Cell-Based Microfluidic Device Utilizing Cell Sheet Technology.

The development of microelectromechanical systems has resulted in the rapid development of polydimethylpolysiloxane (PDMS) microfluidic devices for drug screening models. Various cell functions, such as the response of endothelial cells to fluids, have been elucidated using microfluidic devices. Additionally, organ-on-a-chip systems that include organs that are important for biological circulation, such as the heart, liver, pancreas, kidneys, and brain, have been developed. These organs realize the biological circulation system in a manner that cannot be reproduced by artificial organs; however, the flow channels between the organs are often artificially created by PDMS. In this study, we developed a microfluidic device consisting only of cells, by combining cell sheet technology with microtitanium wires. Microwires were placed between stacked fibroblast cell sheets, and the cell sheets adhered to each other, after which the microwires were removed leaving a luminal structure with a size approximately equal to the arteriolar size. The lumen structure was constructed using wires with diameters of 50, 100, 150, and 200 µm, which were approximations of the arteriole diameters. Furthermore, using a perfusion device, we successfully perfused the luminal structure created inside the cell sheets. The results revealed that a culture solution can be supplied to a cell sheet with a very high cell density. The biofabrication technology proposed in this study can contribute to the development of organ-on-a-chip systems.

Production of scaffold-free cell-based meat using cell sheet technology.

In the production of cell-based meat, it is desirable to reduce animal-derived materials as much as possible to meet the challenges of sustainability. Here, we demonstrate the "cell sheet-based meat": scaffold-free cell-based meat using cell sheet technology and characterize its texture and nutrients. Bovine myoblast cell sheets were prepared using temperature-responsive culture dishes (TRCDs) and 10 stacked cell sheets to fabricate three-dimensional tissue of 1.3-2.7 mm thickness. Hardness was increased by incubation on the TRCD and was further increased by boiling as is characteristic of natural meat. The wet weight percentage of total protein in the cell sheet was about half that of beef. In this method, large-sized items of cell sheet-based meat were also created by simply scaling up the TRCD. This method promises an environment-friendly food product.

Bioengineering of a scaffold-less three-dimensional tissue using net mould.

Tissue engineering has attracted attention worldwide because of its application in regenerative medicine, drug screening, and cultured meat. Numerous biofabrication techniques for producing tissues have been developed, including various scaffold and printing methods. Here, we have proposed a novel tissue engineering method using a net metal mould without the use of a scaffold. Briefly, normal human dermal fibroblasts seeded on a dimple plate were subjected to static culture technique for several days to form spheroids. Spheroids of diameter ⩾200µm were poured into a net-shaped mould of gap ⩽100µm and subjected to shake-cultivation for several weeks, facilitating their fusion to form a three-dimensional (3D) tissue. Through this study, we successfully constructed a scaffold-free 3D tissue having strength that can be easily manipulated, which was difficult to construct using conventional tissue engineering methods. We also investigated the viability of the 3D tissue and found that the condition of the tissues was completely different depending on the culture media used. Collectively, this method allows scaffold-free culture of 3D tissues of unprecedented thickness, and may contribute largely to next-generation tissue engineering products.

Perfusable System Using Porous Collagen Gel Scaffold Actively Provides Fresh Culture Media to a Cultured 3D Tissue.

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.

Invasive Mucinous Adenocarcinoma of the Lung Presenting With Multiple Cavities.

A 78-year-old woman presented to our hospital with a two-week history of productive cough. Chest computed tomography (CT) showed bilateral multiple pulmonary nodules with cavities. Although the cytology of her sputum revealed adenocarcinoma, she refused any treatment. Following supportive care, 30 months later, she presented to our hospital with dyspnea and fever. Chest CT showed progression of multiple pulmonary nodules and cavities. Despite treatment with antibiotics and palliative care, she died on the 10th day of hospitalization. Pathological autopsy confirmed the diagnosis of pulmonary invasive mucinous adenocarcinoma (IMA). The typical CT findings of IMA include multiple consolidations or ground-glass opacities mimicking pneumonia; rarely, cavitary lesions are also observed. Clinicians should consider IMA as a differential diagnosis for lung cavities.

Pulmonary lymphangitic carcinomatosis secondary to ureteral cancer.

