High Density Mammalian Cell Culture Research Articles

Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes

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 (kL a) was determined experimentally and from simulations under various working conditions. We also determined the liquid-phase mass transfer coefficient (kL ) 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 kL . 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.

High-density mammalian cell cultures in stirred-tank bioreactor without external pH control

Maintaining desired pH is a necessity for optimal cell growth and protein production. It is typically achieved through a two-sided pH control loop on the bioreactor controller. Here we investigated cell culture processes with minimum or no pH control and demonstrated that high-density mammalian cell cultures could be maintained for long-term protein production without pH control. The intrinsic interactions between pCO2, lactate, and pH were leveraged to maintain culture pH. Fed-batch cultures at the same lower pH limit of 6.75 but different upper pH limits (7.05, 7.30, 7.45, 7.65) were evaluated in the 3L bioreactors and comparable results were obtained. Neither CO2 sparging nor base addition was required to control pH in the pH range of 6.75–7.65. The impact of sparger configurations (drilled hole sparger vs. frit sparger) and scales (3L vs. 200L) on CO2 accumulation and culture pH was also demonstrated. The same principle was applied in two perfusion cultures with steady state cell densities at 42.5±3.3 or 68.3±6.0×106cells/mL with low cell specific perfusion rates (15±2 to 23±3pL/cell/day), achieving up to 1.9±0.1g/L/day bioreactor productivity. Culture pH level in the 3L perfusion bioreactors was steadily maintained by controlling the residual lactate and pCO2 levels without the requirement of external pH control for up to 40days with consistent productivity and product quality. Furthermore, culture pH could be potentially modulated via adjusting residual glucose levels and CO2 stripping capability in perfusion cultures. To the best of our knowledge, this is the first time a systematic study was performed to evaluate the long-term cell cultivation and protein production in stirred-tank bioreactors without external pH control.

Recombinant human nerve growth factor with a marked activityin vitroandin vivo

Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possess in vivo biochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of approximately 21.5 +/- 2.9 mg/liter recombinant protein. The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphological in vitro studies. This work describes the production on a laboratory scale of high levels of a rhNGF with in vitro and, more important, in vivo biological activity equivalent to the native murine protein.

Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination ofin situ removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis membrane and equipped on the agitator of spinner reactor forin situ removal of ammonium ion. The semi-fed-batch operation was employed to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.

Oxygen Carrying Capacity and Oxygen Supply Rate of Artificial Oxygen Carrier, Neo Red Cell (NRC)

Neo Red Cell (NRC), which is the liposome encapsulated hemolysate, has been developed as an artificial oxygen carrier. Oxygen carrying capacity and oxygen supply rate of NRC were estimated by continuous measurement of dissolved oxygen concentration in a spinner vessel. Oxygen carrying capacity of the medium was risen by adding NRC. The oxygen supply rate of the NRC medium containing hepatocytes was determined by the oxygen consumption rate of hepatocytes. The addition of NRC gave no effect on the oxygen transfer rate from gas phase to liquid phase (or kL a value) of the solution in the spinner vessel. The rate of oxygen absorption to NRC was limited by the oxygen transfer rate from gas phase to liquid phase in the spinner vessel. These results indicate that the oxygen supply from NRC may sustain the high-density culture of mammalian cells.

The vortex wave membrane bioreactor: hydrodynamics and mass transfer

An experimental study of a membrane bioreactor has investigated the dynamics of the vortex wave as an effective aeration technique for application in high density mammalian cell culture. Gas transfer membranes have been employed in order to eliminate the potentially lethal gas\\3-liquid interface found in stirred tank and bubble columns. The diffusion-limited features of oxygen transfer through a membrane have been overcome by harnessing the excellent mixing characteristics of oscillatory flow and vortex formation. The crucial hydrodynamic features of the vortex wave, in a relatively wide channel, have been classified in terms of deflector spacing, Reynolds number and Strouhal number. The dynamic gassing-in of oxygen from a gas phase, across the membrane, to a liquid phase has allowed us to quantify the mass transfer characteristics in terms of the Sherwood number. Significant mass transfer enhancement has been achieved under laminar flow conditions, without a major increase in power dissipation. The Sherwood number has been found to be dependent on both the Reynolds number and the Strouhal number. The Reynolds analogy has been employed to calculate shear rates. The low shear rates (about 300 s{su\ -1}) and maximum theoretical hybridoma cell densities (about 1.0 \\sx 10{su9} cells ml{su\ -1}) indicate that the vortex wave design may be an effective alternative to traditional bioreactors.

Step‐fortifications of nutrients in mammalian cell culture

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated.