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Objectives: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line., Results: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR1 2xHis ) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested., Conclusions: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 10 6 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50-mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high-throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three-dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k L a) was determined experimentally and from simulations under various working conditions. We also determined the liquid-phase mass transfer coefficient (k L ) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k L . High oxygen transfer rates, sufficient for supporting the high-density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192-200, 2017., (© 2016 American Institute of Chemical Engineers.)

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280nm (A(280)) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A(280) was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate-DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection.

For large-scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior system for suspension cultures of mammalian cells based on orbital shaker technology. We found that "square-shaped" bottles (square bottles) provide an inexpensive but efficient means to grow HEK-293 EBNA and CHO-DG44 cells to high density. Cultures in agitated 1-L square bottles exceeded the performance of cultures in spinner flasks, reaching densities up to 7 x 10(6) cells/mL for HEK-293 EBNA cells and 5 x 10(6) cells/mL for CHO-DG44 cells in comparison to (2.5-4) x 10(6) cells/mL for cultures of the same cells grown in spinner flasks. For 1-L square bottles, optimal cell growth and viability were observed with a filling volume of 30-40% of the nominal volume and an agitation speed of 130 rpm at a rotational diameter of 2.5 cm. Transient reporter gene expression following gene delivery by calcium phosphate-DNA co-precipitation was the same or slightly better for HEK-293 EBNA cells grown in square bottles as compared to spinner flasks. Reductions in cost, simplified handling, and better performance in cell growth and viability make the agitated square bottle a new and very promising tool for the cultivation of mammalian cells in suspension.

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.