CHO cells

FIG. 2. Typical set of 23 chromosomes from cell of the R2 clone of Chinese hamster ovary. The original culture had a stemline of 22 chromosomes with a small proportion 21- and 23-chromosome karyotypes. Orcein stain. X 1800.

The Tissue-Type Plasminogen Activator Story Figure 1. Schematic visualization of the molecular interactions regulating physiological fibrinolysis. HISTORY OF DISCOVERY The Tissue-Type Plasminogen Activator Story D. Collen, H.R. Lijnen Figure 1. Schematic visualization of the molecular interactions regulating physiological fibrinolysis. Plasminogen is converted to the proteolytic enzyme plasmin by tissue-type plasminogen activator, but this conversion occurs efficiently only on the fibrin surface, where activator and plasminogen are “assembled.” Free plasmin in the blood is very rapidly inactivated by α2-antiplasmin, but plasmin generated at the fibrin surface is partially protected from inactivation. The lysine-binding sites in plasminogen (represented as the “legs” of the animal) are important for the interaction between plasmin (ogen) and fibrin and between plasmin and α2-antiplasmin. At the Fifth Congress on Fibrinolysis in Malmo, Sweden (1980), where our first results with t-PA were presented, Dr D. Pennica from the Department of Molecular Biology at Genentech Inc. approached us. Collaborations devoted to the cloning and expression of the t-PA gene ensued, with results reported at the Sixth Congress on Fibrinolysis in Lausanne, Switzerland (1982), and published in Nature in January 1983. Nowadays the cloning and expression of t-PA would be a relatively trivial accomplishment, but in 1982 this was quite an achievement for which D. Pennica deserves most of the credit. The cDNA of human t-PA (2530 bp) has first been expressed in E coli. More efficient expression was subsequently obtained in mammalian cells, yielding a properly processed and glycosylated molecule. This recombinant t-PA (rt-PA) was shown to be indistinguishable from the natural activator isolated from human melanoma cell cultures, with respect to biochemical properties, turnover in vivo, and specific thrombolytic activity. The generation of CHO (Chinese Hamster ovary) cells capable of producing single-chain human t-PA has allowed the development of large-scale tissue culture fermentation and purification procedures, yielding rt-PA (alteplase) for commercial purposes (Activase, Genentech Inc.; Actilyse, Boehringer Ingelheim). Figure 1. Schematic visualization of the molecular interactions regulating physiological fibrinolysis. Plasminogen is converted to the proteolytic enzyme plasmin by tissue-type plasminogen activator, but this conversion occurs efficiently only on the fibrin surface, where activator and plasminogen are “assembled.” Free plasmin in the blood is very rapidly inactivated by α2-antiplasmin, but plasmin generated at the fibrin surface is partially protected from inactivation. The lysine-binding sites in plasminogen (represented as the “legs” of the animal) are important for the interaction between plasmin (ogen) and fibrin and between plasmin and α2-antiplasmin.10 Copyright © American Heart Association, Inc. All rights reserved.

Over the 55 year history of CHO cell study, several different lines with different attributes have been derived from the original cell line. A common CHO derivative is CHO-K1, which has, to some extent, less DNA than original CHO cells. A later mutation of CHO-K1 generated CHO-DXB11 (also known as CHO-DUKX), which lacks dihydrofolate reductase (DHFR) activity. Another offshoot of the original CHO cell line was CHO-pro3, which is proline-dependent. CHO-pro3 was then later mutated to create CHO-DG44, which also is DHFR-deficient.

Corning Proculture Spinner Flask Transferring CHO Cells From Monolayer to Suspension Culture Decant medium… Culture should be 50 to 80% confluent to ensure that the cells are actively dividing..add trypsin-EDTA… when cells have rounded up, rap flask on bench top to dislodge the cells... resuspend the cells in 5 ml of medium. Remove an aliquot to determine viable cell count… Determine volume of cell suspension needed for 5 x 105 viable cells/ml. Add an appropriate volume of medium and cell suspension to a sterile spinner or shake flask. Leave sufficient headspace for adequate gas exchange (i.e., 100 ml of medium in 250-ml spinner flask or 75 to 100 ml of medium in a 250-ml shake flask). For serum-free medium that does not already contain a shear protectant, add PLURONIC® F68 to a final concentration of 0.1%. Loosen caps for gas exchange and place at 37oC in a humidified atmosphere of 5 to 10% CO2 in air. Set impeller speed to 75-95 rpm (for Corning Spinners; for paddle-type impellers, speed may need to be decreased). Shake flasks can be rotated on an orbital shaker platform at 125 to 135 rpm. Note:   You can set spinner flask impeller speed to your normal speed (80 to 100 rpm). If the cell viability drops below 85%, reduce the speed in 10 rpm decrements until the viability is good. Check viable cell density daily to establish growth kinetics in suspension culture. When viable cell density reaches 1 x 106 cells/ml (or on day 4 post-planting). Passage cells at 5 x 105 cells/ml centrifugation followed by fresh medium may be necessary if the cell density has not reached 1 x 106 cells/ml by day 4. Once cell density reaches at least 1 x 106 cells/ml with a viability of at least 90% by day 3 post-planting for 3 passages, the cells may be considered adapted to suspension culture. Seeding density can then be reduced to 2 x 105 to 3 x 105cells/ml. Corning Proculture Spinner Flask