claim 1, wherein the second layer further contains dimethyl sulfoxide and albumin.

11. The multilayer matrix according to claim 1, wherein the bioresorbable material is at least one which is selected from a group consisting of collagen, gelatine, cellulose, cellulose acetate, chitosan, chitin, polylactate, hyaluronic acid, and polyglycolic acid.

12. A method for producing a multilayer matrix comprising at least two layers in which the first layer comprises a bioresorbable material and the second layer comprises a bioadhesive material, wherein the second layer contains cells, which comprises steps of; A) preparing a cell suspension and B) applying the cell suspension to the second layer of the multilayer matrix.

13. A method for treatment of lesion or trauma to a tissue or organ of a subject comprising applying the multilayer matrix of claim 1 to the lesion or trauma.

Description

[0122] The invention will now be further described by way of reference to the following examples and drawings which are presented for the purposes of illustration only. In the examples, reference is made to a number of figures in which

[0123] FIG. 1 shows the procedure for cell transplantation using Seprafilm (a single layer material).

[0124] FIG. 2 shows unsuccessful results of cell transplantation using Seprafilm (a single layer material) using 1010.sup.6 rat bone marrow mononuclear cells (BMMNCs). BMMNC-Seprafilm complex was transplanted onto the rat heart surface at 28 days after myocardial infarction (SF-BMMNC group). Control group received sham operation at 28 days after myocardial infarction. Cardiac function was measured by echocardiography at Day 28 after treatment.

[0125] FIG. 3 shows unsuccessful results of cell transplantation using Seprafilm and mesenchymal stem cells. 410.sup.6 rat bone marrow-derived mesenchymal stem cells (MSCs) were transplanted onto the rat heart surface with aid of Seprafilm at 28 days after myocardial infarction (SF-MSC group). Control group received sham operation at 28 days after myocardial infarction. Cardiac function was measured by echocardiography at Day 28 after treatment.

[0126] FIG. 4 shows donor cell leakage after Seprafilm (SF)-aided cell delivery. A certain number of donor cells (labelled with red fluorescent dye, CM-Dil) retained on the heart surface after SF-aided cell delivery (FIGS. 4(a) & 4(b)). Following epicardial placement of Dil (pink)-labelled BMMNCs (FIG. 4(c)) with aid of Seprafilm, cells were collected from the pericardial fluid. Many donor BMMNCs were found leaked (dropped off) in the pericardial cavity (yellow arrows).

[0127] FIG. 5 shows the structure of a TachoSil (multi-layered fibrin sealant patch).

[0128] FIG. 6 shows the method for TachoSil-aided epicardial placement of stem cells.

[0129] FIG. 7 shows therapeutic efficacy of epicardial placement of TachoSil-mesenchymal stem cells (MSC) at Day 28 in a rat acute myocardial infarction model. 110.sup.6 rat fetal membrane-derived MSCs were transplanted onto the rat heart surface with aid of TachoSil at 1 hour after myocardial infarction (TS group). Control group received sham operation at 1 hour after myocardial infarction. Cardiac function was measured by echocardiography at Day 28 after treatment.

[0130] FIG. 8 shows the macroscopic observation at Day 5 after TachoSil-MSC therapy in a rat acute myocardial infarction model. At Day 5 after epicardial placement of 110.sup.6 rat fetal membrane-derived MSCs with aid of TachoSil, TachoSil-MSC complex was found to be firmly adhered to the heart surface.

[0131] FIG. 9 shows the macroscopic observation at Day 28 after TachoSil-MSC therapy in a rat acute myocardial infarction model. At Day 28 after epicardial placement of 110.sup.6 rat fetal membrane MSCs with aid of TachoSil, TachoSil-MSC complex had mostly disappeared. There was no adverse change (complication) found.

[0132] FIG. 10 shows donor cell survival at Day 5 after TachoSil-MSC transplantation on the rat heart. At Day 5 after epicardial placement of 110.sup.6 rat fetal membrane-derived MSCs with aid of TachoSil, a large number of donor MSCs (orange) were observed on the surface of the heart. There was little migration of MSCs into the myocardium, whilst some MSCs settled in the collagen sponge layer.

[0133] FIG. 11 shows donor cell survival at Day 28 after TachoSil-MSC transplantation on the rat heart. At Day 28 after epicardial placement of 110.sup.6 rat fetal membrane-derived MSCs with aid of TachoSil, a number of donor MSCs (orange) were observed on the surface of the heart. There was little migration of MSCs into the myocardium. Collagen of TachoSil appeared to be absorbed or disappeared.

[0134] FIG. 12 shows therapeutic efficacy of epicardial placement of TachoSil-MSC at Day 28 in a rat post myocardial infarction ischaemic heart failure model. Four weeks after left coronary artery ligation in rat (ischaemic heart failure model), TachoSil seeded with a range of rat fetal membrane-derived MSCs, TachoSil only (TS only group), or nothing (Sham group) was placed on the heart surface. Cardiac function was measured by echocardiography (upper lanes) and catheterisation (lower lanes) at Day 28 after treatment.

[0135] FIG. 13 shows improved repair of the damaged myocardium by epicardial placement of TachoSil-MSC in a rat post myocardial infarction ischaemic heart failure model. Four weeks after left coronary artery ligation in rat, TachoSil seeded with 410.sup.6 rat fetal membrane-derived MSCs (TS+MSC group), TachoSil only (TS only group), or nothing (Sham group) was placed on the heart surface. Four weeks after treatment, TS+MSC group showed significantly attenuated pathological fibrosis (A), reduced cardiomyocyte hypertrophy (B; WGA=Wheat germ agglutinin), and improved microvascular formation (C) in both remote and border areas of myocardial infarction, compared to both Sham and TS only groups. Scale bars; 10, 50, 30 mm in A, B, C, respectively.

