No abstract

A single-pass, plug-flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via silicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side-by-side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single-pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale-up.

Controlled secretion processes for the production of secretory proteins in monolayer culture have been described previously (Grampp et al. Adv. Biochem. Eng./ Biotechnol. 1992, 46, 35-62), but little is known about the feasibility of scaling such processes into high-density bioreactors. Two immobilized-cell, ceramic-matrix bioreactor configurations were tested using the beta TC-3 cell model system which, in monolayer culture, can be manipulated to secrete murine insulin in a highly controlled manner. One reactor was configured with an external recirculation reservoir for oxygen transfer and was operated as a conventional immobilized bed/recycle reactor. The other reactor was configured as a single-pass perfusion system with oxygen supplied by diffusion from silicone tubing positioned proximal to the porous walls of the ceramic matrix. After inoculation with beta TC-3 cells, both systems were perfused with serum-supplemented medium to stimulate cell growth, and they ultimately attained high densities (approximately 5 x 10(8) cells/mL of pore volume). To initiate controlled secretion operations, the reactor cores were washed with a serum-free basal medium, then exposed to a serum-free discharging medium containing secretory stimulants. Following several hours of discharging, the reactors were washed again, then switched to a serum-containing medium designed to quench the regulated secretion process. For the single-pass reactor these cycling operations were simple to implement and were effective in promoting the cyclic discharge and recharge of murine insulin. Because of the ability to reduce the perfusion rate in the single-pass reactor independent of oxygen transfer, the discharged insulin was captured in a relatively small volume (2 reactor core hold-up volumes), yielding a mean product concentration 10-fold greater than in the steady-state perfusate. Cyclic operation of the recirculating reactor was more difficult due to the complexity of switching between recirculation reservoirs, and the introduction of air bubbles during such operations resulted in the loss of biomass from the reactor after one cycle. Even in the first discharging cycle, the insulin yield was much lower than in the perfusate from the single-pass reactor, despite the comparable metabolic rates. The single-pass reactor was cycled successfully through four discharging and recharging episodes and maintained its ability to discharge insulin, albeit at a slower rate after the first discharge. Overall, 50-60% of the insulin secreted during the 48 h cycles was recovered during the brief discharging episodes. When insulin secretion rates and discharging yields were normalized to metabolic activity, neither high-density reactor system performed as well as did identically treated control T-flask cultures. It is hypothesized that the productivity and responsiveness of the high-density, pore-immobilized beta TC-3 cells are lower than in monolayer culture.

Disability due to joint pain is increasing as our society ages. This pain often results from cartilage degeneration in joints due to osteoarthritis or trauma. Cartilage damage from sports injuries is common and the normal repair response results in the formation of fibrous tissue that is inferior to normal cartilage in structure and function and eventually degenerates with time. As a result, tens of thousands of total knee replacements and other surgical procedures are performed each year to repair cartilage defects in the knee. A common clinical treatment for cartilage lesions is debridement of the damaged tissue followed by drilling into the subchondral bone to stimulate tissue regeneration. Although this procedure often provides pain relief, it does not restore long-term function and frequently hastens additional degeneration of the injured site. Alternative procedures for repair or regeneration of human articular cartilage are needed.