Alternating electric field application induced non-contact and enzyme-free cell detachment.

Low intensity (< 2 Vpp/cm (peak to peak voltage/cm)), high frequency (10-30 MHz), and 10 min alternating electric fields (sine wave with no DC component) induce non-contact and enzyme-free cell detachment of anchorage-dependent cells directly from commercially available cell culture flasks and stack plates. 0.25 Vpp/cm, 20 MHz alternating electric field for 10 min at room temperature (RT) induced maximum detachment and separated 99.5 ± 0.1% (mean ± SEM, n = 6) of CHO-K1 and 99.8 ± 0.2% of BALB/3T3 cells from the culture flasks. Both vertical and lateral alternating electric field applications for 10 min at RT detach the CHO-K1 cells from 25 cm2 culture flasks. The alternating electric field application induced cell detachment is almost noncytotoxic, and over 90% of the detached cells remained alive. The alternating electric field applied CHO-K1 cells for 90 min showed little or no lag phase and immediately enter exponential phase in cell growth. Combination of the 20 MHz alternating electric field and enzymatic treatment for 4 min at 37 °C showed synergetic effect and quickly detached human induced pluripotent stem cells from a laminin-coated culture flask compared with the only enzymatic treatment. These results indicate that the rapid cell detachment with both the electric field application and the enzymatic treatment could be applied to subcultures of cells that are susceptible to prolonged enzymatic digestion damage for mass culture of sustainable clinical use.

Adequate taylor couette flow-mediated shear stress is useful for dissociating human iPS cell-derived cell aggregates.

Pluripotent stem cell including induced pluripotent stem cells (iPSC) are promising cell sources for regenerative medicine and for three-dimensional suspension culture technologies which may enable the generation of robust numbers of desired cells through cell aggregation. Although manual procedure is widely used for dissociating cell aggregates, the development of non-manual procedures using devices will contribute to efficient cell manufacturing. In the present study, we developed novel cell aggregate dissociation devices with a rotating cylinder inside based on taylor couette flow-mediated shear stress. The shear stress can be increased according to an increase in the size of the rotating cylinder inside the devices and the rotation rate. Adequate device size and suitable rotation rate efficiently dissociated cell aggregates after the undifferentiated expansion and the cardiac differentiation of human iPSC. These finding suggest that non-manual device procedure might be useful for harvesting single cells from human iPSC-derived cell aggregates.

A novel, flexible and automated manufacturing facility for cell-based health care products: Tissue Factory.

INTRODUCTION: Current production facilities for Cell-Based Health care Products (CBHPs), also referred as Advanced-Therapy Medicinal Products or Regenerative Medicine Products, are still dependent on manual work performed by skilled workers. A more robust, safer and efficient manufacturing system will be necessary to meet the expected expansion of this industrial field in the future. Thus, the 'flexible Modular Platform (fMP)' was newly designed to be a true "factory" utilizing the state-of-the-art technology to replace conventional "laboratory-like" manufacturing methods. Then, we built the Tissue Factory as the first actual entity of the fMP. METHODS: The Tissue Factory was designed based on the fMP in which several automated modules are combined to perform various culture processes. Each module has a biologically sealed chamber that can be decontaminated by hydrogen peroxide. The asepticity of the processing environment was tested according to a pharmaceutical sterility method. Then, three procedures, production of multi-layered skeletal myoblast sheets, expansion of human articular chondrocytes and passage culture of human induced pluripotent stem cells, were conducted by the system to confirm its ability to manufacture CHBPs. RESULTS: Falling or adhered microorganisms were not detected either just after decontamination or during the cell culture processes. In cell culture tests, multi-layered skeletal myoblast sheets were successfully manufactured using the method optimized for automatic processing. In addition, human articular chondrocytes and human induced-pluripotent stem cells could be propagated through three passages by the system at a yield comparable to manual operations. CONCLUSIONS: The Tissue Factory, based on the fMP, successfully reproduced three tentative manufacturing processes of CBHPs without any microbial contamination. The platform will improve the manufacturability in terms of lower production cost, improved quality variance and reduced contamination risks. Moreover, its flexibility has the potential to adapt to the modern challenges in the business environment including employment issues, low operational rates, and relocation of facilities. The fMP is expected to become the standard design basis of future manufacturing facilities for CBHPs.