[0136] FIG. 14 shows feasibility of epicardial placement of TachoSil-MSC on the pig heart. Two pieces of TachoSil (25 cm.sup.2) mixed with 1 mL of 510.sup.7 cells/mL pig bone marrow-derived MSCs were placed on to the surface of the beating heart of pigs (20-22 kg). In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1. Scale bar=100 m.

[0137] FIG. 15 shows feasibility of epicardial placement of TachoSil-MSC on the pig heart. Two pieces of TachoSil (25 cm.sup.2) mixed with 1 mL of 810.sup.7 cells/mL human amnion-derived MSCs were placed on to the surface of the beating heart of pigs (20-22 kg). In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1. Scale bar=400 m.

[0138] FIG. 16 shows feasibility of epicardial placement of Integran-Gelfoam-MSC on the pig heart. Integran-Gelfoam (9 cm.sup.2) mixed with 1 mL of 810.sup.7 cells/mL human amnion-derived MSCs were placed on to the surface of the beating heart of pigs (20-22 kg). In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1. Scale bar=400 m.

[0139] FIG. 17 shows feasibility of epicardial placement of INTERCEED-Gelfoam-MSC on the pig heart. INTERCEED-Gelfoam (9 cm.sup.2) mixed with 1 mL of 810.sup.7 cells/mL human amnion-derived MSCs were placed on to the surface of the beating heart of pigs (20-22 kg). In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1. Scale bar=400 m.

EXAMPLE 1: UNSUCCESSFUL TRIAL OF EPICARDIAL CELL PLACEMENT USING A MONOLAYER MATRIX (SEPRAFILM)

[0140] Epicardial placement of stem cells was first tried using a monolayer matrix. A most-widely used clinically-approved matrix, Seprafilm (Sanofi) which is clinically used for prevention of post-surgical adhesion, was tested. Rat bone marrow mononuclear cells (1010.sup.6) or bone marrow-derived mesenchymal stem cells (MSCs; 410.sup.6) were dissolved in 30 L of HBSS and placed onto the 1 cm.sup.2 Seprafilm, which was turned upside down and directly placed onto the surface of the rat heart at 28 days after myocardial infarction (FIG. 1).

[0141] However, this method using either cell type did not achieve significantly-improved cardiac function at Day 28 (measured by echocardiography) compared to sham-treatment group (FIGS. 2 and 3). In addition, it was found that a certain amount of donor cells retained on the heart surface 1 hour after transplantation (FIGS. 4a and 4b), whilst sizeable numbers of donor cells were observed in the pericardial cavity. This result suggests that Seprafilm could not retain donor cells sufficiently on the heart surface and that considerable numbers of transplanted cells were released/dropped off from the heart (FIG. 4c).

EXAMPLE 2: PREPARATION OF MULTILAYER MATRIX CONTAINING CELLS (CELL-DRESSING)

[0142] To solve the issues of a monolayer matrix-aided epicardial placement (Example 1), we invented the use of two-layered (histocompatible and bioresorbable) matrix. TachoSil (produced by TAKEDA/Nycomed; sold by TAKEDA/Baxter/CSL Behring) is an example of such a two-layered matrix (FIG. 5). The front layer of the matrix consists of a collagen sponge, while the back face includes powdered fibrinogen and thrombin. As soon as a cell suspension was dropped onto the back face, fibrinogen and thrombin reacted to produce fibrin, which offers a comfortable scaffold for the donor cells to retain and settle. Fibrin is quite sticky, and thus can adhere to the heart surface firmly without any suture. The front layer of the collagen sponge supports the fibrin-stem cell complex so that the final product of the matrix incorporating stem cells can be easily handled. This layer is also useful to prevent transplanted stem cells from mechanical stress from outside of the heart (i.e. rubbing of the cells by the lung, pericardium, and chest walls).

[0143] Currently, TachoSil is widely used for a different purpose (haemostasis during surgery including cardiac operation). Topical placement and oppression of this product is effective to stop minor bleeding which is not controllable by surgical procedures. Safety of this product in patients has been proven.

[0144] EVARREST (produced by Ethicon; sold by Ethicon/Omrix Biopharmaceuticals N.V.) is also an example of such a multilayer matrix. EVARREST is an oxidized regenerated cellulose and vicryl mesh (made from polylactic acid and poly glycolic acid) supported fibrin patch and is commercially available as a pharmaceutical product. The front layer of the matrix consists of an oxidized regenerated cellulose and vicryl mesh, while the back face includes powdered fibrinogen and thrombin. As soon as a cell suspension was dropped onto the back face, fibrinogen and thrombin reacted to produce fibrin, which offers a comfortable scaffold for the donor cells to retain and settle. Fibrin is quite sticky, and thus can adhere to the heart surface firmly without any suture. The front layer of the oxidized regenerated cellulose and vicryl mesh supports the fibrin-stem cell complex so that the final product of the matrix incorporating stem cells can be easily handled. This layer is also useful to prevent transplanted stem cells from mechanical stress from outside of the heart (i.e. rubbing of the cells by the lung, pericardium, and chest walls). Currently, EVARREST is widely used for a different purpose (haemostasis during surgery including cardiac operation). Topical placement and oppression of this product is effective to stop minor bleeding which is not controllable by surgical procedures. Safety of this product in patients has been proven.

EXAMPLE 3: OPTIMISATION OF MANUFACTURING MSC-TACHOSIL COMPLEX IN VITRO

[0145] We optimised the protocols to produce a TachoSil-MSC complex. A cell pellet was generated by centrifuging MSC suspensions. The pellet was dissolved in different volumes of ordinary solution (i.e. HBSS or PBS) to make appropriate cell suspensions (FIG. 6).

