Diverting the food-freezing technology improves the cryopreservation efficiency of induced pluripotent stem cells and derived neurospheres.

Introduction: Recent advances in induced pluripotent stem (iPS) technology and regenerative medicine require effective cryopreservation of iPSC-derived differentiated cells and three-dimensional cell aggregates (eg. Spheroids and organoids). Moreover, innovative freezing technologies for keeping food fresh over the long-term rapidly developed in the food industry. Therefore, we examined whether one of such freezing technologies, called "Dynamic Effect Powerful Antioxidation Keeping (DEPAK)," could be effective for the cryopreservation of biological materials. Methods: We evaluated the efficiency of cryopreservation using DEPAK and Proton freezers, both of which are used in the food industry, compared with conventional slow-freezing methods using a programmable freezer and a cell-freezing vessel. As they are highly susceptible cells to freeze-thaw damage, we selected two suspension cell lines (KHYG-1 derived from human natural killer cell leukemia and THP-1 derived from human acute monocyte leukemia) and two adherent cell lines (OVMANA derived from human ovarian tumors and HuH-7 derived from human hepatocarcinoma). We used two human iPS cell lines, 201B7-Ff and 1231A3, which were either undifferentiated or differentiated into neurospheres. After freezing using the above methods, the frozen cells and neurospheres were immediately transferred to liquid nitrogen. After thawing, we assessed the cryopreservation efficiency of cell viability, proliferation, neurosphere formation, and neurite outgrowth after thawing. Results: Among the four cryopreservation methods, DEPAK freezing resulted in the highest cell proliferation in suspension and adherent cell lines. Similar results were obtained for the cryopreservation of undifferentiated human iPS cells. In addition, we demonstrated that the DEPAK freezing method sustained the neurosphere formation capacity of differentiated iPS cells to the same extent as unfrozen controls. In addition, we observed that DEPAK-frozen neurospheres exhibited higher viability after thawing and underwent neural differentiation more efficiently than slow-freezing methods. Conclusions: Our results suggest that diversifying food-freezing technologies can overcome the difficulties associated with the cryopreservation of various biological materials, including three-dimensional cell aggregates.

Kasumi leukemia cell lines: characterization of tumor genomes with ethnic origin and scales of genomic alterations.

Kasumi-1 has played an important role in an experimental model with t(8;21) translocation, which is a representative example of leukemia cell lines. However, previous studies using Kasumi-1 show discrepancies in the genome profile. The wide use of leukemia cell lines is limited to lines that are well-characterized. The use of additional cell lines extends research to various types of leukemia, and to further explore leukemia pathogenesis, which can be achieved by uncovering the fundamental features of each cell line with accurate data. In this study, ten Kasumi cell lines established in Japan, including five that were previously unknown, have been characterized by SNP microarray and targeted sequencing. SNP genotyping suggested that the genetic ancestry in four of the ten Kasumi cell lines was not classified as Japanese but covered several different east-Asian ethnicities, suggesting that patients in Japan are genetically diverse. TP53 mutations were detected in two cell lines with complex array profiles, indicating chromosomal instability (CIN). A quantitative assessment of tumor genomes at the chromosomal level was newly introduced to reveal total DNA sizes and Scales of Genomic Alterations (SGA) for each cell line. Kasumi-1 and 6 derived from relapsed phases demonstrated high levels of SGA, implying that the level of SGA would reflect on the tumor progression and could serve as an index of CIN. Our results extend the leukemia cellular resources with an additional five cell lines and provide reference genome data with ethnic identities for the ten Kasumi cell lines.

Endoscopic small-capacity forceps increase the pathological diagnosis of gastric indefinite neoplasia.

