Prediction of antibody production performance change in Chinese hamster ovary cells using morphological profiling.

Monoclonal antibodies (mAbs) represent a significant segment of biopharmaceuticals, with the market for mAb therapeutics expected to reach $200 billion in 2021. Chinese Hamster Ovary (CHO) cells are the industry standard for large-scale mAb production owing to their adaptability and genetic engineering capabilities. However, maintaining consistent product quality is challenging, primarily because of the inherent genetic instability of CHO cells. In this study, we address the need for advanced technologies for quality monitoring of host cells in biopharmaceuticals. We highlight the limitations of traditional cell assessment techniques such as flow cytometry and propose a noninvasive, label-free image-based analysis method. By utilizing advanced image processing and machine learning, this technique aims to non-invasively and quantitatively evaluate subtle quality changes in suspension cells. The research aims to investigate the use of morphological analysis for identifying subtle alterations in mAb productivity of CHO cells, employing cells stimulated by compounds as a model for this study. Our results show that the mAb productivity of CHO cells (day 8) can be predicted only from their early morphological profile (day 3). Our study also discusses the importance of strategic methods for forecasting host cell mAb productivity using morphological profiles, as inferred from our machine learning models specialized in predictive score prediction and anomaly prediction.

The structurally diverse phytocannabinoids cannabichromene, cannabigerol and cannabinol significantly inhibit amyloid ß-evoked neurotoxicity and changes in cell morphology in PC12 cells.

BACKGROUND: Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aß). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aß aggregation and protect against Aß-evoked neurotoxicity in PC12 cells. METHODS: Neuroprotection against lipid peroxidation and Aß-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aß aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. RESULTS: CBD inhibited lipid peroxidation with no significant effect on Aß toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aß1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aß aggregation, whilst Δ9 -THC reduced aggregate density. Aß1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aß1-42 effects on cell morphology. CONCLUSIONS: These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aß-evoked neurite damage and inhibition of amyloid ß aggregation.

Lifestyle parameters of Japanese agricultural and non-agricultural workers aged 60 years or older and less than 60 years: A cross-sectional observational study.

OBJECTIVES: Improving the lifestyle of occupational workers is essential for extending healthy life expectancy. We investigated various lifestyle-related items in a rural Japanese population and compared them between agricultural and non-agricultural workers. METHODS: This cross-sectional study was conducted as a part of the "Iwaki Health Promotion Project." Lifestyle-related items such as sleep, work hours, nutrition, health-related quality of life, and proportion of time spent performing each daily activity were compared between agricultural and non-agricultural workers in the ≥60 years (n = 251) and <60 years (n = 560) age groups. RESULTS: Agricultural workers had significantly lower Pittsburgh Sleep Quality Index total scores than non-agricultural workers in the <60 years group. The proportion of participants with more than 5 weekly working days was high among agricultural workers in both groups. Additionally, the proportion of people who worked more than 8 h per day was high among agricultural workers in both age groups. Energy intake per day was high among agricultural workers in the <60 years group. In both age groups, agricultural workers slept and woke up approximately 40 min earlier than did non-agricultural workers. CONCLUSIONS: Agricultural workers have better sleep habits but work longer than non-agricultural workers, with some differences in energy intake and proportion of time spent on each daily activity. These differences should be considered when planning lifestyle intervention programs for agricultural workers.

Functional analysis of two abnormal antithrombin proteins with different intracellular kinetics.

INTRODUCTION: Hereditary antithrombin (AT) deficiency type I causes venous thrombosis due to decreased levels of AT antigen in the blood. We identified one novel and one known abnormal variant in two unrelated Japanese families with venous thrombosis. In this study, we analyzed the mechanism by which these abnormal variants cause type I AT deficiency. MATERIALS AND METHODS: Wild-type and variant AT expression vectors were constructed and transiently expressed in human embryonic kidney 293 cells, and AT antigen levels and N-glycosylation of cell lysates and culture medium were evaluated by western blot analysis. Subcellular co-localization of AT was also examined using confocal microscopy, and chase experiments with cycloheximide and MG132 were performed to investigate the degradation pathway of AT variants. RESULTS: Genetic analysis identified a novel variant, c.613delC (p.Leu205Trpfs⁎79), and the known variant c.283T>C (p.Tyr95His). These AT variants exhibited significantly reduced extracellular secretion compared with the wild-type; N-glycosylation of the AT protein was normal. Co-localization analysis suggested that the transport of these abnormal AT proteins to the Golgi apparatus was impaired. The c.613delC variant was degraded early by the proteasome, suggesting that the c.283T>C variant is stored in the endoplasmic reticulum (ER). CONCLUSIONS: The AT variants identified here synthesize abnormal AT proteins that exhibit suppressed secretion and impaired transport from the ER to the Golgi apparatus. These results provide clues that could help elucidate the mechanism of type I AT deficiency and facilitate therapy development.