[0146] The cell suspension was gently dropped onto a 1 cm.sup.2 TachoSil (fibrin side) and spread on the surface using a pipette tip or cell-scraper (FIG. 6). This was turned upside down and placed onto a plastic culture dish. Too small volume cannot make a homogenous fibrin layer, while too much volume resulted in leakage of MSCs from the TachoSil when turned upside down.

[0147] Results of these experiments showed that approximately 30 (10) L MSC suspension per 1 cm.sup.2 TachoSil is the optimal condition for the production of the multilayer matrix or cell-dressing. This protocol resulted in appropriate homogeneous fibrin production throughout the TachoSil incorporation/accommodation of donor MSCs in fibrin, and no leakage was observed when turned upside down.

EXAMPLE 4: IN VIVO TRIAL AND OPTIMISATION OF EPICARDIAL CELL PLACEMENT USING TACHOSIL

[0148] TachoSil is a commercially-available two-layer matrix, which is clinically approved to use for haemostasis during surgery. The present inventors have found that this product is suitable for use in a cell-dressing therapy for the treatment of heart diseases as described herein and also for other treatments of lesions or trauma as described herein.

[0149] TachoSil is easy-to handle (soft and easily-cut by scissors). Upon placing a cell suspension on the fibrin glue powder, the fibrinogen/thrombin side becomes wet, generating adhesive fibrin. This holds donor cells and also enables the product to adhere to the heart surface without any suture. In addition to these effects, this method has many advantages as a material to aid epicardial placement of stem cells including potential to prevent post-surgical pericardial adhesion (fibrin is a major player in formation of post-operative adhesions) and to improve functionality/survival of the donor stem cells (FIG. 6).

[0150] A 200 g rat was anaesthetised, and the heart (epicardium) was exposed through left thoracotomy with opening the pericardium. The left coronary artery was ligated with an 8-0 suture to induce myocardial infarction. After confirmation of the infarction by changes in the ventricular colour and motion (1 hour after ligation), one million rat fetal membrane-derived MSCs dissolved in 30 L HBSS was spread on a 1 cm.sup.2 TachoSil to produce MSC-TachoSil complex. This was placed onto the epicardial heart surface (targeting ischaemic areas) with the fibrin/MSC side directly contacting to the heart surface (FIG. 6). For accurate and easy placement, MSC-TachoSil was cut into 3 pieces before placement. To augment the attachment of the MSC-TachoSil complex on the heart, the complex was gently and lightly oppressed (no leakage of cell suspension or adverse cardiac event occurred). All the procedures were straightforward and the MSC-TachoSil complex was firmly fixed on the beating heart.

[0151] At Day 5 after TachoSil-MSC placement, TachoSil-MSC complex was found to be firmly adhered to the heart surface (FIG. 8).

EXAMPLE 5: IN VIVO PROOF-OF-CONCEPT DATA IN A RAT ACUTE MYOCARDIAL INFARCTION MODEL

[0152] One million rat fetal membrane-derived MSCs dissolved in 30 L HBSS was spread on a 1 cm.sup.2 TachoSil to produce MSC-TachoSil complex. A 200 g rat was anaesthetised, and the heart (epicardium) was exposed by left thoracotomy with opening the pericardium. The left coronary artery was ligated with an 8-0 suture to induce myocardial infarction.

[0153] After confirmation of the infarction by changes in the ventricular colour and motion (1 hour after ligation), the MSC-TachoSil was placed onto the epicardial heart surface (targeting ischaemic areas) with the fibrin/MSC side directly contacting to the heart surface. For accurate and easy placement, MSC-TachoSil was cut into 3 pieces before placement. To augment the attachment of the MSC-TachoSil complex on the heart, the complex was gently and lightly oppressed (no leakage of cell suspension or adverse cardiac event occurred).

[0154] For the control group, myocardial infarction was caused but no treatment was added. The chest was closed and the rat was returned to the normal cage.

[0155] All the procedures were straightforward and the MSC-TachoSil complex was firmly fixed on the beating heart. Throughout the post-treatment days studied, there was no adverse event observed (in terms of rat mortality, general behaviour, food intake, body weight changes).

[0156] At 4 weeks post-treatment, echocardiography demonstrated that left ventricular ejection fraction (=LVEF, an indicator of global cardiac function) was significantly improved in the TS group (MSC-TachoSil treatment) compared to the Control group (FIG. 7; n=7 in each group). Post-myocardial infarction cardiac dilatation (LVDs=left ventricular systolic dimension) was attenuated. There was a tendency of reduction of diastolic cardiac size (LVDd=left ventricular diastolic dimension), but this was not statistically significant (p=0.0979).

[0157] The MSC-TachoSil complex was not clearly detected by direct vision at Day 28 (FIG. 9), suggesting the most of TachoSil had been degraded and absorbed by this time. No adverse event such as fluid collection or post-operative pericardial adhesion was observed, demonstrating the safety of the treatment.

[0158] Immunohistological analysis demonstrated a great retention and survival of donor cells (MSCs were labelled with an orange fluorescent dye before transplantation) on the surface of the heart at Day 7 and 28 post-treatment (FIGS. 10 and 11). The majority of MSCs were retained on the heart surface.