BACKGROUND: Pulmonary lymphangitic carcinomatosis (PLC) is a metastatic lung disease of malignant tumors that spread through pulmonary lymphatic vessels. Although prompt diagnosis and specific treatment of PLC are required due to the poor prognosis associated with this disease, it is often challenging to determine the primary cancer site. CASE PRESENTATION: A 67-year-old Japanese woman presented to our hospital with a 10-day history of cough and dyspnea on exertion. Chest radiography and computed tomography (CT) revealed diffuse nodular opacities with interlobular septal thickening. Both bronchoalveolar lavage (BAL) and transbronchial lung biopsy (TBLB) revealed carcinoma cells with unknown origin. Contrast-enhanced CT depicted a mass in the right ureter with hydronephrosis, and retrograde urography showed a narrowing of the right ureter. Urine cytology from her right ureter via ureteral catheter also revealed atypical cells, highly suggestive of malignancy. Immunohistochemical examination of lung specimens via TBLB showed results consistent with lung metastasis of ureteral cancer. Therefore, we arrived at a diagnosis of PLC secondary to ureteral cancer. CONCLUSIONS: This case encouraged multidisciplinary discussion and a whole-body examination, including TBLB with immunohistochemistry, to determine the origin of PLC.

Higher enterococcus counts indicate a lower risk of colorectal adenomas: a prospective cohort study.

Intestinal bacteria play an important role in human health. This prospective cohort study aimed to investigate the relationship between the abundance of different intestinal bacteria and the risk of developing colorectal cancer (CRC). Fecal samples from CRC patients (n = 157) were collected at the start of the study wherein patients subsequently underwent endoscopy to remove polyps. Gut bacteria were isolated by using specific culture methods and the fecal counts of various bacteria were quantified by reverse-transcription-quantitative-PCR (RT-qPCR) assays. The obtained data were subjected to cohort analysis in relation to the incidence of colorectal adenomas after 4 years of intervention. No relationship was detected between the counts of major intestinal bacteria and the incidence of colorectal adenomas. However, interestingly, a significant negative correlation was noted between colorectal adenoma incidence and the counts of bacteria grown on Columbia blood agar base (COBA) (P = 0.007). The risk ratio of colorectal adenomas was 0.58 (95% CI: 0.35-0.96) in the group with the highest bacterial count compared to the lowest. Bacteria grown on COBA were more abundant in older patients, non-smoking patients, and patients with a lower body mass index. The RT-qPCR results revealed a significantly lower colorectal adenoma incidence in subjects with higher enterococcal count as compared to subjects with a lower count, with a risk ratio of 0.47 (95% CI: 0.30-0.76). Correlation of a higher enterococci count with a lower risk of CRC development suggests that certain Enterococcus strains may have adenoma suppressive effects.

Mother-to-infant transmission of intestinal bifidobacterial strains has an impact on the early development of vaginally delivered infant's microbiota.

OBJECTIVES: Bifidobacterium species are one of the major components of the infant's intestine microbiota. Colonization with bifidobacteria in early infancy is suggested to be important for health in later life. However, information remains limited regarding the source of these microbes. Here, we investigated whether specific strains of bifidobacteria in the maternal intestinal flora are transmitted to their infant's intestine. MATERIALS AND METHODS: Fecal samples were collected from healthy 17 mother and infant pairs (Vaginal delivery: 12; Cesarean section delivery: 5). Mother's feces were collected twice before delivery. Infant's feces were collected at 0 (meconium), 3, 7, 30, 90 days after birth. Bifidobacteria isolated from feces were genotyped by multilocus sequencing typing, and the transitions of bifidobacteria counts in infant's feces were analyzed by quantitative real-time PCR. RESULTS: Stains belonging to Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium longum subsp. longum, and Bifidobacterium pseudocatenulatum, were identified to be monophyletic between mother's and infant's intestine. Eleven out of 12 vaginal delivered infants carried at least one monophyletic strain. The bifidobacterial counts of the species to which the monophyletic strains belong, increased predominantly in the infant's intestine within 3 days after birth. Among infants delivered by C-section, monophyletic strains were not observed. Moreover, the bifidobacterial counts were significantly lower than the vaginal delivered infants until 7 days of age. CONCLUSIONS: Among infants born vaginally, several Bifidobacterium strains transmit from the mother and colonize the infant's intestine shortly after birth. Our data suggest that the mother's intestine is an important source for the vaginal delivered infant's intestinal microbiota.

Reduction of enhanced rabbit intraocular pressure by instillation of pyroglutamic acid eye drops.