Comparison of the OPTIVUS-Complex PCI Multivessel Cohort With the Historical CREDO-Kyoto Registry Cohort-3.

BACKGROUND: There is a paucity of data on the effect of optimal intravascular ultrasound (IVUS)-guided percutaneous coronary intervention (PCI) compared with standard PCI or coronary artery bypass grafting (CABG) in patients with multivessel disease.Methods and Results: The OPTIVUS-Complex PCI study multivessel cohort was a prospective multicenter single-arm study enrolling 1,021 patients undergoing multivessel PCI including the left anterior descending coronary artery using IVUS aiming to meet the prespecified criteria for optimal stent expansion. We conducted propensity score matching analyses between the OPTIVUS group and historical PCI or CABG control groups from the CREDO-Kyoto registry cohort-3 (1,565 and 899 patients) fulfilling the inclusion criteria for this study. The primary endpoint was a composite of death, myocardial infarction, stroke, or any coronary revascularization. In the propensity score-matched cohort (OPTIVUS vs. historical PCI control: 926 patients in each group; OPTIVUS vs. historical CABG control: 436 patients in each group), the cumulative 1-year incidence of the primary endpoint was significantly lower in the OPTIVUS group than in the historical PCI control group (10.4% vs. 23.3%; log-rank P<0.001) or the historical CABG control group (11.8% vs. 16.5%; log-rank P=0.02). CONCLUSIONS: IVUS-guided PCI targeting the OPTIVUS criteria combined with contemporary clinical practice was associated with superior clinical outcomes at 1 year compared with not only the historical PCI control, but also the historical CABG control.

Identification and functional analysis of three novel genetic variants resulting in premature termination codons in three unrelated patients with hereditary antithrombin deficiency.

Hereditary antithrombin (AT) deficiency is an autosomal dominant inherited thrombophilia. In three pedigrees of hereditary type I AT deficiency, we identified novel variants c.126delC (p.Lys43Serfs*7), c.165C > G (p.Tyr55*), and c.546delA (p.Lys182Asnfs*102) in the open reading frame encoding AT in each patient. Each of these aberrant variants leads to premature termination of AT protein synthesis. To investigate whether these abnormal variants are involved in the pathogenesis of type I AT deficiency, we analyzed the function of these variants in HEK293 cells. Results of western blot analysis and immunofluorescence microscopy showed that all abnormal variants were expressed intracellularly, but p.Lys43Serfs*7 and p.Tyr55* protein were aggregated in the cells. These three variants were not detected in the spent culture medium, indicating that these novel variants affect protein secretion. In summary, we suggest that these variants in the AT-encoding gene are translated in the cell, but form abnormal proteins that form aggregates and/or inhibit secretion. These results provide insight into novel mechanisms of type I AT deficiency and potential therapies for the condition.

Label-free morphological sub-population cytometry for sensitive phenotypic screening of heterogenous neural disease model cells.

Label-free image analysis has several advantages with respect to the development of drug screening platforms. However, the evaluation of drug-responsive cells based exclusively on morphological information is challenging, especially in cases of morphologically heterogeneous cells or a small subset of drug-responsive cells. We developed a novel label-free cell sub-population analysis method called "in silico FOCUS (in silico analysis of featured-objects concentrated by anomaly discrimination from unit space)" to enable robust phenotypic screening of morphologically heterogeneous spinal and bulbar muscular atrophy (SBMA) model cells. This method with the anomaly discrimination concept can sensitively evaluate drug-responsive cells as morphologically anomalous cells through in silico cytometric analysis. As this algorithm requires only morphological information of control cells for training, no labeling or drug administration experiments are needed. The responses of SBMA model cells to dihydrotestosterone revealed that in silico FOCUS can identify the characteristics of a small sub-population with drug-responsive phenotypes to facilitate robust drug response profiling. The phenotype classification model confirmed with high accuracy the SBMA-rescuing effect of pioglitazone using morphological information alone. In silico FOCUS enables the evaluation of delicate quality transitions in cells that are difficult to profile experimentally, including primary cells or cells with no known markers.