EXAMPLE 6: IN VIVO PROOF-OF-CONCEPT DATA OF EPICARDIAL CELL PLACEMENT USING TACHOSIL IN A RAT POST MYOCARDIAL INFARCTION ISCHAEMIC CARDIOMYOPATHY MODEL

[0159] Four weeks after induction of myocardial infarction by left coronary artery ligation in rat, TachoSil (1 cm.sup.2) mixed with different numbers (0.5, 1, 2, 410.sup.6 cells respectively) of rat fetal membrane-derived MSCs dissolved in 30 L HBSS (TS+MSC group), TachoSil only (TS only group), or nothing (Sham group) was placed onto the epicardial heart surface (targeting ischaemic areas) with the fibrin/MSC side directly contacting to the heart surface (FIG. 6). For accurate and easy placement, MSC-TachoSil was cut into 3 pieces before placement. All the procedures were straightforward and the MSC-TachoSil complex was firmly fixed on the beating heart.

[0160] Four weeks after treatment, echocardiography (FIG. 12 upper panels) and cardiac catheterisation (FIG. 12 lower panels) demonstrated that epicardial placement of the MSC-TachoSil complex improved cardiac function and structure in proportion to the cell numbers. Functional improvement by intramyocardial injection (IM) occurred to a lesser extent compared to the TachoSil-aided placement of the same number of MSCs.

[0161] Four weeks after treatment, TS+MSC group showed significantly attenuated pathological fibrosis (FIG. 13A), reduced cardiomyocyte hypertrophy (FIG. 13B; WGA=Wheat germ agglutinin), and improved microvascular formation (FIG. 13C) in both remote and border areas of myocardial infarction, compared to both Sham and TS only groups.

EXAMPLE 7: EVALUATION OF SURVIVAL RATE OF THE CELLS APPLIED TO MULTILAYER MATRIX

[0162] Human amnion-derived MSCs were obtained in the following steps. The fetal appendage (amnion) was aseptically collected from pregnant patients with informed consent. The resulting amnion and was placed in a sterile vat containing physiological saline solution. Amnion was washed with Hanks' balanced salt solution (Ca.Math.Mg-free) to remove the adhered blood and clots. 240 PU/mL collagenase and 200 PU/mL dispase I were added to the amnion, and stirred under conditions of 50 rpm for 90 min at 37 C. The resulting cell suspension containing amnion MSC was filtered with nylon mesh filter (pore size: 95 m) and remaining undigested amnion was removed. A cell population containing amnion MSC obtained above were cultured in Cell Stack (6,000 cells/cm.sup.2) with MEM containing 10% FBS until cells were being subconfluent. Then, cells were treated with TrypLE Select and were detached from the Cell Stack. The resulting cell suspension was cultured in MEM containing 10% FBS with cell density of 6,000 cells/cm.sup.2. This cell culture and passage were repeated 5 times (totally 6 passage).

[0163] 6 passaged human amnion-derived MSCs obtained in the above procedure were suspended in a mixed solution (CP-1 solution) of a saline containing 5 wt % DMSO, 6 wt % hydroxyethyl starch, 4% human serum albumin and 50 wt % PRM11640 at a density of 810.sup.7cells/mL, and were frozen and stored in a liquid nitrogen. It can be suitably used as amniotic MSC drugs. After thawing of this drug, 30 L (2.410.sup.6 cells) were collected and were spread on fibrin layer of TachoSil (1 cm.sup.2) to prepare TachoSil-MSC complex. Then, MSC TachoSil-MSC complex was applied to 12 well plate containing RPM11640 (200 L), so that the fibrin layer is upper. The 12 well plate was covered with a lid, and left to stand at 37 C. After 3, 6 and 10 hours, TachoSil-MSC complex which was taken out from the 12 well plate was added to Eppendorf tube containing saline (500 L) added with 10 IU/mL nattokinase, and was incubated at 37 C. for 10 minutes to release the cells. TachoSil was removed from the Eppendorf tube, and PRM11640 (500 L) was added. Then, the tube was centrifuged at 400 g for 3 minutes, and the supernatant was removed. Further, PRM11640 was added to prepare a cell suspension (100 L) of 110.sup.6 cells/mL, and PRM11640 was added in the same way to prepare a cell suspension (100 L) of 110.sup.6 cells/mL. 7-AAD (Via Probe, BD bioscience) (10 L) was added thereto, and the cell suspension was left stand at dark place for 15 minutes. Then, 7-AAD positive ratio (death cell ratio) of (A) the cell immediately after thawing and (B) the cells after preparation of TachoSil-MSC complex was measured by using flow cytometer. The cell survival ratio was calculated by the following formula from the obtained values. As a result, the cell survival ratios after 3, 6, and 10 hours were 95%, 96%, and 92% respectively. (the cell survival ratio)=[100(7-AADpositive ratio of (B))%/(100(7-AAD positive rate of (A))%]

[0164] These results showed Tachosil could retain cells with a high cell viability. Therefore, fibrinogen/thrombin can be preferably used as a bioadhesive material and collagen can be preferably used as a bioresorbable material.

EXAMPLE 8: IN VIVO FEASIBILITY DATA OF EPICARDIAL CELL PLACEMENT USING TACHOSIL IN A PIG MODEL

[0165] Two pieces of TachoSil (25 cm.sup.2) mixed with 1 mL of 510.sup.7 cells/mL pig bone marrow-derived MSCs were placed on to the surface of the beating heart of pigs (20-22 kg) through thoracotomy under general anaesthesia and mechanical ventilation.

[0166] In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1 (FIG. 14). The TachoSil-MSC complex strongly adhered to the heart surface. Also, no acute adverse event, including arrhythmia occurrence and immunological reaction, was observed in these studies.

[0167] Histological analysis demonstrated a great retention and survival of donor cells (MSCs were labelled with an orange fluorescent dye before transplantation) on the surface of the heart at day 1 (FIG. 14). The majority of MSCs retained on the heart surface. Scale bar=100 m.

[0168] In the previous experiments in a large animal model (using a hydrogel-aided epicardial placement), it has been revealed that mechanical rubbing is the major problem for the donor cells to be retained on the heart surface. The multilayer matrix of this invention enabled successful epicardial placement of stem cells onto the heart surface (as below), which could not be achieved using other films such as a monolayer film, Seprafilm, Integran, Gelfoam and INTEGRAN.

