Vol.1 No.3 2009

6/69

Research paper : Development of regenerative medical technology working toward practical application (H. Ohgushi)−157 Synthesiology - English edition Vol.1 No.3 (2009) elaborate on this due to space limitation. 3.2 Technology for selecting target cell (verification of cell growth)Cell growth process is the first process in any regenerative medicine. However, since the harvested cells contain various types of cells, separation of target cell from the mix of cells is necessary. For example, we select and grow mesenchymal stem cell (MSC) from fresh bon marrow that contains hematopoietic cells such as red and white blood cells as well as others. Fresh bone marrow is sowed on culture dish, and the floating blood cells are removed by replacing the culture medium. Thereafter, cells that adhere to the culture surface grow and can be collected. In fact, the cell population collected by this method presents various marker expression usually seen in MSC. However, even at this stage, the cultured cells consists of cell population with different proliferative ability and is not homogenous. This means it is difficult to determine whether the MSC collected at this moment will grow as expected.In our experience in clinical application, we observed that the growth rate decreases when the nucleus of MSC in culture became thin and the cells flattened in shape. Therefore, we decided to estimate the proliferative ability by measuring this phenomenon quantitatively. We investigated the correlation between the thickness of MSC measured using atomic force microscope and the cell proliferation activity. We found that compared to MSCs with low proliferative ability, the cells with high proliferative ability were smaller and had increased thickness of the nuclear region[3]. However, the atomic force microscope is extremely expensive, difficult to operate, and takes time to make measurements. Therefore, we worked with Olympus Corporation to investigate whether evaluation of cell proliferation activity level in culture was possible by observing the thickness of area of MSC nucleus and the shape of cell (plane) using light microscope image, and then evaluated cell proliferation activity and developed device to measure proliferative activity using these indices. To measure the thickness of cell by light microscope, the phase image of MSC that adhered to the culture dish was obtained, and numerical information corresponding to cell thickness and cell surface area were obtained by image processing and analysis software. As shown in Figure 4, the MSC in culture were displayed 3-dimensionally, and the thickness could be measured automatically. Using this device, proliferative ability of the cultured cell could be estimated non-invasively, and it could also check proper growth of the cells. We succeeded in developing technology that allows cell cultivation with higher efficacy. This device can be linked to existing light microscope, and can be accessory device to microscopes that are installed in hospitals and research centers. The device we developed has excellent cost performance, and it is expected that it will be used in various places in the future.3.3 Verification of cell differentiation (verification of differentiation on material in case cells are hybridized with biomaterials)In the development of regenerative medical technology, we have worked on the technologies for bone regeneration. Bone regeneration involves the method for regenerative tissue-engineered bone in which MSCs are differentiated into osteoblasts with bone formation ability by cell cultivation, and the bone matrix is formed on biomaterial by these osteoblasts[4][5]. Various types of biomaterials are used to create tissue-engineered bone. Particularly, materials with porous structure to anchor cells are useful. However, evaluation of whether a biomaterial can anchor cells efficiently and whether it has ability to form new bone in vivo are necessary. Therefore, we compared the activities of MSC regarding properties of the biomaterial used for tissue-engineered cells, and tried to establish a methodology for assessing new bone formation in vivo. To standardize this evaluation method, we employed universal source for cell (in this case bone marrow from rat femur) as well as fixed procedure.Specific procedures were as follows. Bone marrow of 7-week old rat were cultured in flask to grow MSC, and cell concentration was adjusted to 1×106 cell/ml. Porous materials were placed on the culture plate and immersed in adjusted cell suspension. The samples were cultured for 2 weeks using osteogenic culture condition. Detection of differentiated bone cells (osteoblasts) after completion of the culture was conducted by alkaline phostaphase staining. As shown in top photograph of Figure 5, comparing the two materials (porous synthetic hydroxyapatite and hydroxyapatite derived from coral skeleton), bone differentiation was observed only in the pores of surface of the synthetic material. In contrast, cells grew inside the pores in the coral material, and good bone differentiation was observed. The engineered cells were transplanted to rats of same strain. As shown in lower photograph of Figure 5, new bone formation (shown in red) was observed inside the material for coral hydroxyapatite. (3)−Edge of cell is detected and planar form of cell is tracedOlympus Corp.Development of proliferation activity measurement deviceCameraElectromotive stageZ-axisSample (culture container)Cell thickness distribution is measured from multiple image data obtained by scanning along z-axisCondenserLight source(Inverted light microscope + image processor)Composition of deviceAISTCorrelation between cell thickness and cell proliferation NucleusNucleusFig. 4 Development of cell thickness measurement device (evaluation technology for cell proliferation activity and development of the device).

※このページを正しく表示するにはFlashPlayer9以上が必要です