BACKGROUND/AIMS: A definitive biopsy-based diagnosis of gastric cancer is sometimes difficult, and some cases are pathologically diagnosed as gastric indefinite neoplasia (GIN). The most appropriate forceps size for gastric biopsy has yet to be determined. In this study, we investigated the relation between the forceps size and the frequency of GIN diagnosis. MATERIALS AND METHODS: The records of patients from two historical groups were reviewed. The first group comprised patients evaluated during the period when standard biopsy forceps (StF) were used (April 2010-March 2011), and the second group comprised patients evaluated during the period when small biopsy forceps (SmF) were used (April 2011-March 2013). Patients in whom GIN lesions were diagnosed with biopsy were identified, and pertinent data were compared between the two groups of patients. RESULTS: Among the 8,420 patients who underwent esophagogastroduodenoscopy (EGD) during the first period, 2,584 (30.7%) underwent gastric biopsy with StF. Among the 15,968 patients who underwent EGD during the second period, 4,204 (26.3%) underwent gastric biopsy with SmF. GIN was diagnosed in a significantly greater number of patients in the SmF group than in the StF group (52 [1.25%] vs. 19 [0.73%]; p=0.048). The mean minor-axis lengths of the biopsy samples were 1.50±0.50 mm and 1.38±0.40 mm in the StF group and the SmF group, respectively, with the SmF group samples tending to be shorter (p=0.088). CONCLUSION: Because the SmF use may increase the rate of GIN diagnosis, the use of SmF with a standard-caliber endoscope should be avoided.

Screening for 15 pathogenic viruses in human cell lines registered at the JCRB Cell Bank: characterization of in vitro human cells by viral infection.

Human cell lines have been used in a variety of research fields as an in vitro model. These cells are all derived from human tissue samples, thus there is a possibility of virus infection. Virus tests are routinely performed in clinical practice, but are limited in cell lines. In this study, we investigated 15 kinds of viruses in 844 human cell lines registered at the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Our real-time PCR analysis revealed that six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in 43 cell lines. Of them, 20 cell lines were transformed by intentional infection in vitro with EBV or HTLV-1. Viruses in the other 23 cell lines and one EBV transformed cell line are derived from an in vivo infection, including five de novo identifications of EBV, B19V or HHV-7 carriers. Among them, 17 cell lines were established from patients diagnosed with virus-associated diseases. However, the other seven cell lines originated from in vivo cells unrelated to disease or cellular tropism. Our approach to screen for a set of 15 viruses in each cell line has worked efficiently to identify these rare cases. Virus tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral infection which can be a characteristic feature of cell lines.

HuH-7 reference genome profile: complex karyotype composed of massive loss of heterozygosity.

Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines.

The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture.

Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral genome was similar to the HHV-6B HST strain. FISH analysis using a HHV-6 DNA probe showed one signal in each cell, detected at the distal end of the long arm of chromosome 9. This was consistent with a digital PCR assay, validating one copy of the viral DNA. Because exposure of HUV-EC-C to chemicals did not cause viral reactivation, long term cell culture of HUV-EC-C was carried out to assess the stability of viral integration. The growth rate was altered depending on passage numbers, and morphology also changed during culture. SNP microarray profiles showed some differences between low and high passages, implying that the HUV-EC-C genome had changed during culture. However, no detectable change was observed in chromosome 9, where HHV-6B integration and the viral copy number remained unchanged. Our results suggest that integrated HHV-6B is stable in HUV-EC-C despite genome instability.

Aborted fetal sizes of Thoroughbred horses in Hidaka, Japan, between 2005 and 2015.

The degree of fetal growth restriction has been unclear in equine reproduction. In this study, 2,195 fetuses from 2,137 abortions during 11 seasons were examined to determine the causes of abortion, and fetal size dimensions (crown rump length and body weight) were measured. In total, 900 cases (42.1%) of abortion were identified as caused by viral infection (215, 10.1%), bacterial infection (156, 7.3%), fungal infection (25, 1.2%), circulation failure (406, 19.0%), multiple causes (66, 3.1%), deformity (13, 0.6%), placental abnormality (12, 0.6%), and other causes (7, 0.3%). All viral infections originated from equine herpes virus. Of all abortions, 94.3% occurred between 181-360 days of pregnancy, and the gestational ages at abortion were different based on the causes. Fetal sizes in viral abortions were considerably larger than those due to other reasons. Compared with viral infection, the crown rump length size dimension of fetuses aborted from multiple and fungal infection was affected. In addition, bacterial infection, circulation failure, and unknown causes of abortions also contributed to growth restriction in terms of body weight. In conclusion, the present study showed details of equine abortion and the relationships between causes of abortion and fetal size. Most of the aborted fetuses showed restrictions in their growth. The manifestations of growth restriction were more related to weight than skeletal length.

