Characterization of the cell growth analysis for detection of immortal cellular impurities in human mesenchymal stem cells.

The analysis of in vitro cell senescence/growth after serial passaging can be one of ways to show the absence of immortalized cells, which are frequently tumorigenic, in human cell-processed therapeutic products (hCTPs). However, the performance of the cell growth analysis for detection of the immortalized cellular impurities has never been evaluated. In the present study, we examined the growth rates of human mesenchymal stem cells (hMSCs, passage 5 (P = 5)) contaminated with various doses of HeLa cells, and compared with that of hMSCs alone. The growth rates of the contaminated hMSCs were comparable to that of hMSCs alone at P = 5, but significantly increased at P = 6 (0.1% and 0.01% HeLa) or P = 7 (0.001% HeLa) within 30 days. These findings suggest that the cell growth analysis is a simple and sensitive method to detect immortalized cellular impurities in hCTPs derived from human somatic cells.

[Development of guidance for the approval process of brand-new medical products and regenerative medicine products].

Ministry of Health, Labour and Weltare has been conducting development of guidance for the approval process of brand-new medical products/development of guidance for medical devices in collaboration with Ministry of Economy, Trade and Industry as part of measures to promote practical use of brand-new medical products since 2005. The objective of this project is to expedite the processes from developmental process of medical devices to approval review and to introduce the medical devices to medical front quickly.. Ministry of Health, Labour and Welfare side has been making guidance for the guide in approval process of brand-new medical products and regeneration medicine products to aim at acceleration and facilitation of development and approval process of innovative medical products. Twenty-two of the guidance have been issued as director of the evaluation and licensing division. The evaluation index about safety and efficacy required for medical devices and regenerative medicine products in progress were put together in these guidance and useful for medical devices developer to understand the point at the approved review. Therefore, I think that the evaluation index could also contribute to the efficient product development. The guidance about implantable artificial heart is issued as the representative example which was useful in the approved review.

[Polyploidy induction by spherical size standard polystyrene particles in a Chinese hamster cell line CHL].

To investigate relationships between particle (as a model of aggregates) size in a nanomaterial test suspension and its cytotoxicity, a series of eleven sizes of polystyrene (PS) particles were tested in the cytotoxicity test and the chromosome aberration test by using a Chinese hamster cell line CHL. The PS particles were spheres with defined diameters ranging from 0.1 to 9.2 µm. A series of eight sizes of particles with diameters ranging from 0.92 to 4.45 µm showed stronger cytotoxicity than the others. There was a marked difference in cytotoxicity between the 4.45- and 5.26-µm particles. The 0.92- to 4.45-µm particles did not induce structural chromosome aberrations but induced a high frequency of polyploidy in the chromosome aberration test. The 5.26-µm particles showed very weak induction of polyploidy. The incorporation of the 4.45-µm particles into CHL cells was observed by scanning electron microscopy (SEM). Some cells incorporated more than 10 particles. The semi-quantitative measurement of incorporation of particles into cells was performed by flow cytometry with a parameter of side scattered light (SSC) intensity. It showed that CHL cells preferably incorporated the 4.45-µm particles to the 5.26-µm particles. These findings suggest that CHL cells may have a kind of size-recognition ability and incorporate a particular size of particles. The particles may prevent a normal cytokinesis resulting in polyploidy induction. Nanomaterials also may show size-dependent toxicity. Data on particle (or aggregate) size distribution in the test suspension should be provided to evaluate properly the results of toxicity tests of nanomaterials.

[Projects to accelerate the practical use of innovative medical devices to collaborate with TWIns, Center for Advanced Biomedical Sciences, Waseda University and School of Engineering, The University of Tokyo].

Division of Medical Devices has been conducting the projects to accelerate the practical use of innovative medical devices to collaborate with TWIns, Center for Advanced Biomedical Sciences, Waseda University and School of Engineering, The University of Tokyo. The TWIns has been studying to aim at establishment of preclinical evaluation methods by "Engineering Based Medicine", and established Regulatory Science Institute for Medical Devices. School of Engineering, The University of Tokyo has been studying to aim at establishment of assessment methodology for innovative minimally invasive therapeutic devices, materials, and nanobio diagnostic devices. This report reviews the exchanges of personnel, the implement systems and the research progress of these projects.