Quantitative evaluation of upper limb ataxia in spinocerebellar ataxias.

OBJECTIVE: To quantitatively evaluate upper limb ataxia using a novel pen-like sensor device in patients with spinocerebellar ataxia (SCA) and to assess its validity, reliability, and sensitivity to disease progression. METHODS: We designed a cross-sectional and longitudinal study of patients with SCA and healthy controls. Upper limb ataxia was evaluated using a device that measures the three-dimensional position every 10 msec. Participants were instructed to move a pen-like part of the device iteratively between two buttons. We evaluated the time, length, velocity, and variation coefficient of the stroke, and calculated the distortion index using the mean squared error. The following scales were also evaluated: Scale for the Assessment and Rating of Ataxia (SARA), the International Cooperative Ataxia Rating Scale (ICARS), and the nine-hole pegboard test. Subjects were followed 12 months after the baseline evaluation. RESULTS: A total of 42 patients with SCA and 33 healthy controls were enrolled and evaluated. For all ataxia indices measured using the device there were significant differences between healthy controls and patients with SCA. Among the ataxia indices, the distortion index showed the strongest correlation with the SARA and ICARS upper limb score (Pearson's r = 0.647 and 0.722, respectively). Test-retest reliability was high for most of the ataxia indices. In the longitudinal analysis, the distortion index showed high standardized response mean and adjusted effect size, regardless of disease severity. INTERPRETATION: Our study demonstrated that the distortion index is a reliable functional marker that is sensitive to longitudinal change in patients with SCA.

Late onset of coronary ostial stenosis following surgical aortic valve replacement in a patient with anomalous origin of the right coronary artery.

Surgical aortic valve replacement (SAVR) in patients with anomalous origination of a coronary artery from the opposite sinus is associated with risk for myocardial ischemia during the perioperative period. [1] However, iatrogenic coronary ostial stenosis (ICOS) generally occurs within the first 6 months after SAVR. We present an unusual case of a 74-year-old man with anomalous origination of the right coronary artery from the left coronary sinus, who developed effort angina due to ICOS 19 months following SAVR and ascending aorta replacement. Angiography and computed tomography were utilized to perform a comparison before and after the procedure. From the results, it was evident that the flattened mild stenosis preoperatively was caused by anomalous origination of a coronary artery from the opposite sinus and progressed to severe stenosis by ICOS after the procedure. The patient was successfully treated with percutaneous coronary intervention. .

Morphological heterogeneity description enabled early and parallel non-invasive prediction of T-cell proliferation inhibitory potency and growth rate for facilitating donor selection of human mesenchymal stem cells.

BACKGROUND: Within the extensively developed therapeutic application of mesenchymal stem cells (MSCs), allogenic immunomodulatory therapy is among the promising categories. Although donor selection is a critical early process that can maximize the production yield, determining the promising candidate is challenging owing to the lack of effective biomarkers and variations of cell sources. In this study, we developed the morphology-based non-invasive prediction models for two quality attributes, the T-cell proliferation inhibitory potency and growth rate. METHODS: Eleven lots of mixing bone marrow-derived and adipose-derived MSCs were analyzed. Their morphological profiles and growth rates were quantified by image processing by acquiring 6 h interval time-course phase-contrast microscopic image acquisition. T-cell proliferation inhibitory potency was measured by employing flow cytometry for counting the proliferation rate of peripheral blood mononuclear cells (PBMCs) co-cultured with MSCs. Subsequently, the morphological profile comprising 32 parameters describing the time-course transition of cell population distribution was used for explanatory parameters to construct T-cell proliferation inhibitory potency classification and growth rate prediction models. For constructing prediction models, the effect of machine learning methods, parameter types, and time-course window size of morphological profiles were examined to identify those providing the best performance. RESULTS: Unsupervised morphology-based visualization enabled the identification of anomaly lots lacking T-cell proliferation inhibitory potencies. The best performing machine learning models exhibited high performances of predictions (accuracy > 0.95 for classifying risky lots, and RMSE < 1.50 for predicting growth rate) using only the first 4 days of morphological profiles. A comparison of morphological parameter types showed that the accumulated time-course information of morphological heterogeneity in cell populations is important for predicting the potencies. CONCLUSIONS: To enable more consistent cell manufacturing of allogenic MSC-based therapeutic products, this study indicated that early non-invasive morphology-based prediction can facilitate the lot selection process for effective cell bank establishment. It was also found that morphological heterogeneity description is important for such potency prediction. Furthermore, performances of the morphology-based prediction models trained with data consisting of origin-different MSCs demonstrated the effectiveness of sharing morphological data between different types of MSCs, thereby complementing the data limitation issue in the morphology-based quality prediction concept.