Successful endoscopic fragmentation of large hardened fecaloma using jumbo forceps.

We present a rare case of fecaloma, 7 cm in size, in the setting of systemic scleroderma. A colonoscopy revealed a giant brown fecaloma occupying the lumen of the colon and a colonic ulcer that was caused by the fecaloma. The surface of the fecaloma was hard, large and slippery, and fragmentation was not possible despite the use of various devices, including standard biopsy forceps, an injection needle, and a snare. However, jumbo forceps were able to shave the surface of the fecaloma and break it successfully by repeated biting for 6 h over 2 d. The ability of the jumbo forceps to collect large mucosal samples was also appropriate for achieving fragmentation of the giant fecaloma.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution.

Microbiological survey of mice (Mus musculus) purchased from commercial pet shops in Kanagawa and Tokyo, Japan.

Information regarding the prevalence of infectious agents in mice in pet shops in Japan is scarce. This information is particularly useful for minimizing the risk of potential transmission of infections to laboratory mice. Therefore, we surveyed infectious agents in mice from pet shops in Kanagawa and Tokyo, Japan. The survey was conducted in 28 mice from 5 pet shops to screen for 47 items (17 viruses, 22 bacteria and fungi, 10 parasites) using culture tests, serology, PCR, and microscopy. The most common viral agent detected was murine norovirus (17 mice; 60.7%), followed by Theiler's murine encephalomyelitis virus (13 mice; 46.4%), and mouse hepatitis virus (12 mice; 42.8%). The most common agent amongst the bacteria and fungi was Pasteurella pneumotropica (10 mice; 35.7%), followed by Helicobacter ganmani and Pneumocystis murina (8 mice; 28.5%, for both). Tritrichomonas muris was the most common parasite (19 mice; 67.8%), followed by Spironucleus muris (13 mice; 46.4%), Aspiculuris tetraptera, and Syphacia obvelata (8 mice each; 28.5%). Remarkably, a zoonotic agent, Hymenolepis nana, was found in 7 mice (25%). Given these results, we suggest that the workers in laboratory animal facilities should recognize again the potential risks of mice outside of the laboratory animal facilities as an infectious source, and avoid keeping mice as pets or as feed for carnivorous reptiles as much as possible for risk management.

Current microbiological status of laboratory mice and rats in experimental facilities in Japan.

On the basis of our 2011 microbiological monitoring tests, we report here the current microbiological status of mice and rats housed in experimental facilities in Japan. We tested more than 14,000 mice, 6,000 serum samples, 500 fecal or cecal samples, and 200 lung samples from 3,549 mouse facilities within Japanese universities and institutes (U/I), pharmaceutical companies and contract research organizations (P/C). We also tested more than 1,500 rats, 1,600 serum samples, and 20 fecal or cecal samples from 772 U/I and P/C rat facilities. Bacterial cultures, serology, microscopy, PCR, and DNA analysis using DNA chips were performed. Staphylococcus aureus (18.8% in mouse facilities, 58.6% in rat facilities) was the most prevalent agent in both the mouse and rat facilities. The next most prevalent agents in the mouse facilities were murine norovirus (11.97%), intestinal protozoa (0.05-8.49%, from various species), Pasteurella pneumotropica (5.32%), and Helicobacter hepaticus (3.17%), while intestinal protozoa (0.74-6.84% from various species), Syphacia muris (6.20%), Pseudomonas aeruginosa (3.61%), and Pasteurella pneumotropica (3.05%) were the subsequent most prevalent agents in the rat facilities. These results suggest that the currently prevalent microbes in laboratory mice and rats in Japan are mainly opportunistic pathogens, intestinal protozoa, and microbes with low pathogenicity.