Spontaneous Thrombosis of a Hepatic Pseudoaneurysm Following Blunt Liver Injury.

Hepatic pseudoaneurysm (HPA) is a rare complication of liver injury in children. Prophylactic embolization is preferable to prevent life-threatening hemorrhage due to pseudoaneurysm rupture. We present the case of a four-year-old boy who sustained a grade III liver injury from blunt abdominal trauma. He was conservatively managed since he was hemodynamically stable. Follow-up contrast-enhanced computed tomography (CECT) performed 10 days following the injury revealed an HPA measuring 4 mm × 4 mm × 3 mm. Herein, we chose conservative treatment for HPA as the patient was asymptomatic and hemodynamically stable. Conservative treatment was successful, and HPA spontaneously resolved 23 days following the injury without radiologic or surgical intervention. Although there are studies reporting asymptomatic HPAs that have spontaneously resolved, the natural history of HPAs remains unknown. Conservative treatment may be an option for asymptomatic HPA; however, to identify factors contributing to spontaneous thrombosis, further evaluation is needed.

[Determination of the relative light scattering intensity of aggregates induced by stirring of humanized monoclonal antibody product using dynamic light scattering].

To evaluate the usefulness of dynamic light scattering for estimation of the relative level of aggregates in the manufacturing process of monoclonal antibody substance and its final product, the particle sizes and relative light scattering intensities of monomer and aggregates induced by stirring of humanized monoclonal antibody product were determined by dynamic light scattering. The particle sizes of monomer and aggregates were approximately 5 and 500 nm, respectively. When aggregates and monomer were mixed at the ratio of 1 to 6, the relative light scattering intensity of aggregates was approximately 50%. These findings indicate the relative light scattering intensity of aggregates is approximately 7 times higher than that of monomer. Furthermore, these findings suggest that dynamic light scattering may be useful for the estimation of relative content of aggregates in the case that the relationship between the particle sizes of monomer and aggregates, and their relative light scattering intensities has been already examined.

Estimated Gestational Age From Infant's Foot Length in Japanese.

Aim In developed countries including Japan, gestational age (GA) is predicted by the last menstrual period (LMP) and/or fetal ultrasound. In some developing countries, GA is predicted by infant's foot length (FL). Pregnant women who did not have pregnancy check-up is not infrequent in Japan, therefore there are sometimes opportunities to estimate the GA from infants after the delivery. The aim of this study is to determine the estimated GA formula from infant's FL in Japanese. Methods This study was a prospective cohort study. Infants between May 2021 and August 2021 at Iizuka Hospital and Tagawa Hospital or transferred from other hospitals within 24 hours of birth were collected. GA was determined using LMP and/or fetal ultrasound. The infant's FL was measured with a digital caliper within 24 hours of birth. The relationship between FL and GA was analyzed by simple regression analysis to determine the coefficient of determination (R2). The infant's FL of males and females, infant's FL of preterm and term, and infant's FL of low birth weight and appropriate weight infants were performed by the t-test as independent samples. A statistically significant difference was p < 0.05. Statistical analysis was performed using JMP Pro 16 (SAS Institute Japan Co., Ltd., Minato-ku, Tokyo). Results Ninety of the 135 infants were enrolled. The average GA was 38.2 ± 1.8 weeks, the average infant's FL was 7.230 ± 0.411 centimeter (cm), and the range of the infant's FL was 5.385 to 8.089 cm. The estimated GA formula, GA = 18.49 + 0.27 x infant's FL (R2 = 0.39), was determined. Conclusions We determined the estimated GA formula from the infant's FL. There are some limitations and care should be taken in the use.

Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges.

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing ß-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 µm in diameter and elongated pores 250-450 µm in length and 100-150 µm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 µm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.

[Determination of the particle size and relative light scattering intensity of aggregates of human IgG and humanized monoclonal antibody product induced by various stress using dynamic light scattering].

To evaluate the usefulness of dynamic light scattering for the analysis of aggregates in the manufacturing process of monoclonal antibody and its final product, the particle size and relative light scattering intensity of aggregates of human IgG and humanized monoclonal antibody product induced by various stress such as stirring, increasing temperature, shaking and freeze-thaw were determined using dynamic light scattering. It was found that 1.3% of relative light scattering intensity as the minimum range and the range of particle size from 28.91nm to 3,000 nm of aggregates induced were determined by dynamic light scattering. These findings indicate that dynamic light scattering is useful for the in-process control tests of aggregates during the manufacturing and evaluation tests of aggregates for final products including the formulation and the storage. Some points well taken at the application of dynamic light scattering are discussed from the viewpoint of its merits and demerits.