[Civil engineering education at the Imperial College of Engineering in Tokyo: an analysis based on Ayahiko Ishibashi's memoirs].

The Imperial College of Engineering (ICE or Kobu-Daigakko) in Tokyo, founded in 1873 under the auspices of the Ministry of Public Works, was one of the most prominent modern institutions of engineering education in early Meiji Japan. Previous studies have revealed that the ICE offered large scale practical training programs at enterprises of the Ministry, which sometimes lasted several months, and praised their ideal combination of theory and practice. In reality, it has been difficult to evaluate the quality of education at the ICE mainly because of scarcity of sources. ICE students published a collection of memoirs for alumni members, commemorating the fiftieth-year of the history of the Tokyo Imperial University. Drawing on the previously neglected collection of students' memoires, this paper appraises the education of civil engineering offered by the ICE. The paper also compares this collection with other official records of the college, and confirms it as a reliable source, even though it contains some minor errors. The author particularly uses the memoirs by Ayahiko Ishibashi, one of the first graduates from its civil engineering course, who left sufficient reminiscences on education that he received. This paper, as a result, illustrates that the main practical training for the students of civil engineering was limited to designing process, including surveying. Furthermore, practical training that Ishibashi received at those enterprises often lacked a plan, and its effectiveness was questionable.

Influence of antiplatelet therapy on postoperative recurrence of chronic subdural hematoma: a multicenter retrospective study in 719 patients.

OBJECTIVE: The present study tested the hypothesis of whether antiplatelet agents (APA) induce chronic subdural hematoma (CSDH) recurrence via a platelet aggregation inhibitory effect. METHOD: We examined risk factors for CSDH recurrence, focusing on APA, in 719 consecutive patients who admitted to three tertiary hospitals and underwent burr-hole craniostomy and irrigation for CSDH. This was a multicenter, retrospective, observational study. RESULTS: Age, sex, history of diabetes mellitus, hypertension, chronic renal failure, alcohol consumption habits, consciousness disturbance on admission, or preoperative CT density was not associated with recurrence. Subdural drainage was significantly associated with less recurrence. Preoperative oral APA administration was significantly associated with more recurrence. The recurrence rate of CSDH in non-APA group was 11% if surgery was performed on admission. However, if surgery was performed immediately after discontinuation of oral APA administration, the recurrence rate in APA group significantly increased to 32% (p value<0.0001; odds ratio, 3.77; 95% confidence interval, 1.72-8.28). The effect of APA on CSDH recurrence gradually diminished as the number of days until initial surgery, after stopping APA, increased. CONCLUSION: Antiplatelet therapy significantly influences the recurrence of CSDH.

Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique.

Automated manufacturing is a key for tissue-engineered therapeutic products to become common-place and economical. Here, we developed an automatic cell sheet stacking apparatus to fabricate 3-dimensional tissue-engineered constructs exploiting our cell sheet manipulator technique, where cell sheets harvested from temperature-responsive culture dishes are stacked into a multilayered cell sheet. By optimizing the stacking conditions and cell seeding conditions, the apparatus was eventually capable of reproducibly making five-layer human skeletal muscle myoblast (HSMM) sheets with a thickness of approximately 70-80 µm within 100 min. Histological sections and confocal topographies of the five-layer HSMM sheets revealed a stratified structure with no delamination. In cell counts using trypsinization, the live cell numbers in one-, three- and five-layer HSMM sheets were equivalent to the seeded cell numbers at 1 h after the stacking processes; however, after subsequent 5-day static cultures, the live cell numbers of the five-layered HSMM sheets decreased slightly, while one- and three-layer HSMM sheets maintained their live cell numbers. This suggests that there are thickness limitations in maintaining tissues in a static culture. We concluded that by combining our cell sheet manipulator technique and industrial robot technology we can create a secure, cost-effective manufacturing system able to produce tissue-engineered products from cell sheets.

Effectiveness of deep vein thrombosis screening on admission to a rehabilitation hospital: a prospective study in 1043 consecutive patients.