[0169] Gelfoam (produced by Pfizer) is an example of a bioadhesive material. Gelfoam is a sterile compressed sponge made from gelatine and is commercially available as a medical product. Currently, this commercial product is widely used for a different purpose (hemostatic). Safety of this product in patients has been proven.

[0170] Integran (produced by Koken) is an example of a bioresorbable material. Integran is a topical hemostat sheet made from collagen and is commercially available as a medical product. Currently, this commercial product is widely used for a different purpose (hemostatic). Safety of this product in patients has been proven.

[0171] INTERCEED (produced by Ethicon) is an example of a bioresorbable material. INTERCEED is an anti-adhesion sheet made of regenerated oxidized cellulose and is commercially available as a medical product. Currently, this commercial product is widely used for a different purpose (post-operative adhesion prevention). Safety of this product in patients has been proven.

EXAMPLE 9: IN VIVO FEASIBILITY DATA OF EPICARDIAL CELL PLACEMENT USING TACHOSIL IN A PIG MODEL

[0172] Two pieces of TachoSil (25 cm.sup.2) mixed with 1 mL of 810.sup.7 cells/mL human amnion-derived MSC drug which were obtained according to example 7, were placed on to the surface of the beating heart of pigs (20-22 kg) through thoracotomy under general anaesthesia and mechanical ventilation.

[0173] In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1 (FIG. 15). The TachoSil-MSC complex strongly adhered to the heart surface.

[0174] Histological analysis demonstrated a great retention and survival of donor cells (MSCs were labelled with an orange fluorescent dye before transplantation) on the surface of the heart at Day 1 (FIG. 15). The majority of MSCs were retained on the heart surface. Scale bar=400 m.

[0175] No acute adverse event, including arrhythmia occurrence and immunological reaction, did not occur as far as we studied.

[0176] With regard to the used human amnion-derived MSCs, the ratio of cells exhibiting positive to the surface antigens (CD73, CD90, CD105, CD34, CD45, CD106, and CD142) was analyzed as follows, under measurement conditions, the number of cells to be analyzed was 10,000 cells, and a flow rate setting of Slow (14 L/min), using BD Accuri C6 Flow Cytometer manufactured by Becton, Dickinson and Company (BD).

[0177] (1) The measurement results were shown in the form of a histogram, in which the longitudinal axis indicates the number of cells and the horizontal axis indicates the fluorescence intensity of an antibody labeling dye.

[0178] (2) The fluorescence intensity, at which a cell population with stronger fluorescence intensity accounts for 0.1% to 1.0% of all of the cells measured with the antibody used as an isotype control, was determined.

[0179] (3) The ratio of cells having fluorescence intensity higher than the fluorescence intensity determined in (2) above to all of the cells measured with antibodies against various types of antigens was calculated.

[0180] As a result, the CD73-positive rate was 50% or more (specifically, 100%), the CD90-positive rate was 50% or more (specifically, 100%), the CD105-positive rate was 50% or more (specifically, 99%), the CD34-positive rate was less than 5% (specifically, 0%), the CD45-positive rate was less than 5% (specifically, 1%), the CD106-positive rate was less than 5% (specifically, 0%), and the CD142-positive rate was 50% or more (specifically, 98%). In the present measurement, as isotype control antibodies, PE Mouse IgG1, Isotype Control (BD/model number: 555749), FITC Mouse IgG1, Isotype Control (BD/model number: 550616), FITC Mouse IgG2a, Isotype Control, REA Control

[0181] (S)-PE isotype control antibody (Miltenyi Biotec/130-104-612) were used. As an antibody against the CD73 antigen, PE Mouse Anti-Human CD73 (BD/model number: 550257) was used; as an antibody against the CD90 antigen, FITC Mouse Anti-Human CD90 (BD/model number: 555595) was used; as an antibody against the CD105 antigen, Anti-Human Antibodies FITC Conjugate (AnCell/model number: 326-040) was used; as an antibody against the CD34 antigen, PE Mouse Anti-Human CD34 (BD/model number: 343505) was used; as an antibody against the CD45 antigen, FITC Mouse Anti-Human CD45 (BD/model number: 555482) was used; as an antibody against the CD106 antigen, CD106-PE, human monoclonal (Miltenyi Biotec/130-104-163) was used; and as an antibody against the CD142 antigen, PE Mouse Anti-Human CD142 (BD/model number: 561713) was used.

EXAMPLE 10: EVALUATION OF SURVIVAL RATE OF THE HUMAN AMNION-DERIVED MSCS APPLIED TO MULTILAYER MATRIX

[0182] Human amnion-derived MSCs which were obtained according to example 7, were suspended in a mixed solution (CP-1 solution) of a saline containing 5 wt % DMSO, 6 wt % hydroxyethyl starch, 4% human serum albumin and 50 wt % PRM11640 at a density of 110.sup.7 cells/mL, were frozen and stored in a liquid nitrogen. After thawing, 30 L (0.310.sup.6 cells) were collected and were spread on fibrin layer of TachoSil (1 cm.sup.2) to prepare TachoSil-MSC complex. Then, MSC TachoSil-MSC complex was applied to 12 well plate containing RPMI1640 (200 L), so that the fibrin layer is upper. The 12 well plate was covered with a lid, and left to stand at 37 C. After 3, 6 and 10 hours, TachoSil-MSC complex which was taken out from the 12 well plate was added to Eppendorf tube containing saline (500 L) added with 10 IU/mL nattokinase, and was incubated at 37 C. for 10 minutes to release the cells. TachoSil were removed from the Eppendorf tube, and PRM11640 (500 L) was added. Then, the tube was centrifuged at 400 g for 3 minutes, and the supernatant was removed. Further, PRM11640 was added to prepare a cell suspension (100 L) of 110.sup.6 cells/mL, and PRM11640 was added in the same way to prepare a cell suspension (100 L) of 110.sup.6 cells/mL. 7-AAD (Via Probe, BD bioscience) (10 L) was added thereto, and the cell suspension was left stand at dark place for 15 minutes. Then, 7-AAD positive ratio (death cell ratio) of (A) the cell immediately after thawing and (B) the cells after preparation of TachoSil-MSC complex was measured by using flow cytometer. The cell survival ratio was calculated by the following formula from the obtained values. As a result, the cell survival ratios after 3, 6, and 10 hours were 96%, 82%, and 83% respectively. (the cell survival ratio)=[100(7-AADpositive ratio of (B))%/(100(7-AAD positive rate of (A))%]