Triple combinations of lower and longer alkyl gallates and oxacillin improve antibiotic synergy against methicillin-resistant Staphylococcus aureus.

Using liposome systems, we found that gallates with short alkyl chains were located in the external medium and those with longer alkyl chains were located in the surface region of lipid bilayer. Combinations of these gallates remarkably reduced oxacillin MICs against methicillin-resistant Staphylococcus aureus to below the antibiotic breakpoint (< or = 2 microg/ml).

Elucidation of the statistical factors that influence anti-drug antibody cut point setting through a multi-laboratory study.

Aim: Appropriateness of anti-drug antibody (ADA) assay is critical for immunogenicity assessment of biopharmaceuticals. Although cut point setting in ADA assay has a large impact on the results, a standard statistical approach for its setting has not been well established. Methodology: In this multi-laboratory study, to elucidate factors influencing the cut point setting, we compared the statistical approaches and calculated cut points for multiple datasets of ADA assays using the individual procedure employed at each laboratory. Conclusion: We showed that outlier exclusion, false-positive rate and investigating data distribution have the greatest impact on both screening and confirmatory cut points. Our results would be useful for industry researchers and regulators engaged in immunogenicity assessment of biopharmaceuticals.

Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration.

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.

Collaborative study using common samples to evaluate the performance of anti-drug antibody assays constructed by different companies.

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.

Annexin A3-expressing cellular phenotypes emerge from necrotic lesion in the pericentral area in 2-acetylaminofluoren/carbon tetrachloride-treated rat livers.

Recently we found a small hepatocyte-specific protein, annexin A3 (AnxA3), in fractionated adult rat hepatocytes. Here we describe the results of an in vivo demonstration of AnxA3-expressing cellular phenotypes in the liver with 2-acetylaminofluoren (2-AAF)/carbon tetrachloride (CCl(4))-injury. In association with an elevation of alanine amino transferase (ALT) and aspartic acid amino transferase (AST) activities, hepatic AnxA3 mRNA increased markedly. AnxA3-positive cells were detected in clustered cells present in or emerging from the pericentral region. These albumin-expressed cells were histologically similar to cells expressing CD34, a hematopoietic cell marker protein. The number of clusters decreased in the days following CCl(4) treatment, and annexin-negative, but albumin-positive, oval cells appeared. We concluded that the agent-induced liver defect initially recruits bone marrow-derived cells, and that it promotes differentiation of these cells into AnxA3-positive cells, followed by emergence of the oval cells, which might have a role in the restitution of the damaged liver.

Practice of Regulatory Science (Development of Medical Devices).

Prototypes of medical devices are made in accordance with the needs of clinical practice, and for systems required during the initial process of medical device development for new surgical practices. Verification of whether these prototypes produce the intended performance specifications is conducted using basic tests such as mechanical and animal tests. The prototypes are then improved and modified until satisfactory results are obtained. After a prototype passes through a clinical trial process similar to that for new drugs, application for approval is made. In the approval application process, medical devices are divided into new, improved, and generic types. Reviewers judge the validity of intended use, indications, operation procedures, and precautions, and in addition evaluate the balance between risk and benefit in terms of efficacy and safety. Other characteristics of medical devices are the need for the user to attain proficiency in usage techniques to ensure efficacy and safety, and the existence of a variety of medical devices for which assessment strategies differ, including differences in impact on the body in cases in which a physical burden to the body or failure of a medical device develops. Regulatory science of medical devices involves prediction, judgment, and evaluation of efficacy, safety, and quality, from which data result which can become indices in the development stages from design to application for approval. A reduction in the number of animals used for testing, improvement in efficiency, reduction of the necessity for clinical trials, etc. are expected through rational setting of evaluation items.

A biological study establishing the endotoxin limit of biomaterials for bone regeneration in cranial and femoral implantation of rats.