Predicting quality decay in continuously passaged mesenchymal stem cells by detecting morphological anomalies.

With rapid advances in cell therapy, technologies enabling both consistency and efficiency in cell manufacturing are becoming necessary. Morphological monitoring allows practical quality maintenance in cell manufacturing facilities, but relies heavily on human skill. For more reproducible and data-driven quality evaluation, image-based morphological analysis provides multiple advantages over manual observation. Our group has investigated the performance of multiple morphological parameters obtained from time-course images to non-invasively and quantitatively predict cellular quality using machine learning algorithms. Although such morphology-based computational models succeeded in early cell quality predictions, it was difficult to introduce our approach in cell manufacturing facilities owing to data variation issues. Since manufacturing facilities have fixed their protocol to minimize anomalies as much as possible, most accumulated data are normal, and anomalies are scarce. Thus, our morphological analysis had to adapt to such practical situation where it was difficult to observe a wide range of data variations, including both normal samples and anomalies, which is typically essential to improve most machine learning models' performance. In the present study, we introduce a practical morphological analysis concept by investigating the performance of anomalous quality decay discrimination during the continuous passaging of human mesenchymal stem cells (hMSCs). Combining the visualization method and asymmetric statistic discrimination, we describe an effective morphology-based, in-process quality monitoring concept to detect quality anomalies throughout cell culture process. Our results showed that the use of morphological parameters to reflect cellular population heterogeneity can predict hMSC quality decay within 6 h after seeding.

The importance of scoring recognition fitness in spheroid morphological analysis for robust label-free quality evaluation.

Because of the growing demand for human cell spheroids as functional cellular components for both drug development and regenerative therapy, the technology to non-invasively evaluate their quality has emerged. Image-based morphology analysis of spheroids enables high-throughput screening of their quality. However, since spheroids are three-dimensional, their images can have poor contrast in their surface area, and therefore the total spheroid recognition by image processing is greatly dependent on human who design the filter-set to fit for their own definition of spheroid outline. As a result, the reproducibility of morphology measurement is critically affected by the performance of filter-set, and its fluctuation can disrupt the subsequent morphology-based analysis. Although the unexpected failure derived from the inconsistency of image processing result is a critical issue for analyzing large image data for quality screening, it has been tackled rarely. To achieve robust analysis performances using morphological features, we investigated the influence of filter-set's reproducibility for various types of spheroid data. We propose a new scoring index, the "recognition fitness deviation (RFD)," as a measure to quantitatively and comprehensively evaluate how reproductively a designed filter-set can work with data variations, such as the variations in replicate samples, in time-course samples, and in different types of cells (a total of six normal or cancer cell types). Our result shows that RFD scoring from 5000 images can automatically rank the best robust filter-set for obtaining the best 6-cell type classification model (94% accuracy). Moreover, the RFD score reflected the differences between the worst and the best classification models for morphologically similar spheroids, 60% and 89% accuracy respectively. In addition to RFD scoring, we found that using the time-course of morphological features can augment the fluctuations in spheroid recognitions leading to robust morphological analysis.

Genetic analysis of a compound heterozygous patient with congenital factor X deficiency and regular replacement therapy with a prothrombin complex concentrate.

