Expansion of human mesenchymal stem cells on poly(vinyl alcohol) microcarriers.

Microcarriers provide a high surface-area-to-volume ratio that can realize high yields of cell products, including human mesenchymal stem cells (hMSCs). Here, we report a novel poly(vinyl alcohol) (PVA)-based microcarrier for hMSC expansion in suspension culture. PVA microcarriers were prepared as collagen-coated PVA hydrogels 181 µm in size and a high surface-area-to-weight ratio of 2945 cm2/g. The PVA microcarriers supported a 2.6-fold expansion of hMSCs in a 30-mL single-use stirred bioreactor after a 7 d culture period, comparable to that of commercially available microcarriers. Interestingly, we observed that hMSCs on PVA microcarriers adhered to adjacent microcarriers, resulting in the aggregation of hMSC-PVA microcarriers. Therefore, we conducted a long-term expansion culture using a bead-to-bead cell transfer method with PVA microcarriers. Fresh microcarriers were added to the cell-populated microcarriers in the bioreactor on days 7 and 14. hMSCs on PVA microcarriers continued to grow for 21 d using the bead-to-bead cell transfer method. Furthermore, magnetic PVA (PVA-mag) microcarriers were developed by loading magnetic nanoparticles into PVA microcarriers, and we demonstrated that these PVA-mag microcarriers enabled cell recovery by magnetic separation. These results suggest that these PVA microcarriers can contribute to the large-scale culture of hMSCs for regenerative medicine and cell therapy.

Transplantable cell-encapsulation device using a semipermeable ethylene-vinyl alcohol copolymer membrane in a mouse diabetic model.

Cell-based therapy is an attractive approach, and encapsulation of therapeutic cells is a promising strategy because it prevents immune responses and allows transplanted cells to be retrieved in case of dysfunction. Bioartificial pancreas, in which insulin-secreting cells are encapsulated in a semipermeable membrane bag, is a new class of medical device for treating type-I diabetes. In this study, we developed a macroencapsulation device in which the pancreatic beta cell line MIN6 was encapsulated in a semipermeable bag made of an ethylene-vinyl alcohol copolymer membrane. In vitro evaluation of ATP and insulin levels revealed that MIN6 cells grown in Matrigel within the device secreted insulin in response to glucose levels. Transplantation of the device lowered blood glucose levels for 30 days in diabetic mice. Histological observation revealed that MIN6 cells formed spheroids in Matrigel, and no host cells were detected within the device. Blood levels of inflammatory cytokines in the transplanted mice were similar to those in non-transplanted mice, and antibody levels in the device were lower than those in the intraperitoneal fluid. These results suggest that the semipermeable ethylene-vinyl alcohol copolymer membrane developed in this study is useful for cell encapsulation in cell-based therapies, including beta-cell macroencapsulation for type-1 diabetes.

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system.

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.

Dynamics of yeast prion aggregates in single living cells.

Prions are propagating proteins that are ordered protein aggregates, in which the phenotypic trait is retained in the altered protein conformers. To understand the dynamics of the prion aggregates in living cells, we directly monitored the fate of the aggregates using an on-chip single-cell cultivation system as well as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed that the visible foci of yeast prion Sup35 fused with GFP are dispersed throughout the cytoplasm during cell growth, but retain the prion phenotype. FCS showed that [PSI+] cells, irrespective of the presence of foci, contain diffuse oligomers, which are transmitted to their daughter cells. Single-cell observations of the oligomer-based transmission provide a link between previous in vivo and in vitro analyses of the prion and shed light on the relationship between the protein conformation and the phenotype.