The purpose of this study was to accurately quantify the risk of endotoxin contamination in biomaterials for bone regeneration in order to establish the acceptable endotoxin limit. Collagen sheets containing varying amounts of purified endotoxin from Escherichia coli and dried, heat-killed E. coli or Staphylococcus aureus cells were implanted into cranial or femoral defects in rats. These defects were artificially prepared to a size of 5 × 5 mm or a diameter of 1 mm, respectively. The degree of osteoanagenesis was assessed by soft X-ray radiography and histopathology at 1 and 4 weeks after implantation. The collagen sheet containing the dried E. coli cells showed a dose-dependent delay in cranial and/or femoral osteoanagenesis at endotoxin activities of more than 33.6 EU/mg, at which no inflammatory response was observed. In contrast, no such observation occurred with the collagen sheet containing S. aureus cells. These results suggest that endotoxins may affect the process of osteoanagenesis. Additionally, the no-observed-adverse-effect level was 9.6 EU/mg, corresponding to 255 EU/kg body weight in rats. Interestingly, no delay in osteoanagenesis was induced by the implantation of collagen sheets containing purified endotoxin at any dose tested. This suggested that pure endotoxin implanted into tissues having poor circulation of bodily fluids without bleeding may not be recognized as a foreign substance and may not induce a significant biological response. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1514-1524, 2017.

Impact of lipid-induced degradation on the mechanical properties of ultra-high molecular weight polyethylene for joint replacements.

Gamma or electron beam irradiation of ultra-high molecular weight polyethylene (UHMWPE) used in artificial joints for sterilization and/or crosslinking purposes generates free radicals in the material, which causes long-term oxidative degradation of UHMWPE. Recently, another mechanism for the degradation of UHMWPE by the absorption of lipids during in vivo clinical use was proposed. However, knowledge on lipid-induced degradation is quite limited, compared with that on radical-induced degradation. In this study, lipid-induced degradation was simulated using squalene absorption and subsequent accelerated aging, and its impact on the mechanical properties of UHMWPE was evaluated. The simulated lipid-induced degradation caused an increased elastic modulus and decreased elongation with maximum degradation at the surfaces. These results imply that degradation of UHMWPE may occur during in vivo long-term use, even if free radicals are completely eliminated. Therefore, further investigation is required to clarify the impact of lipid-induced degradation on clinical outcomes, such as the wear and fatigue characteristics of UHMWPE components.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

Thrombomodulin enhances the invasive activity of mouse mammary tumor cells.

Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells that converts thrombin from a procoagulant to an anticoagulant. Thrombin promotes invasion by various tumor cells, and positive or negative correlations are found between the expression of TM and tumorigenesis in some patients. In this study, we used an invasion assay to investigate the effect of TM on the invasive activity of a mouse mammary tumor cell line, MMT cells, and the effects of TM were compared with those of thrombin as a positive control. In the presence of 1% fetal calf serum (FCS), TM significantly stimulated MMT cell invasion in a dose-dependent manner, resulting in an approximately 3-fold increase at 1-10 pg/ml over the untreated control. Thrombin also caused a similar degree of stimulation at 50 ng/ml. Since thrombin activity was detected in the components of the assay system, an invasion assay was also performed in a thrombin-activity-depleted assay system constructed to eliminate the effect of thrombin activity; TM (10 pg/ml) plus thrombin (1 pg/ml) stimulated invasion by approximately 3.5-fold in this assay system. Hirudin, a specific thrombin inhibitor, inhibited stimulation by TM as well as by thrombin in both the presence and absence of 1% FCS. Investigations of the effects of TM on proliferation, adhesion and chemotaxis to clarify the mechanism of stimulation by TM revealed that TM does not affect proliferation or adhesion in the presence of 1% FCS, but stimulates chemotaxis by approximately 2.3-fold. Similar results were obtained in experiments using thrombin. TM (10 pg/ml) plus thrombin (1 pg/ml), on the other hand, stimulated chemotaxis by approximately 2.3-fold in the thrombin-activity-depleted assay system. Binding studies using [125I]-thrombin revealed that the cells have specific saturable binding sites for thrombin. These results show that TM stimulates the invasive activity of MMT cells, probably by acting as a cofactor for the thrombin-stimulated invasion of the cells via its receptor and lowering the effective concentration of thrombin. The findings also indicate that the stimulation of invasive activity in the presence of 1% FCS and in the thrombin-activity-depleted assay system may mainly be mediated by the stimulation of chemotaxis.