Organoid models are used to study kidney physiology, such as the assessment of nephrotoxicity and underlying disease processes. Personalized human pluripotent stem cell-derived kidney organoids are ideal models for compound toxicity studies, but there is a need to accelerate basic and translational research in the field. Here, we developed an automated continuous imaging setup with the "read-on-ski" law of control to maximize temporal resolution with minimum culture plate vibration. High-accuracy performance was achieved: organoid screening and imaging were performed at a spatial resolution of 1.1 μm for the entire multi-well plate under 3 min. We used the in-house developed multi-well spinning device and cisplatin-induced nephrotoxicity model to evaluate the toxicity in kidney organoids using this system. The acquired images were processed via machine learning-based classification and segmentation algorithms, and the toxicity in kidney organoids was determined with 95% accuracy. The results obtained by the automated "read-on-ski" imaging device, combined with label-free and non-invasive algorithms for detection, were verified using conventional biological procedures. Taking advantage of the close-to-in vivo-kidney organoid model, this new development opens the door for further application of scaled-up screening using organoids in basic research and drug discovery.
Humans ; Kidney ; Organoids ; Pluripotent Stem Cells

Erythromycin is a macrolide antibiotic produced by Saccharopolyspora erythraea. Its yield is greatly affected by the fermentation conditions and the bioreactor configurations. In this study, a novel scale-up method for erythromycin fermentation was developed based on computational fluid dynamics (CFD) and time constant analysis. Firstly, the dissolved oxygen (DO) was determined as a key parameter according to the physiological properties of S. erythraea cultivated in a 50 L bioreactor. It was found that the time constant of oxygen supply (t_mt) in a 500 m³ bioreactor should be less than 6.25 s in order to satisfy the organism's oxygen uptake rate (OUR). Subsequently, a 500 m³ bioreactor was designed using the time constant method combined with empirical correlations. The impeller combination with one BDT8 impeller at bottom and two MSX4 impellers at upper part was determined, and then validated by numerical simulation. The results indicated that the t_mt of the bioreactor (< 6.25 s) and the fluid properties, including gas hold-up, shear stress and fluid vector, met the requirements of erythromycin fermentation. Finally, the industrial production of erythromycin in the 500 m³ showed the design method was applicable in large scale fermentation.
Erythromycin ; Saccharopolyspora/genetics* ; Bioreactors ; Fermentation ; Anti-Bacterial Agents

Objective:To explore the application of scenario simulation and rapid film reading training in the standardization of cervical vertebra DR examination in radiology department.Methods:Using the method of scene simulation in the examination room to take DR films of cervical vertebra, two senior technicians in charge evaluated and guided the deficiencies of junior technicians from the aspects of patient check, exposure conditions, body position placement, film printing, inspection safety and radiation protection. Every morning, DR images of cervical vertebra were randomly selected and read quickly, and the junior technicians should quickly browse the images within one minute and make quality control evaluation. Then the senior technician in charge pointed out the shortcomings of the evaluation and related improvement methods. After that, the technical specification manual of cervical vertebra DR was compiled based on the deficiencies and problems found by each technician, and 200 patients aged 18-55 years, without abnormal movement and no internal fixation before and after the establishment of standardization were randomly selected in group A (before standardization) and group B (after standardization). The image quality and repetition projection rate of group A and group B before and after the standardization were compared. SPSS 25.0 was used to analyze the data.Results:The image quality score of group B (8.83±0.33) was higher than that of group A (8.45±0.30), and the repetition projection rate of group B (2%) was lower than that of group A (9%), with statistical significance ( P<0.05). Conclusion:Through the training mode of rapid film reading and scenario simulation, the standardization of cervical vertebra DR examination technology suitable for the situation of the department has been established, which effectively solves the problem of abstraction in the technical teaching of radiology department. The standardization establishment plays an important role in standardized training, for which, the technicians can quickly grasp the work process of the department and the corresponding examination technology during participating in the standardization establishment and training process.

Objective:To observe the ultrasonographic features of cervical lymph node metastasis and analyze their relationship with the expression of cell proliferation-associated nuclear antigen (Ki-67).Methods:The clinical data of 92 patients with cervical lymph node metastasis who received treatment in Zhejiang Quhua Hospital, China between June 2017 and July 2019 were retrospectively analyzed. The correlation between ultrasonographic features of cervical lymph node metastasis and Ki-67 expression was analyzed.Results:Among the 92 patients, 158 metastatic lymph nodes were confirmed by pathological examination. The main ultrasonic imaging features were the length ratio of long diameter to short diameter < 2 in 119 (75.32%) lymph nodes, disappearance of portal hyperechoic signal in 127 (80.38%) lymph nodes, punctate hyperechoic signal in 108 (68.35%) lymph nodes, cystic degeneration in 57 (36.08%) lymph nodes, mixed types of peripheral blood flow signal in 124 (78.48%) lymph nodes, microcalcification in 123 (77.85%) lymph nodes. The length ratio of long diameter to short diameter < 2, punctate hyperechoic signal and mixed types of peripheral blood flow signal were correlated with high expression of Ki-67 ( χ2 = 24.252, 15.732, 17.033, all P < 0.05). Conclusion:High expression of Ki-67 is correlated with the length ratio of long diameter to short diameter < 2, punctate hyperechoic signal and mixed types of peripheral blood flow signal in cervical lymph node metastasis.