EXAMPLE 11: IN VIVO PROOF-OF-CONCEPT DATA OF EPICARDIAL CELL PLACEMENT USING TACHOSIL IN A RAT POST MYOCARDIAL INFARCTION ISCHAEMIC CARDIOMYOPATHY MODEL

[0183] Human amnion-derived MSCs were suspended in a mixed solution (CP-1 solution) of a saline containing 5 wt % DMSO, 6 wt % hydroxyethyl starch, 4% human serum albumin and 50 wt % PRM11640 at a density of 810.sup.7 cells/mL, were frozen and stored in a liquid nitrogen. Just after thawing, 25 L (210.sup.6 cells) were collected and were spread on fibrin layer of TachoSil (1 cm.sup.2) to prepare TachoSil-MSC complex. TachoSil-MSC complex (TS+MSC group) or nothing (Sham group) was placed onto the epicardial heart surface (targeting ischaemic areas) with the fibrin/MSC side directly contacting to the heart surface of rat which has been passed four weeks after induction of myocardial infarction by left coronary artery ligation. For accurate and easy placement, MSC-TachoSil was cut into 3 pieces before placement. All the procedures were straightforward and the MSC-TachoSil complex was firmly fixed on the beating heart. Four weeks after treatment, echocardiography and cardiac catheterisation demonstrated that epicardial placement of the MSC-TachoSil complex improved cardiac function and structure compared to the sham group. TS+MSC group showed approximately 10% increase in LVEF. With regard to the used human amnion-derived MSCs, the ratio of cells exhibiting positive to the surface antigens (CD73, CD90, CD105, CD34, CD45, CD106, and CD142) was analyzed as follows, the CD73-positive rate was 50% or more (specifically, 100%), the CD90-positive rate was 50% or more (specifically, 100%), the CD105-positive rate was 50% or more (specifically, 100%), the CD34-positive rate was less than 5% (specifically, 0%), the CD45-positive rate was less than 5% (specifically, 0%), the CD106-positive rate was less than 5% (specifically, 1%), and the CD142-positive rate was 50% or more (specifically, 95%).

EXAMPLE 12: IN VIVO FEASIBILITY DATA OF EPICARDIAL CELL PLACEMENT USING INTEGRAN-GELFOAM MATRIX IN A PIG MODEL

[0184] 9 cm.sup.2 of Integran (as a bioresorbable material) was put on the 9 cm.sup.2 of Gelfoam (as a bioadhesive material), and their four corners were fixed with suture thread. Then, 1 mL of 810.sup.7 cells/mL human amnion-derived MSC drug which were obtained according to example 7 (but the donor amnion was different from example 7), was put on the Gelfoam side and this was placed on to the surface of the beating heart of pigs (20-22 kg) through thoracotomy under general anaesthesia and mechanical ventilation.

[0185] In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1 (FIG. 16). The Integran-Gelfoam-MSC complex strongly adhered to the heart surface. Also, no acute adverse event, including arrhythmia occurrence and immunological reaction, did not occur as far as we studied.

[0186] Histological analysis demonstrated a great retention and survival of donor cells (MSCs were labelled with an orange fluorescent dye before transplantation) on the surface of the heart at day 1 (FIG. 16). The majority of MSCs were retained on the heart surface. Scale bar=400 m.

[0187] Human amnion MSC drug used for the in vivo study was obtained from a different donor from Example 7. The ratio of cells exhibiting positive to the surface antigens (CD73, CD90, CD105, CD34, CD45, CD106, and CD142) was analyzed according to example 7.

[0188] As a result, the same characteristics as the donor in Example 7 were observed. The CD73-positive rate was 50% or more (specifically, 100%), the CD90-positive rate was 50% or more (specifically, 100%), the CD105-positive rate was 50% or more (specifically, 99%), the CD34-positive rate was less than 5% (specifically, 0%), the CD45-positive rate was less than 5% (specifically, 0%), the CD106-positive rate was less than 5% (specifically, 1%), and the CD142-positive rate was 50% or more (specifically, 95%). In the present measurement, as isotype control antibodies, PE Mouse IgG1, Isotype Control (BD/model number: 555749), FITC Mouse IgG1, Isotype Control (BD/model number: 550616), FITC Mouse IgG2a, Isotype Control, REA Control (S)-PE isotype control antibody (Miltenyi Biotec/130-104-612) were used. As an antibody against the CD73 antigen, PE Mouse Anti-Human CD73 (BD/model number: 550257) was used; as an antibody against the CD90 antigen, FITC Mouse Anti-Human CD90 (BD/model number: 555595) was used; as an antibody against the CD105 antigen, Anti-Human Antibodies FITC Conjugate (AnCell/model number: 326-040) was used; as an antibody against the CD34 antigen, PE Mouse Anti-Human CD34(BD/model number: 343505) was used; as an antibody against the CD45 antigen, FITC Mouse Anti-Human CD45 (BD/model number: 555482) was used; as an antibody against the CD106 antigen, CD106-PE, human monoclonal (Miltenyi Biotec/130-104-163) was used; and as an antibody against the CD142 antigen, PE Mouse Anti-Human CD142 (BD/model number: 561713) was used.