L-Pyroglutamic acid (PGA) is an endogenous molecule derived from l-glutamate. We demonstrate the effects of PGA on intraocular pressure (IOP) in experimentally induced ocular hypertension in rabbits. In the in vitro and in vivo transcorneal penetration studies, the PGA solution (PGA in saline) did not penetrate the rabbit cornea. On the other hand, the penetration of PGA was improved by the addition of zinc chloride and 2-hydroxypropyl-ß-cyclodextrin (HPCD), and PGA penetration was enhanced with increasing HPCD concentration. Therefore, PGA solutions containing 0.5% zinc chloride and 5% or 10% HPCD (PGA/HPCD(5% or 10%) eye drops) were used to investigate the effects for IOP in this study. An elevation in IOP was induced by the rapid infusion of 5% glucose solution (15 mL/kg of body weight) through the marginal ear vein or maintaining under dark phase for 5 h. In the both models, the induced elevation in IOP was prevented by the instillation of PGA/HPCD eye drops, and the IOP-reducing effect enhanced with increasing HPCD concentration in the drops. Nitric oxide (NO) levels elevated in the aqueous humor following the infusion of 5% glucose solution, and this increase was also suppressed by the instillation of PGA/HPCD eye drops. In conclusion, the present study demonstrates that the instillation of PGA/HPCD eye drops has an IOP-reducing effect in rabbits with experimentally induced ocular hypertension, probably as a result of the suppression of NO production.

Ethnic diversity of gut microbiota: species characterization of Bacteroides fragilis group and genus Bifidobacterium in healthy Belgian adults, and comparison with data from Japanese subjects.

The composition of the human gut microbiota is related to host health, and it is thought that dietary habits may play a role in shaping this composition. Here, we examined the population size and prevalence of six predominant bacterial genera and the species compositions of genus Bifidobacterium (g-Bifid) and Bacteroides fragilis group (g-Bfra) in 42 healthy Belgian adults by quantitative PCR (qPCR) over a period of one month. The population sizes and prevalence of these bacteria were basically stable throughout the study period. The predominant g-Bifid species were Bifidobacterium adolescentis and Bifidobacterium longum ss. longum, and the predominant g-Bfra species were Bacteroides vulgatus, Bacteroides uniformis, and Bacteroides ovatus. The Belgian gut microbiota data were then compared with gut microbiota data from 46 Japanese subjects collected according to the same protocol (Matsuki et al., Appl. Environ. Microbiol. 70, 167-173, 2004). The population size and prevalence of Bifidobacterium catenulatum group were significantly lower in the Belgian gut microbiota than in the Japanese gut microbiota (P < 0.001); however, the population size and prevalence of g-Bifid did not differ. This species-level qPCR analysis will be helpful for investigating the diversity of gut microbiota among ethnic groups.

Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant, determined by multilocus sequencing typing and amplified fragment length polymorphism.

The gastrointestinal tracts of neonates are colonized by bacteria immediately after birth. It has been discussed that the intestinal microbiota of neonates includes strains transferred from the mothers. Although some studies have indicated possible bacterial transfer from the mother to the newborn, this is the first report confirming the transfer of bifidobacteria at the strain level. Here, we investigated the mother-to-infant transmission of Bifidobacterium longum subsp. longum by genotyping bacterial isolates from the feces of mothers before delivery and of their infants after delivery. Two hundred seven isolates from 8 pairs of mothers and infants were discriminated by multilocus sequencing typing (MLST) and amplified fragment length polymorphism (AFLP) analysis. By both methods, 11 strains of B. longum subsp. longum were found to be monophyletic for the feces of the mother and her infant. This finding confirms that these strains were transferred from the intestine of the mother to that of the infant. These strains were found in the first feces (meconium) of the infant and in the feces at days 3, 7, 30, and 90 after birth, indicating that they stably colonize the infant's intestine immediately after birth. The strains isolated from each family did not belong to clusters derived from any of the other families, suggesting that each mother-infant pair might have unique family-specific strains.

Beneficial effects of probiotic bifidobacterium and galacto-oligosaccharide in patients with ulcerative colitis: a randomized controlled study.

BACKGROUND/AIM: In previous studies, we described the beneficial effects of bifidobacteria-fermented milk in patients with ulcerative colitis (UC). Here, we examined the effects of a live Bifidobacterium breve strain Yakult, a probiotic contained in bifidobacteria-fermented milk, and galacto-oligosaccharide (GOS) as synbiotics in UC patients. METHODS: Forty-one patients with mild to moderate UC were assigned to two groups; one group was treated with the synbiotics and the other was not (control). The treatment group ingested 1 g of the probiotic powder (10(9) CFU/g) three times a day, and 5.5 g of GOS once a day for one year. At the start and the end of this study, colonoscopic index and the amount of myeloperoxidase in a lavage solution were used as disease activity indices. Bacterial counts in the feces at the start and the end of this study were also examined. RESULTS: After a one-year treatment with the synbiotics, the clinical status of the UC patients as assessed by colonoscopy, significantly improved. Furthermore, the amount of myeloperoxidase in the lavage also decreased in these patients after the synbiotic treatment. The synbiotics significantly reduced the fecal counts of Bacteroidaceae and fecal pH. CONCLUSION: Administration of live B. breve strain Yakult and GOS can improve the clinical condition of patients with UC. These results encouraged us to perform a large-scale randomized, placebo-controlled trial.