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.
Animals ; Batch Cell Culture Techniques ; Bioreactors ; standards ; CHO Cells ; Cell Count ; Computer Simulation ; Cricetinae ; Cricetulus ; Industrial Microbiology ; instrumentation ; methods

A rapid quantitative evaluation method for Siraitia grosvenorii cells was successfully developed based on plant cells' capacitance value detected by a viable cell mass monitor and the cryopreservation of S. grosvenorii suspension cells was optimized. The survival rate of S. grosvenorii cells was quantitatively measured by viable cell mass monitor and 2, 3, 5-triphenyltetrazolium chloride (TTC). An optimum cryoprotectant recipe is that the growth medium contained 10% sucrose and 10% DMSO. The experimental results also showed higher cell survival rates and cell viabilities were achieved when suspension cells were treated with pretreatment of 0.2 mol/L sucrose. With the increase of concentration of sucrose, however, the cell survival rate was decreased. And the cell survival rate represented a bell shape with the increase of pretreatment time. The highest cell survival rate and cell viability were obtained with the 9 h' s pretreatment. In addition, there was a good correlation between the cell survival rate measured by cell recovery test and that measured by viable cell mass monitor, while there were no significant differences in the cell morphology and the ability of mogrosides V production by S. grosvenorii cells cultured in suspension after cryopreservation. Therefore, the evaluation method developed based on the viable cell mass monitor has good feasibility and reliability.

To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
Agrobacterium tumefaciens ; Ascomycota ; genetics ; metabolism ; DNA, Bacterial ; Malates ; metabolism ; Polymerase Chain Reaction ; Polymers ; metabolism ; Transformation, Genetic

The aim of this study is to develop a synthetic medium suitable for 13C metabolic flux analysis (13C-MFA) of Streptomyces rimosus. The cell growth rate and oxytetracycline production by S. rimosus M4018 were compared when M4018 cells were growth on the optimized chemically defined media with organic nitrogen sources or inorganic nitrogen sources. First, a synthetic medium contained KNO3 as the main nitrogen source was screened, then optimized by a response surface method. Using this new medium, the oxytetracycline yield was increased from 75.2 to 145.6 mg/L. Furthermore, based on the 13C-MFA, we identified that Entner-Doudoroff pathway does not exist in S. rimosus cells cultured in a chemically defined medium with feed of 100% 1-13C labeled glucose. This study is helpful for subsequent 13C-MFA application of S. rimosus.
Carbon Isotopes ; analysis ; Culture Media ; chemistry ; Metabolic Flux Analysis ; Nitrogen ; chemistry ; Oxytetracycline ; biosynthesis ; Streptomyces rimosus ; metabolism

Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat's testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated in vitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine, amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2-4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressure above 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors for SCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.
Amino Acids ; metabolism ; Animals ; Animals, Newborn ; Cell Culture Techniques ; Cells, Cultured ; Culture Media ; Male ; Rats ; Sertoli Cells ; cytology ; metabolism ; Testis ; cytology

Objective To study cancer metastasis in mesorectum and ischiorectal loss in cases of ultra-low rectal cancer and evaluate the rationale of Miles procedure. Methods Whole-mount slice and tissue mieroarray technique were used to study the dissected specimen from 23 cases of uhra-low rectal cancer for metastatic lymph nodes. Result 415 lymph nodes were harvested in 23 mesorectum specimen, 169 and 59 lymph nodes were metastasic and micrometastasie respectively. 12 eases were diagnosed with metastasis, 4 cases were found to have micrometastasis. Metastatic lymph nodes in the lateral and anterior mesorectum were 29.0% (49/169) and 17.2% (29/169) respectively. There were 2 patients with metastasis and 1 with micrometsstssis in ischiorectal fossa lymph nodes, accounting for 13% patients. Conclusion Regional metastasis exists in ultra-low rectal cancer and its incidence varies in different location of mesorectum and ischiorectal fessa. The value of Miles procedure as the standard therapy for ultra-low rectal cancer should undergo an evaluation.