An Electrical Stimulation Culture System for Daily Maintenance-Free Muscle Tissue Production.

Low-labor production of tissue-engineered muscles (TEMs) is one of the key technologies to realize the practical use of muscle-actuated devices. This study developed and then demonstrated the daily maintenance-free culture system equipped with both electrical stimulation and medium replacement functions. To avoid ethical issues, immortal myoblast cells C2C12 were used. The system consisting of gel culture molds, a medium replacement unit, and an electrical stimulation unit could produce 12 TEMs at one time. The contractile forces of the TEMs were measured with a newly developed microforce measurement system. Even the TEMs cultured without electrical stimulation generated forces of almost 2 mN and were shortened by 10% in tetanic contractions. Regarding the contractile forces, electrical stimulation by a single pulse at 1 Hz was most effective, and the contractile forces in tetanus were over 2.5 mN. On the other hand, continuous pulses decreased the contractile forces of TEMs. HE-stained cross-sections showed that myoblast cells proliferated and fused into myotubes mainly in the peripheral regions, and fewer cells existed in the internal region. This must be due to insufficient supplies of oxygen and nutrients inside the TEMs. By increasing the supplies, one TEM might be able to generate a force up to around 10 mN. The tetanic forces of the TEMs produced by the system were strong enough to actuate microstructures like previously reported crawling robots. This daily maintenance-free culture system which could stably produce TEMs strong enough to be utilized for microrobots should contribute to the advancement of biohybrid devices.

Murine serum obtained from bone marrow-transplanted mice promotes the proliferation of hematopoietic stem cells by co-culture with MS-5 murine stromal cells.

To examine whether serum obtained from bone marrow-transplanted mice can selectively expand hematopoietic stem cells (HSCs) among whole bone marrow cells in vitro, whole bone marrow cells were cultured with or without MS-5 murine stromal cells in the presence of serum obtained from transplanted mice on day 3 (day 3 serum) or serum from normal mice for 7 days. When whole bone marrow cells and MS-5 cells were co-cultured in day 3 serum for 7 days, the c-kit-positive, Sca-1-positive, lineage marker-negative cells (KSL cells) expanded approximately 25 times; however, when they were co-cultured in normal serum for 7 days, the KSL cells expanded approximately 1.3 times. Direct contact between the whole bone marrow cells and MS-5 cells was essential for expansion of KSL cells in the co-culture, and it upregulated the expression of some cytokines in MS-5. Above all, the day 3 serum specifically upregulated the expression of SCF, SDF-1 alpha, G-CSF, IL-11 and IL-6 in MS-5. The level of testosterone in the day 3 serum was higher than normal serum and the addition of the testosterone in the culture expanded the KSL cells among whole bone marrow cells on MS-5 cells and also upregulated the expression of SDF-1 alpha, IL-11 and IL-6 in MS-5. These data indicates that the serum of bone marrow-transplanted mice contains a factor(s) that induced changes in the expression levels of various cytokines in MS-5 stromal cells and enabled the MS-5 cells to expand the KSL cells among whole bone marrow cells.

Cloning of hibernation-related genes of bullfrog (Rana catesbeiana) by cDNA subtraction.

Seven genes specifically expressed during hibernation in the bullfrog (Rana catesbeiana) were cloned from a subtracted cDNA library constructed from livers of winter bullfrogs. Those genes were fibrinogen alpha-subunit, fibrinogen gamma-subunit, complement component C3, alpha-1-microglobulin/bikunin precursor (AMBP), transferrin, apoferritin middle subunit and one novel gene. Northern hybridization has indicated that these seven genes were specifically induced or enhanced in winter. Above all, expression of the novel gene was specifically induced in winter in liver, though the expression of that was neither induced in bullfrog nor Xenopus laevis by cold treatment. The novel gene, which was designated as rc-hirp (Rana catesbeiana hibernation-related protein), encoded 420 base pairs length and a putative protein of 139 amino acid residues. Annual analyses of the expression of these genes have suggested that the seven winter-specific genes are playing an important role in hibernation processes.