[0189] Also, the survival rate of the cells applied to Integran-Gelfoam matrix was evaluated according to example 7. As a result, the cell survival ratios after 3, 6, and 10 hours were 97%, 95%, and 90% respectively.

[0190] These results showed the great adhesion ability of Integran-Gelfoam matrix to biological tissues, as well as Tachosil, and Integran-Gelfoam matrix could retain cells with a high cell viability. As described above, the use of a monolayer film such as Gelfoam and Integran resulted in unsuccessful epicardial placement of stem cells onto the pig heart surface, however, Integran-Gelfoam matrix which is made by combining these monolayer films to form a multi-layer matrix achieved great retention of stem cells onto the pig heart surface. As the results of this example, the multilayer matrix made of collagen and gelatin layer containing cells can be preferably used as drugs for heart diseases such as ischemic cardiomyopathy.

EXAMPLE 13: IN VIVO FEASIBILITY DATA OF EPICARDIAL CELL PLACEMENT USING INTERCEED-GELFOAM IN A PIG MODEL

[0191] 9 cm.sup.2 of INTERCEED (as a bioresorbable material) was put on the 9 cm.sup.2 of Gelfoam (as a bioadhesive material), and their four corners were fixed with suture thread. Then, 1 mL of 810.sup.7 cells/mL human amnion-derived MSC drug (amnion donor was same as example 12) which were obtained according to example 12, was put on the Gelfoam side and this was placed on to the surface of the beating heart of pigs (20-22 kg) through thoracotomy under general anaesthesia and mechanical ventilation.

[0192] In addition to the technical feasibility, great retention and survival of MSCs (labelled orange with CM-Dil) was histologically confirmed at Day 1 (FIG. 16). The INTERCEED-Gelfoam-MSC complex strongly adhered to the heart surface. Also, no acute adverse event, including arrhythmia occurrence and immunological reaction, did not occur as far as we studied.

[0193] Histological analysis demonstrated a great retention and survival of donor cells (MSCs were labelled with an orange fluorescent dye before transplantation) on the surface of the heart at day 1 (FIG. 16). The majority of MSCs were retained on the heart surface. Scale bar=400 m.

[0194] Also, the survival rate of the cells applied to INTERCEED-Gelfoam matrix was evaluated according to example 12. As a result, the cell survival ratios after 3, 6, and 10 hours were 95%, 92%, and 90% respectively.

[0195] These results showed the great adhesion ability of INTERCEED-Gelfoam matrix to biological tissues, as well as Tachosil, and INTERCEED-Gelfoam matrix could retain cells with a high cell viability. As described above, the use of a monolayer film such as INTERCEED and Gelfoam resulted in unsuccessful epicardial placement of stem cells onto the pig heart surface, however, Integran-Gelfoam matrix which is made by combining these monolayer films to form a multi-layer matrix achieved great retention of stem cells onto the pig heart surface. As the results of this example, the multilayer matrix made from regenerated oxidized cellulose and gelatin layer containing cells can be preferably used as drugs for heart diseases such as ischemic cardiomyopathy.

EXAMPLE 14: EVALUATION OF SURVIVAL RATE OF THE HUMAN ADIPOSE TISSUE-DERIVED MSCS APPLIED TO MULTILAYER MATRIX

[0196] Human adipose tissue-derived stem cells (Lonza, PT-5006) were thawed and cultured in a density of 6,000 cells/cm.sup.2 with aMEM containing 5% human platelet lysate until cells were being subconfluent. Then, cells were treated with TrypLE Select and were detached from the dish and washed with aMEM containing 5% human platelet lysate. The cell suspension was centrifuged and cell pellet was suspended in a mixed solution (CP-1 solution) of a saline containing 5 wt % DMSO, 6 wt % hydroxyethyl starch, 4% human serum albumin and 50 wt % PRM11640 at a density of 810.sup.7 cells/mL, were frozen and stored in a liquid nitrogen. The ratio of cells exhibiting positive to the surface antigens (CD73, CD90, CD105, CD34, CD45, CD106, and CD142) was analyzed according to example 7. As a result, the CD73-positive rate was 50% or more (specifically, 100%), the CD90-positive rate was 50% or more (specifically, 95%), the CD105-positive rate was 50% or more (specifically, 99%), the CD34-positive rate was less than 5% (specifically, 0%), the CD45-positive rate was less than 5% (specifically, 0%). After thawing of this adipose tissue-derived MSC, 30 L (2.410.sup.6 cells) were collected and were spread on fibrin layer of TachoSil (1 cm.sup.2) to prepare TachoSil-MSC complex. Then, TachoSil-MSC complex was applied to 12 well plate containing RPM11640 (200 L), so that the fibrin layer is upper. The 12 well plate was covered with a lid, and left to stand at 37 C. After 3, 6 and 10 hours, TachoSil-MSC complex which was taken out from the 12 well plate was added to Eppendorf tube containing saline (500 L) added with 10 IU/mL nattokinase and was incubated at 37 C. for 10 minutes to release the cells. TachoSil was removed from the Eppendorf tube, and PRM11640 (500 L) was added. Then, the tube was centrifuged at 400 g for 3 minutes, and the supernatant was removed. Further, PRM11640 was added to prepare a cell suspension (100 L) of 110.sup.6 cells/mL, and PRM11640 was added in the same way to prepare a cell suspension (100 L) of 110.sup.6 cells/mL. 7-AAD (Via Probe, BD bioscience) (10 L) was added thereto, and the cell suspension was left to stand at dark place for 15 minutes. Then, 7-AAD positive ratio (death cell ratio) of (A) the cells just thawed and (B) the cells after preparation of TachoSil-MSC complex was measured by using flow cytometer. The cell survival ratio was calculated by the following formula from the obtained values. As a result, the cell survival ratios after 3, 6, and 10 hours were 92%, 90%, and 81% respectively. (the cell survival ratio)=[100(7-AADpositive ratio of (B))%/(100(7-AAD positive rate of (A))%]

[0197] This result indicated that adipose tissue-derived MSCs can also be preferably used with multi-layer matrix.

