Cellular aggregate capture by fluidic manipulation device highly compatible with micro-well-plates.

This paper proposes a capture device to manipulate and transport a cellular aggregate in a micro-well. A cellular aggregate (a few hundreds µm in diameter) is currently manipulated by a pipette. The manual manipulation by a pipette has problems; low reliability, low throughput, and difficulty in confirmation of task completion. We took into account of compatibility with existing methods such as a micro-well-plate and designed for the capture device of a cellular aggregate in a micro-well. A newly developed capture device flows and carries a cellular aggregate from a bottom of a well to a trap of the capture device. We designed a curved surface at the bottom of the capture device to form a space to act as a channel between the inner wall of the micro-well. This paper presents concept, design, fabrication, and of the proposed cellular aggregate capture, followed by successful experimental results.

Preparation of cell aggregates incorporating gelatin hydrogel microspheres containing bone morphogenic protein-2 with different degradabilities.

The objective of this study is to evaluate the survival and functions of cells in cell aggregates incorporating gelatin hydrogel microspheres (GM) containing bone morphogenic protein-2 (BMP2). Gelatin was dehydrothermally crosslinked in a water-in-oil emulsion state at 140 °C for various time periods to prepare GM with different degradabilities. BMP2 was dropped onto the GM freeze dried, followed by leaving at 25 °C to obtain GM containing BMP2 (GM-BMP2). MC3T3-E1 cells were cultured with GM-BMP2 and GM in round U-bottom wells of 96-multiwell microplates which had been coated with poly (vinyl alcohol) (PVA), to allow to form cell aggregates containing GM-BMP2 and GM, respectively. Higher MC3T3-E1 cell proliferation and the L-lactic acid/glucose ratio were observed for MC3T3-E1 cell aggregates cultured with the GM of slower degradation. The runt-related transcription factor 2 (RUNX2) messenger ribonucleic acid (mRNA) expression, alkaline phosphatase (ALP) activity, and calcium content of MC3T3-E1 cells in cell aggregates were assayed to evaluate their osteogenic differentiation. When cultured for 7 days with GM-BMP2 or free BMP2, the RUNX2 mRNA expression and ALP activity were higher for MC3T3-E1 cell aggregates cultured with the GM-BMP2 of faster degradation than those of free BMP2 added into the medium. After 21 days culture, the ALP activity and calcium content were higher for the GM-BMP2 of medium degradation compared with other experimental groups. It is concluded that BMP2 of GM-BMP2 incorporated in the cell aggregates enhanced the osteogenic differentiation of cells compared with free BMP2 added externally. The degradability of GM-BMP2 affected the extent of osteogenic differentiation.

Preparation of EpH4 and 3T3L1 cells aggregates incorporating gelatin hydrogel microspheres for a cell condition improvement.

The objective of this study is to prepare three dimensional (3D) of mouse mammary epithelial EpH4 and mouse preadipocyte 3T3L1 cells in the presence of gelatin hydrogel microspheres (GM) and evaluate the effect of GM presence on the survival and functions of cells in the 3D cell aggregates. Gelatin was dehydrothermally crosslinked at 140 °C for 48 h in a water-in-oil emulsion state to obtain the GM with average diameters of 50 and 200 µm, followed by treatment with fibronectin (FN). EpH4 and/or 3T3L1 cells were cultured with or without the FN-treated GM in round U-bottom wells of 96-multiwell culture plates which had been coated with poly (vinyl alcohol) (PVA) to allow the cells to form their aggregates. On the other hand, EpH4 cells were precultured with the FN-treated GM, and then continued to culture with 3T3L1 cells in the same condition described above. The EpH4 cells attached onto the GM in the cell number dependent manner, irrespective of their size. When 3T3L1 cells were incubated with the original and GM-preincubated EpH4 cells in the presence of both the FN-treated GM, the number of alive cells in the aggregates was significantly high compared with that for the absence of FN-treated GM. In addition, higher ß-casein expression level of EpH4 cells in EpH4/3T3L1 cells aggregates in the presence of FN-treated GM was observed than that of cells in the absence of FN-treated GM. Laminin secretion was also promoted for the cells aggregates cultured with FN-treated GM. It is concluded that the presence of FN-treated GM in the EpH4/3T3L1 cells aggregates gave a better condition to cells, resulting in an enhanced generation of ß-casein from EpH4 cells in the aggregates.

Preparation of epithelial cell aggregates incorporating matrigel microspheres to enhance proliferation and differentiation of epithelial cells.

The objective of this study is to investigate the effect of matrigel microspheres (MM), gelatin hydrogel microspheres (GM), and matrigel-coated GM on the proliferated and biological functions of epithelial cells in cell aggregates incorporating the microspheres. The MM were prepared by a coacelvation method. When mammary epithelial EpH4 cells were cultured with the MM, GM, and matrigel-coated GM in round U-bottom wells of 96-multiwell culture plates which had been coated with poly (vinyl alcohol) (PVA) to suppress the cell adhesion, EpH4 cell aggregates with each microspheres homogeneously incorporated were formed. Higher EpH4 cells proliferation was observed for cell aggregates incorporating MM, GM, and matrigel-coated GM compared with the conventional 3-dimensional (3D) culture method. When examined to evaluate the epithelial differentiation of EpH4 cells, the ß-casein expression was significantly higher for the cell aggregates incorporating MM than that of aggregates incorporating GM and matrigel-coated GM or the conventional 3D culture method. It is concluded that the proliferation and differentiation of mammary epithelial EpH4 cells were promoted by the incorporation of MM.

