CYCLIC OPERATION OF CERAMIC-MATRIX ANIMAL-CELL BIOREACTORS FOR CONTROLLED SECRETION OF AN ENDOCRINE HORMONE - A COMPARISON OF SINGLE-PASS AND RECYCLE MODES OF OPERATION

Controlled secretion processes for the production of secretory protein s in monolayer culture have been described previously (Grampp et al. A dv. Biochem. Eng./Biotechnol. 1992, 46, 35-62), but little is known ab out the feasibility of scaling such processes into high-density biorea ctors. 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 cont rolled manner. One reactor was configured with an external recirculati on reservoir for oxygen transfer and was operated as a conventional im mobilized bed/recycle reactor. The other reactor was configured as a s ingle-pass perfusion system with oxygen supplied by diffusion from sil icone tubing positioned proximal to the porous walls of the ceramic ma trix. After inoculation with beta TC-3 cells, both systems were perfus ed with serum-supplemented medium to stimulate cell growth, and they u ltimately attained high densities (similar to 5x10(8) cells/mL of pore volume). To initiate controlled secretion operations, the reactor cor es were washed with a serum-free basal medium, then exposed to a serum -free discharging medium containing secretory stimulants. Following se veral hours of discharging, the reactors were washed again, then switc hed to a serum-containing medium designed to quench the regulated secr etion process. For the single-pass reactor these cycling operations we re simple to implement and were effective in promoting the cyclic disc harge and recharge of murine insulin. Because of the ability to reduce the perfusion rate in the single-pass reactor independent of oxygen t ransfer, the discharged insulin was captured in a relatively small vol ume (2 reactor core hold-up volumes), yielding a mean product concentr ation 10-fold greater than in the steady-state perfusate. Cyclic opera tion of the recirculating reactor was more difficult due to the comple xity of switching between recirculation reservoirs, and the introducti on of air bubbles during such operations resulted in the loss of bioma ss from the reactor after one cycle. Even in the first discharging cyc le, the insulin yield was much lower than in the perfusate from the si ngle-pass reactor, despite the comparable metabolic rates. The single- pass reactor was cycled successfully through four discharging and rech arging episodes and maintained its ability to discharge insulin, albei t at a slower rate after the first discharge. Overall, 50-60% of the i nsulin secreted during the 48 h cycles was recovered during the brief discharging episodes. When insulin secretion rates and discharging yie lds were normalized to metabolic activity, neither high-density reacto r 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.