[0198] Also, great therapeutic effect can be expected by applying complex of adipose tissue-derived MSCs and multilayer matrix to the heart, as it is well known that adipose tissue-derived MSC is useful for heart diseases such as ischemic cardiomyopathy (D. Mori et al., Cell Spray Transplantation of Adipose-Derived Mesenchymal Stem Cell Recovers Ischemic Cardiomyopathy in a Porcine Model, Transplantation 2018;).

EXAMPLE 15: EVALUATION OF SURVIVAL RATE OF THE HUMAN AMNION-DERIVED MSCS APPLIED TO BESCHITIN-GELFOAM

[0199] Beschitin (produced by Nipro) is a wound protective agent made from chitin and is commercially available as a medical product in Japan. 1 cm.sup.2 of Beschitin (W-A type as a bioresorbable material) was put on the 1 cm.sup.2 of Gelfoam (as a bioadhesive material), and their diagonal two corners were fixed with suture thread. Then, 30 L of 810.sup.7 cells/mL human amnion-derived MSC drug (amnion donor was same as example 12) which were obtained according to example 12, was put on the Gelfoam side. Then, the survival rate of the cells applied to Beschitin-Gelfoam matrix was evaluated according to example 7. As a result, the cell survival ratios after 3, 6, and 10 hours were 96%, 94%, and 90% respectively. These results showed Beschitin-Gelfoam matrix could retain cells with a high cell viability. As the results of this example, the multilayer matrix made from chitin and gelatin layer containing cells can be preferably used as drugs for cell therapies.

EXAMPLE 16: EVALUATION OF SURVIVAL RATE OF THE HUMAN AMNION-DERIVED MSCS APPLIED TO NEOVEIL-GELFOAM

[0200] NEOVEIL (produced by Gunze) is a tissue reinforcement agent made from polyglycolic acid and is commercially available as a medical product in Japan. 1 cm.sup.2 of NEOVEIL (S type as a bioresorbable material) was put on the 1 cm.sup.2 of Gelfoam (as a bioadhesive material), and their diagonal two corners were fixed with suture thread. Then, 30 L of 810.sup.7 cells/mL human amnion-derived MSC drug (amnion donor was same as example 12) which were obtained according to example 12, was put on the Gelfoam side. Then, the survival rate of the cells applied to NEOVEIL-Gelfoam matrix was evaluated according to example 7. As a result, the cell survival ratios after 3, 6, and 10 hours were 96%, 94%, and 91% respectively. These results showed NEOVEIL-Gelfoam matrix could retain cells with a high cell viability. As the results of this example, the multilayer matrix made from polyglycolic acid and gelatin layer containing cells can be preferably used as drugs for cell therapies.

EXAMPLE 17: EVALUATION OF SURVIVAL RATE OF THE HUMAN AMNION-DERIVED MSCS APPLIED TO INTERCEED-TachoSil

[0201] 1 cm.sup.2 of INTERCEED (as a bioresorbable material) was put on the 1 cm.sup.2 of TachoSil (as a bioadhesive material), and their diagonal two corners were fixed with suture thread. Then, 30 L of 810.sup.7 cells/mL human amnion-derived MSC drug (amnion donor was same as example 12) which were obtained according to example 12, was put on the fibrinogen-thrombin side of TachoSil. Then, the survival rate of the cells applied to INTERCEED-TachoSil matrix was evaluated according to example 7. As a result, the cell survival ratios after 3, 6, and 10 hours were 96%, 96%, and 93% respectively. These results showed NEOVEIL-TachoSil matrix could retain cells with a high cell viability. As the results of this example, the multilayer (three-layer) matrix made from regenerated oxidized cellulose, collagen and fibrinogen layer containing cells can be preferably used as drugs for cell therapies.

SUMMARY

[0202] The examples herein show that a cell-dressing multilayer matrix can be prepared which is suitable for the treatment of acute myocardial infarction as well as chronic heart failure. Although monolayer matrix could not achieve this, we found that a two-layer matrix is able to establish epicardial placement of stem cells. Epicardial placement of this composition was feasible and effective to retain and engraft donor cells to the heart. Therapeutic efficacy of this composition was also confirmed in two different heart disease models. Feasibility in a large animal was also confirmed.

[0203] This method allows instant and on-site production of a tissue-engineering multilayer matrix, which can be immediately applied to the patient (onto the heart surface). All procedures required for the cell-dressing therapy are extremely simple and practical by any ordinary clinician/hospital staff without requirement of specific equipment or expertise. The cell-dressing can be generated in a surgical theatre without requirement of any specific equipment such as high-grade cell processing centre, when the donor cells are supplied externally. The whole process for generation/placement will complete in half an hour, causing little additional burden to the clinical staff or to the patients. Thus, it is very easy and reasonable to add this treatment to the routine heart surgery (it is known that cell therapy enhances the effects of coronary artery bypass graft surgery). It is also possible to perform this treatment solely (with open chest procedure or using endoscope and so on).