Bioreactors For Suspension Cell Cultures Small Scale Culture Systems

Small-scale culture systems are characterized by a relatively simple design and low level of instrumentation and control. Traditionally, roller bottles and spinner flasks have been used for small-scale suspension culture although even T-flasks, Petri dishes, multiwell plates, and other stationary culture systems are applicable for suspension cell propagation in small-scale. Spinner flasks (Techne, Integra Biosciences, etc.) are available from 125 mL to 5L working volume for operation in humidified CO2 incubators. Larger flasks up to 36 L require access to warm rooms or can be operated with heating belts (Belco). They are generally made of glass but flasks made of plastics (NalgeNunc) are also available. The most primitive design consists of a flask with two side ports for inoculum and medium addition and a magnetically coupled stirrer either of a bar type on a central axis or a conical pendulum (20). The stirrer rate is maintained between 30 and 100 rpm, frequently as low as possible to prevent sedimentation of cells. The flasks are commonly kept in a humidified CO2 incubator and gas transfer takes place via the headspace through slightly opened caps. Oxygen transfer coefficients in the rage of 0.1-4 h_1 are reported for spinners (21). A general drawback of this design is the very low oxygen transfer resulting in oxygen limitation already at relatively low cell densities. To maximize oxygen transfer in spinners the height to surface ratio should be kept low; i.e. the working volume should be minimized. To overcome this limitation but still keeping it a simple culture system a special floating stirrer has been suggested (20). Another solution to overcome gas transfer limitations is to submerge gas permeable membranes in the culture fluid [Biott Spinner (22)]. Heidemann et al. developed the so-called Superspinner (23) where microporous polypropylene membranes are fixed on a pendulum stirrer in a standard 1 L Schott glass bottle and the incubator air/CO2 mixture is pumped through the membrane and the headspace using a simple air pump. This spinner flask set-up is also used in a recently developed small-scale screening system where pH and oxygen electrodes are introduced for monitoring and control (DASGIP) (24). Spinner flasks have been widely used for the cultivation of many different suspension cell lines (primary and transformed mammalian and insect cell lines) for a variety of applications within preclinical research, process development, and for inoculum expansion in production. In addition, shaker flasks are commonly used for the propagation of suspension cells, not only insect cells (25) but also hybridoma or CHO cells (26,27). It is also possible to culture suspension cells in sterile plastic tubes; e.g., centrifuge tubes, placed in a modified shaker apparatus or roller drums (New Brunswick Scientific and models from other manufacturers) inside a humidified CO2 incubator. This set-up is commonly used for simple screening purposes in media development trials where numerous parallel experiments are undertaken. Microtiter plates are increasingly used for screening and optimization purposes in cell culture (28), in the area of high-throughput protein expression, and biopharma-ceutical cell and process development. However, little knowledge is currently available on the fundamental characteristics affecting culture conditions in this system (29,30). Generally, scale-down techniques are becoming more important in industrial process development and optimization. A review by Palomares and Ramirez (31) summarizes the approach and provides examples and experimental configurations.

Another simple culture system is the WaveBioreactorâ„¢ (32) made for suspension cell culture of 100 mL up to 500 L volumes in disposable plastic bags (see Fig. 1). Oxygen is transferred through the gas permeable wall. Due to the rocking motion of the bag on a rocker base oxygen transfer is considerably improved compared to spinner culture. The system can be operated in CO2 incubators or standalone in combination with a heater and a CO2 control unit. Singh (32) describes the application of the system for monoclonal antibody production, adenovirus production with HEK 293 cells, and baculovirus production with insect cells. Due to the disposable nature of the bioreactor it offers clear advantages for cell therapy related to patient safety.

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