Implementation of soft microfingers for a hMSC aggregate manipulation system.

This paper describes a pneumatic balloon actuator (PBA) composed of polydimethylsiloxane (PDMS) for cellular aggregate manipulation. We evaluated the ability of the microdevice to manipulate a tiny and sensitive cellular aggregate without causing serious damage. We used human mesenchymal stem cells (hMSCs) for the cellular aggregate. We describe the design, fabrication, characterization and operation of the soft microfingers to pinch and release a spherical hMSC aggregate (φ200 µm), and we employed a PBA to serve as an artificial muscle to drive the microfingers. A design of the microfingers in terms of dimensions, generated force and contact conditions was accomplished. The designed dimensions of a single finger were 560 µm×900 µm. In summary, we demonstrate the utility of the surface modification of a fingertip for pinching and releasing a cellular aggregate and describe a manipulation system that was constructed to drive and control the microfingers. The implemented manipulation system, which is composed of microfingers and a positioning mechanism, was tested and verified in a series of operations.

Efficient long-term survival of cell grafts after myocardial infarction with thick viable cardiac tissue entirely from pluripotent stem cells.

Poor engraftment of cells after transplantation to the heart is a common and unresolved problem in the cardiac cell therapies. We previously generated cardiovascular cell sheets entirely from pluripotent stem cells with cardiomyocytes, endothelial cells and vascular mural cells. Though sheet transplantation showed a better engraftment and improved cardiac function after myocardial infarction, stacking limitation (up to 3 sheets) by hypoxia hampered larger structure formation and long-term survival of the grafts. Here we report an efficient method to overcome the stacking limitation. Insertion of gelatin hydrogel microspheres (GHMs) between each cardiovascular cell sheet broke the viable limitation via appropriate spacing and fluid impregnation with GHMs. Fifteen sheets with GHMs (15-GHM construct; >1 mm thickness) were stacked within several hours and viable after 1 week in vitro. Transplantation of 5-GHM constructs (≈2 × 10(6) of total cells) to a rat myocardial infarction model showed rapid and sustained functional improvements. The grafts were efficiently engrafted as multiple layered cardiovascular cells accompanied by functional capillary networks. Large engrafted cardiac tissues (0.8 mm thickness with 40 cell layers) successfully survived 3 months after TX. We developed an efficient method to generate thicker viable tissue structures and achieve long-term survival of the cell graft to the heart.

Preparation and functional evaluation of cell aggregates incorporating gelatin microspheres with different degradabilities.

The objective of this study was to investigate the viability and biological functions of cells in their aggregates incorporating gelatin microspheres with different degradabilities. After being prepared by a water-in-oil emulsion procedure, the gelatin microspheres were dehydrothermally crosslinked at 140°C for various time periods. In vitro degradation tests showed that the gelatin microspheres were slowly degraded slowly with an increase in the crosslinking time. When MC3T3-E1 cells were cultured with the gelatin hydrogel microspheres in the round U-bottom wells of 96-well microplates which had been coated with poly(vinyl alcohol), cell aggregates with homogeneously distributed gelatin microspheres were formed. A large amount of slowly degraded gelatin microspheres remained in the cell aggregates for long time periods, while a higher proliferation of MC3T3-E1 cells was observed. When evaluated as a measure of aerobic glycolysis, the ratio of l-lactic acid production:glucose consumption of MC3T3-E1 cells was lower for MC3T3-E1 cells in the cell aggregates incorporating slowly degraded gelatin microspheres than for aggregates incorporating rapidly degraded ones. The alkaline phosphatase activity and calcium content of MC3T3-E1 cells were higher for cell aggregates incorporating slowly degraded gelatin microspheres. It is possible that the incorporation of gelatin hydrogel microspheres with slow degradability enabled the permeation of oxygen and nutrients into the cell aggregates for longer time periods, resulting in better culture conditions for the survival, proliferation and differentiation of the cells.

Angiogenesis induced by controlled release of neuropeptide substance P.

The in vivo recruitment of circulating host cells to the site to be regenerated is one of the promising strategies for therapeutic angiogenesis. Substance P (SP), a member of neuropeptides, mediates pain perception and regulates wound healing, inflammation, tumor cell proliferation, and angiogenesis. This SP enhanced the migration, adhesion, and angiogenic gene expression of granulocytes in vitro. A biodegradable hydrogel was prepared from an anionic derivative of gelatin to achieve the controlled release of SP in vivo. When the anionic gelatin hydrogels incorporating SP were subcutaneously implanted into the mouse back, significant angiogenesis was induced around the site implanted, in contrast to the injection of SP solution. In vivo accumulation of granulocytes around the implanted sites was observed. It is concluded that the controlled release of SP efficiently induced the recruitment and the subsequent activation of granulocytes, one of the circulating cells with angiogenic activities, from the blood circulation into the site implanted, resulting in enhanced angiogenesis.