Introduction Recently,bone marrow mesenchymal stem cells(MSCs) have been reported to repair chronically infracted myocardium with direct injection.However,it is very difficult to localize the injected cells onto the ischemic area to regenerate sufficient cardiac mass in the thinned scar area.To overcome the problem,we have utilized our cell sheet technology based on temperature-responsive culture dishes.When the culture temperature is reduced from 37℃to 20℃,all cells connected via cell-cell junction proteins are harvested as a single sheet without using proteolytic enzymes.This technology allows us to transplant stem cells in vivo for treatment heart disease without the problems mention in the previous.Methods Male Clawn mini pigs were used in this study.Bone marrow(5-7 mL)was collected under general anesthesia. Histopaqe-1077(15 mL),were added to bone marrow and centrifuged.The cells were collected and cultured for 7 days.We seeded the bone marrow-derived MSCs at the concentration of(6×10~5/ml) on 60 mm diameter temperature -responsive dishes for 7 days.As the culture temperature decreased from 37℃to 20℃,MSC sheet detached itself spon-taneously and floated up into the culture medium. Triple layers were stacked together repeatedly forming special multiplayer.Myocardial infarction was created by the ligation of the left anterior descending branch of the left coronary artery.A cell sheets was transplanted onto the ischemia area.The echocardiography was performed two and four weeks after transplantation.The heart tissue with cell sheets were removed and fixed with 10%formalin for histological analysis one month after the transplantation of cell sheets.Results Most MSCs are positive for CD29,CD90,CD146 and CD73.These mean the culture cell sheets were composed of undifferentiated MSCs and remained multipotent.Monolayers(20- 30μm) and multilayer(120μm) cell sheets were produced,which retained all cell-to-cell contaction. Histological analyses show the cell sheets become closely contacted with the heart tissue after cell sheets transplantation.The adhesion between cell sheets and ischemia heart tissue has been significantly detected.Ejection fraction(EF) and%fractional shortening(FS)were changed and cardiac function was improved after cell sheets transplantation. Conclusions Using this cell sheet technology, we have made a multilayer MSCs cell sheet derived from bone marrow.For transplantation,the cell sheets improved the cardiac function in pigs with myocardial infarction.The present study represents progress toward the clinical application of multi-layered cell sheet of MSCs as a powerful treatment vehicle for human heart disease. Introduction Recently, bone marrow mesenchymal stem cells (MSCs) have been reported to repair chronically infracted myocardium with direct injection. However, it is very difficult to localize the injected cells onto the ischemic area to regenerate sufficient cardiac mass in the thinned scar area. To overcome the problem, we have utilized our cell sheet technology based on temperature-responsive culture dishes. Whilst the culture temperature is reduced from 37 ° C to 20 ° C, all cells connected via cell-cell junction proteins are harvested as a single sheet without using proteolytic enzymes. This technology allows us to transplant stem cells in vivo for treatment heart disease without the problems mention in the previous. Methods Male Clawn mini pigs were used in this study. Bone marrow (5-7 mL) was collected under general anesthesia. Histopaqe -1077 (15 mL), were added to bone marrow and centrifuged.The cells were collected and cultured for 7 days. We seeded the bone marrow-derived MSCs at the concentration of (6 × 10 ~ 5 / ml) on 60 mm diameter temperature -responsive dishes for 7 days. As the culture temperature decreased from 37 ℃ to 20 ℃, MSC sheet was detached itself spon- taneously and floated up into the culture medium. Triple layers were stacked together repeatedly repeatedly forming special multiplayer. Myocardial infarction was created by the ligation of the left anterior descending branch of the left coronary artery. A cell sheets was transplanted onto the ischemia area. The echocardiography was performed two and four weeks after transplantation. heart tissue with cell sheets were removed and fixed with 10% formalin for histological analysis one month after transplantation of cell sheets. Results Most MSCs are positive for CD29, CD90, CD146 and CD73. These mean the culture cell sheets were composed of undifferentiated MSCs and remained multipotent. Monolayers (20-30 μm) and multilayer (120 μm) cell sheets were produced, which retained all cell-to-cell contactions. Histological analyzes show the cell shee ts become closely contacted with the heart tissue after cell sheets transplantation. The adhesion between cell sheets and ischemia heart tissue has been significantly detected. Ejection fraction (EF) and% fractional shortening (FS) were changed and cardiac function was improved after cell sheets transplantation. Conclusions Using this cell sheet technology, we have made a multilayer MSCs cell sheet derived from bone marrow. For transplantation, the cell sheets improved the cardiac function in pigs with myocardial infarction. The present study represents progress toward the clinical application of multi-layered cell sheet of MSCs as a powerful treatment vehicle for human heart disease.