INTRODUCTION: The diagnosis and treatment of deep vein thrombosis/pulmonary embolism (DVT/PE) are important issues not only in acute-care hospitals, but also in rehabilitation hospitals. To test the hypothesis that DVT/PE occurring at rehabilitation hospitals is carried over from acute-care hospitals, we evaluated a method of DVT screening on admission that combined D-dimer (D-D) measurement and compression ultrasound (CUS). MATERIAL AND METHODS: This prospective single-center observational study included 1043 patients who were admitted to our rehabilitation hospital between August 1, 2007, and August 1, 2011, after excluding those meeting the exclusion criteria. We screened patients on admission and observed the occurrence of DVT/PE until discharge. RESULTS: Of the 1043 patients, 152 (14.6%) had a D-D level of ≥ 3.0 µg/mL on admission. CUS was performed for these patients and indicated the presence of DVT in 15 patients (1.4%), who were subsequently treated. Of these 15 patients, six (40%) had no DVT symptoms, and five of these six patients had spinal cord injury. Of 137 patients who were CUS negative, two developed DVT/PE within 8 days of hospitalization, and recovery was achieved by treatment. No subsequent occurrence was observed. CONCLUSIONS: These results indicated all cases were carried over from acute-care hospitals. Six out of 15 patients had no symptoms of DVT/PE. Thus, this method of DVT screening on admission to a rehabilitation hospital is useful for risk management.

In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels.

In vitro fabrication of functional vascularized three-dimensional tissues has been a long-standing objective in the field of tissue engineering. Here we report a technique to engineer cardiac tissues with perfusable blood vessels in vitro. Using resected tissue with a connectable artery and vein as a vascular bed, we overlay triple-layer cardiac cell sheets produced from coculture with endothelial cells, and support the tissue construct with media perfused in a bioreactor. We show that endothelial cells connect to capillaries in the vascular bed and form tubular lumens, creating in vitro perfusable blood vessels in the cardiac cell sheets. Thicker engineered tissues can be produced in vitro by overlaying additional triple-layer cell sheets. The vascularized cardiac tissues beat and can be transplanted with blood vessel anastomoses. This technique may create new opportunities for in vitro tissue engineering and has potential therapeutic applications.

Creation of human cardiac cell sheets using pluripotent stem cells.

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.

Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.

Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1 × 10(8) cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5 × 10(8). Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.

Genotyping of thiopurine methyltransferase using pyrosequencing.

Thiopurine methyltransferase (TPMT) metabolizes thiopurine drugs which are used in the treatment of leukemia and some autoimmune diseases. Previously, 11 mutant alleles of TPMT gene (TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8) have been reported. These mutant alleles may cause life-threatening toxicity in patients exposed to thiopurine drugs, 6-mercaptopurine and azathioprine. We have developed a rapid and accurate protocol for TPMT genotype determination using Pyrosequencing(TM) technology in 96 Japanese subjects. Five fragments of the TPMT gene (exon 4, 5, 7, 8, 10) were amplified by PCR, and the 10 single-nucleotide polymorphisms (SNPs) for TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8 were sequenced. The results of this pyrosequencing method corresponded exactly with those of the DNA sequencing method using BigDye terminator chemistry. We have demonstrated that typing of 10 SNPs can be performed within 30 min. Pyrosequencing has a wide application in the large-scale identification of individual TPMT genotypes.

Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi.

Ethanol was produced by simultaneous saccharification and fermentation (SSF) from beech wood chips after bioorganosolve pretreatments by ethanolysis and white rot fungi, Ceriporiopsis subvermispora, Dichomitus squalens, Pleurotus ostreatus, and Coriolus versicolor. Beech wood chips were pretreated with the white rot fungi for 2-8 weeks without addition of any nutrients. The wood chips were then subjected to ethanolysis to separate them into pulp and soluble fractions (SFs). From the pulp fraction (PF), ethanol was produced by SSF using Saccharomyces cerevisiae AM12 and a commercial cellulase preparation, Meicelase, from Trichoderma viride. Among the four strains, C. subvermispora gave the highest yield on SSF. The yield of ethanol obtained after pretreatment with C. subvermispora for 8 weeks was 0.294 g g(-1) of ethanolysis pulp (74% of theoretical) and 0.176 g g(-1) of beech wood chips (62% of theoretical). The yield was 1.6 times higher than that obtained without the fungal treatments. The biological pretreatments saved 15% of the electricity needed for the ethanolysis.