New polar constituents in the pupae of the silkworm Bombyx mori L. (II): developmental changes of the constituents.

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. (1)H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.

Alistipes indistinctus sp. nov. and Odoribacter laneus sp. nov., common members of the human intestinal microbiota isolated from faeces.

Two anaerobic, non-spore-forming, non-motile, Gram-negative-staining bacteria, strains YIT 12060(T) and YIT 12061(T), were isolated from human faeces. Cells of strain YIT 12060(T) were coccoid to rod-shaped with round ends, positive for catalase, negative for indole and oxidase production, produced succinic and acetic acids as end products of glucose metabolism in peptone/yeast extract/glucose medium and had a DNA G+C content of 55.2 mol%. The main respiratory quinones were MK-10 (40%) and MK-11 (57%). Fatty acid analysis demonstrated the presence of a high concentration of iso-C(15 : 0) (56%). Following 16S rRNA gene sequence analysis, this strain was found to be most closely related to species of the genus Alistipes, with 90.9-92.6% gene sequence similarities to type strains of this species. Phylogenetic analysis and biochemical data supported the affiliation of strain YIT 12060(T) to the genus Alistipes of the family 'Rikenellaceae'. Strain YIT 12060(T) therefore represents a novel species of the genus Alistipes for which the name Alistipes indistinctus sp. nov. is proposed; the type strain is YIT 12060(T) (=DSM 22520(T)=JCM 16068(T)). Cells of the other isolate, strain YIT 12061(T), were pleomorphic rods that were asaccharolytic, catalase- and oxidase-negative, positive for gelatin hydrolysis and indole production, produced small amounts of succinic, acetic and iso-valeric acids as end products of metabolism in peptone/yeast extract medium and had a DNA G+C content of approximately 42.4 mol%. On the basis of 16S rRNA gene sequence similarity values, this strain was shown to belong to the family 'Porphyromonadaceae' and related to the type strains of Odoribacter splanchnicus (89.6%) and Odoribacter denticanis (86.2%); similarity values with strains of recognized species within the family 'Porphyromonadaceae' were less than 84 %. Biochemical data supported the affiliation of strain YIT 12061(T) to the genus Odoribacter. Strain YIT 12061(T) therefore represents a novel species for which the name Odoribacter laneus sp. nov. is proposed; the type strain is YIT 12061(T) (=DSM 22474(T)=JCM 16069(T)).

Bacteroides clarus sp. nov., Bacteroides fluxus sp. nov. and Bacteroides oleiciplenus sp. nov., isolated from human faeces.

Three Gram-stain-negative, obligately anaerobic, non-spore-forming, rod-shaped bacteria (strains YIT 12056T, YIT 12057T and YIT 12058T) were isolated from human faeces. These strains were characterized by phylogenetic analyses based on 16S rRNA gene sequence and phenotypic tests. 16S rRNA gene sequence analyses revealed that strains YIT 12056T, YIT 12057T and YIT 12058T were most closely related to the type strains of Bacteroides gallinarum, Bacteroides uniformis and Bacteroides intestinalis with approximate similarity values of 96.6, 95.0 and 96.7%, respectively. The DNA G+C contents of the novel strains were 45.3 (YIT 12056T), 45.2 (YIT 12057T) and 43.6 mol% (YIT 12058T) and the major respiratory quinones of all three isolates were menaquinones MK-10 and MK-11. These properties were typical for members of the genus Bacteroides. The results of the other phenotypic analyses also supported the affiliation of these strains to the genus Bacteroides. The 16S rRNA gene sequence analysis, analysis of the major cellular fatty acids and other biochemical tests enabled the genotypic and phenotypic differentiation of the three new strains. Based on these data, three novel species, Bacteroides clarus sp. nov., Bacteroides fluxus sp. nov. and Bacteroides oleiciplenus sp. nov. are proposed. The type strains of B. clarus, B. fluxus and B. oleiciplenus are YIT 12056T (=JCM 16067T=DSM 22519T), YIT 12057T (=JCM 16101T=DSM 22534T) and YIT 12058T (=JCM 16102T=DSM 22535T), respectively.

Early use of probiotics is important therapy in infants with severe congenital anomaly.