Congenital factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million. The proband, a 2-year-old girl, exhibited easy bruising and a history of umbilical cord bleeding at birth. Prothrombin time (> 40 s) and activated partial thromboplastin time (65.0 s) were prolonged. Marked declines in FX activity (< 1%) and FX antigen levels (5%) were also observed. Genetic analysis of the proband identified two types of single-base substitutions, c.353G>A (p.Gly118Asp) and c.1303G>A (p.Gly435Ser), indicating compound heterozygous congenital FX deficiency. Genetic analysis of family members revealed that her father and older sister (5-year-old) were also heterozygous for p.Gly118Asp, and that her mother was heterozygous for p.Gly435Ser. To improve the bleeding tendency, the proband received regular replacement of 500 units of PPSB-HT, a prothrombin complex concentrate (PCC). Following continued regular replacement of 500 units of PPSB-HT once per week, the proband has exhibited no bleeding tendencies and no new bruises have been observed. There are no previous report of the use of PPSB-HT for regular FX replacement. Regular replacement therapy with PPSB-HT may be an effective method for preventative control of bleeding tendencies in FX deficiency.

Effect of mechanical vibration stress in cell culture on human induced pluripotent stem cells.

The development of induced pluripotent stem cell (iPSC) techniques has solved various limitations in cell culture including cellular proliferation and potency. Hence, the expectations on wider applications and the quality of manufactured iPSCs are rapidly increasing. To answer such growing expectations, enhancement of technologies to improve cell-manufacturing efficiency is now a challenge for the bioengineering field. Mechanization of conventional manual operations, aimed at automation of cell manufacturing, is quickly advancing. However, as more processes are being automated in cell manufacturing, it is need to be more critical about influential parameters that may not be as important in manual operations. As a model of such parameters, we focused on the effect of mechanical vibration, which transmits through the vessel to the cultured iPSCs. We designed 7 types of vertical vibration conditions in cell culture vessels using a vibration calibrator. These conditions cover a wide range of potential situations in cell culture, such as tapping or closing an incubator door, and examined their effects by continuous passaging (P3 to P5). Detailed evaluation of cells by time-course image analysis revealed that vibrations can enhance cell growth as an early effect but can negatively affect cell adhesion and growth profile after several passages as a delayed effect. Such unexpected reductions in cell quality are potentially critical issues in maintaining consistency in cell manufacturing. Therefore, this work reveals the importance of continuous examination across several passages with detailed, temporal, quantitative measurements obtained by non-invasive image analysis to examine when and how the unknown parameters will affect the cell culture processes.

In-process evaluation of culture errors using morphology-based image analysis.

INTRODUCTION: Advancing industrial-scale manufacture of cells as therapeutic products is an example of the wide applications of regenerative medicine. However, one bottleneck in establishing stable and efficient cell manufacture is quality control. Owing to the lack of effective in-process measurement technology, analyzing the time-consuming and complex cell culture process that essentially determines cellular quality is difficult and only performed by manual microscopic observation. Our group has been applying advanced image-processing and machine-learning modeling techniques to construct prediction models that support quality evaluations during cell culture. In this study, as a model of errors during the cell culture process, intentional errors were compared to the standard culture and analyzed based only on the time-course morphological information of the cells. METHODS: Twenty-one lots of human mesenchymal stem cells (MSCs), including both bone-marrow-derived MSCs and adipose-derived MSCs, were cultured under 5 conditions (one standard and 4 types of intentional errors, such as clear failure of handlings and machinery malfunctions). Using time-course microscopic images, cell morphological profiles were quantitatively measured and utilized for visualization and prediction modeling. For visualization, modified principal component analysis (PCA) was used. For prediction modeling, linear regression analysis and the MT method were applied. RESULTS: By modified PCA visualization, the differences in cellular lots and culture conditions were illustrated as traits on a morphological transition line plot and found to be effective descriptors for discriminating the deviated samples in a real-time manner. In prediction modeling, both the cell growth rate and error condition discrimination showed high accuracy (>80%), which required only 2 days of culture. Moreover, we demonstrated the applicability of different concepts of machine learning using the MT method, which is effective for manufacture processes that mostly collect standard data but not a large amount of failure data. CONCLUSIONS: Morphological information that can be quantitatively acquired during cell culture has great potential as an in-process measurement tool for quality control in cell manufacturing processes.