Newly established cell lines derived from Chinese hamster for production of biologics

The Chinese hamster (Cricetulus griseus) is a species of hamster that was used as a laboratory animal more than 50 years ago. The Chinese hamster ovary (CHO) cell line was established in 1957 by Puck et al. and maintained in ex vivo conditions (1). CHO cells are now a workhorse for recombinant biopharmaceutical production. Puck et al. continuously cultivated lung, kidney, spleen and ovary-derived cells for more than 10 months using serum medium. In this study, we established a cell line from female Chinese hamster tissues: lung, kidney and ovary. Primary cells were obtained from these tissues and maintained for several months or more (Figure 1). We were ultimately able to construct three immortal cell lines, CHL-YN (fibroblast) from lung, CHK-Q (epitheliocyte) from kidney and CHO-MK (epitheliocyte) from ovary, respectively. Infinite proliferation of these cell lines is obtained by spontaneous transformation. Please click Additional Files below to see the full abstract

Newly-established Chinese hamster-derived cell line for protein production

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3-D Modeling of Tomato Canopies Using a High-Resolution Portable Scanning Lidar for Extracting Structural Information

In the present study, an attempt was made to produce a precise 3D image of a tomato canopy using a portable high-resolution scanning lidar. The tomato canopy was scanned by the lidar from three positions surrounding it. Through the scanning, the point cloud data of the canopy were obtained and they were co-registered. Then, points corresponding to leaves were extracted and converted into polygon images. From the polygon images, leaf areas were accurately estimated with a mean absolute percent error of 4.6%. Vertical profile of leaf area density (LAD) and leaf area index (LAI) could be also estimated by summing up each leaf area derived from the polygon images. Leaf inclination angle could be also estimated from the 3-D polygon image. It was shown that leaf inclination angles had different values at each part of a leaf

Intracellular secretion analysis of therapeutic antibodies in engineered high- producible CHO cells

The Chinese Hamster Ovary (CHO) cell is the most commonly used cell line for the production of therapeutic recombinant proteins. The improvements in target gene amplification and culture method have contributed in achieving a very high productivity. Some studies have focused on post-translational secretion processes, and overexpression of proteins which work in the secretion pathway successfully increased the productivity [1]. However, those studies were performed based on the knowledge obtained from the normal, adherent cultured cells, and the detailed secretion processes of recombinant proteins in engineered, suspension cultured cells is still unclear. To clarify problems and to find new targets for a more efficient establishment of high producers, the basic analyses about the secretion in engineered, high-producible CHO cells were performed. Please click Additional Files below to see the full abstract

Cre-loxP-controlled cell-cycle checkpoint engineering in Chinese Hamster ovary cells

The gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the productive cell line construction of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering1). Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system2). A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems

Use of virtual assisted lung mapping (VAL-MAP), a bronchoscopic multispot dye-marking technique using virtual images, for precise navigation of thoracoscopic sublobar lung resection

ObjectiveWe have developed a novel bronchoscopic multiple marking technique to assist resection of hardly palpable lung tumors. Because 3-dimensional virtual images were used and multiple markings made on the lung surface to provide “geometric” information, we termed this technique “virtual assisted lung mapping” (VAL-MAP). The safety and efficacy of VAL-MAP were evaluated.MethodsVirtual bronchoscopy was used to select 2 to 4 appropriate bronchial branches for marking. Bronchoscopy was conducted with the patient under local anesthesia. A metal-tip catheter was inserted into a selected bronchus and advanced to the pleura. The location of the catheter tip was fluoroscopically confirmed, and 1 mL of indigo carmine was injected. This procedure was repeated to complete all the planned markings. Post–VAL-MAP computed tomography was used to visualize the localization of the multiple markings on 3-dimensional virtual images, which were used as references in the subsequent operation.ResultsOf the 95 marking attempts made for 37 tumors in 30 patients, 88 (92.6%) were identified and contributed to the surgery. No clinically evident complications were associated with the procedure. A total of 15 wedge resections and 18 segmentectomies were thoracoscopically conducted, with a successful resection rate of 100%. Multiple markings of the VAL-MAP were complementary, enabling us to achieve complete resection even when 1 of the markings failed. The markings were visible even on interlobar fissures, at the apex, and on the diaphragm, which conventional percutaneous marking can hardly reach.ConclusionsVAL-MAP was safely conducted with satisfactory outcomes in our early experience. Additional confirmation of its safety and efficacy is necessary

Seed germination and seedling emergence of three Artemisia species (Asteraceae) inhabiting desert sand dunes in China

Abstract Artemisia ordosica, A. arenaria and A. sphaerocephala are semi-shrubs inhabiting desert sand dunes in China and often used to rehabilitate desertified areas. Improvement of dune rehabilitation success by sowing requires better understanding of the processes involved in the control of seed germination and seedling emergence in these species. Thus, (1) effects of temperature, light and osmotica (polyethylene glycol-6000) on seed germination, and (2) effects of seed burial depth in sand and irrigation regime on seedling emergence, were studied under controlled conditions. Seeds of the three species required light for germination, and the light fluence needed for germination was dependent on temperature. Seedling emergence of the three species was maximal (70-94%) for seeds sown at a depth of 2.5 mm, and decreased with increasing seed burial depth when the pots were initially and subsequently treated with 16 mm and 3 mm irrigation at 1-d intervals. However, when the pots were initially and subsequently treated with 8 mm and 3 mm irrigation at 2-d intervals, seedling emergence was almost completely suppressed due to water deficiency in sand. It is suggested that the probability of seed germination and seedling emergence of the three species in the field is very limited, because the light requirement restricts seed germination to shallow sand layers where water is lost rapidly due to evaporation. Temperature appeared to have secondary effects on seed germination, by modifying the light sensitivity of seeds