BACKGROUND: Infants with severe congenital anomaly often need to undergo operation followed by antibiotic therapy. As a result they inevitably acquire abnormal intestinal microbiota, which cause severe infections such as necrotizing enterocolitis. Also, intestinal function deteriorates and their nutritional state is very poor. In order to prevent these situations probiotic therapy is proposed as an effective supporting treatment. Probiotic therapy were therefore applied to infants with severe congenital anomaly as early as possible to ascertain its efficacy. METHODS: As probiotics, two bacteria were used: Bifidobacterium breve Yakult and Lactobacillus casei Shirota. Probiotic therapy was used in four infants with severe congenital anomaly as early as possible after surgery. Their intestinal microbiota and physical growth were followed through the treatment course. RESULTS: Two patients suffered from meconium peritonitis with ileal atresia. One patient was born with complex anomalies (omphalocele, bladder exstrophy, myelomeningocele). The fourth patient suffered from complete urorectal septum malformation. The intestinal microbiota of these four patients was first induced to be probiotic dominant and finally changed to commensal anaerobe dominant that was similar to normal intestinal microbiota. Pathogenic bacteria were seldom detected. The patients' physical growth was excellent despite short bowel and pulmonary hypoplasia. CONCLUSION: Probiotic therapy was effective in inducing probiotic dominant intestinal microbiota and normal intestinal microbiota in infants with severe congenital anomalies. As a result their intestinal absorptive functions were activated and severe infections were completely prevented. All of the infants grew well despite their physical disadvantages.

Effect of Lactobacillus casei on Streptococcus bovis in faecal flora.

Bacteraemia caused by Streptococcus bovis is often associated with colorectal tumours. Also, experimental studies have been proposed that S bovis acts as a promoter of tumours. We report the case of a man with colon adenoma who had a high proportion of S bovis in his faecal flora. He was treated with a Lactobacillus casei preparation (BLP) and the effect on the faecal flora was examined. L casei reduced the proportion of S bovis (from 43% down to 9%), and the effect continued after the administration of BLP was stopped. Our data indicate that BLP can repress the excessive colonisation of S bovis.

Succinatimonas hippei gen. nov., sp. nov., isolated from human faeces.

A novel strictly anaerobic, non-spore-forming, non-motile, non-flagellated, rod-shaped, Gram-negative bacterium (YIT 12066T) was isolated from human faeces. The isolate was negative for catalase, oxidase, urease, hydrolysis of aesculin and gelatin, nitrate reduction and indole production. The major end products of glucose metabolism were succinate and acetate. The major cellular fatty acids (>10%) were C14:0, C18:1omega7c, C18:1omega9c, C16:1omega7c and C16:0. The G+C content of the DNA was 40.3 mol%. 16S rRNA gene sequence analysis showed that strain YIT 12066T was most closely related to members of the family Succinivibrionaceae, with sequence similarity of 92-87%. However, some phenotypic characteristics such as cellular morphology and the major fatty acid profile of strain YIT 12066T were markedly different from those of other members of the family Succinivibrionaceae. On the basis of both phylogenetic and phenotypic evidence, it is suggested that strain YIT 12066T represents a novel species in a new genus, for which the name Succinatimonas hippei gen. nov., sp. nov. is proposed; the type strain of Succinatimonas hippei is YIT 12066T (=DSM 22608T =JCM 16073T).

Paraprevotella clara gen. nov., sp. nov. and Paraprevotella xylaniphila sp. nov., members of the family 'Prevotellaceae' isolated from human faeces.

Two anaerobic, non-spore-forming, pleomorphic, Gram-negative rods, designated YIT 11840T and YIT 11841T, were isolated from human faeces. The organisms were catalase-negative, produced succinic and acetic acids as end products of glucose metabolism and had DNA G+C contents of approximately 48-49 mol%. Although the phenotypic characteristics of these two strains were very similar, analysis of their 16S rRNA gene sequences showed that they are only distantly related (93.8%), indicating that they represent two different species. A comparative sequence analysis revealed that these two species are members of the family 'Prevotellaceae' but are phylogenetically distant (<88% sequence similarity) from the known genera belonging to this family, including Prevotella, Hallela and Xylanibacter. On the basis of the phylogenetic analysis and physiological tests, strains YIT 11840T and YIT 11841T represent two novel species of a new genus, for which the names Paraprevotella clara gen. nov., sp. nov. (type strain YIT 11840T=JCM 14859T=DSM 19731T), the type species, and Paraprevotella xylaniphila sp. nov. (type strain YIT 11841T=JCM 14860T=DSM 19681T) are proposed.