The search for more productive mammalian cell culture techniques has been primarily driven by the need to increase both the cell density and product concentration. In the technique used in our laboratory, microencapsulation, hybridoma cells were grown and monoclonal antibody was entrapped within microcapsules, which had a controlled membrane molecular weight cutoff. In our studies it was shown that using the alginate‐poly‐l‐lysine (PLL) microcapsule system and protein diffusion experiments, the capsule membrane molecular weight cutoff could be controlled from 20 × 103 to 300 × 103. This was achieved by increasing the viscosity average molecular weight (Mv) of the LL, used in the encapsulation procedure, from 14 × 103 to 525 × 103 and by decreasing the alginate — PLL reaction time from 40 minutes to 6 minutes. Cell culture studies with microencapsulated mouse hybridoma cells indicated that while the conventional (single‐membrane) microcapsules produced a maximum intracapsular cell density of about 2 × 107 cells/mL and a monoclonal antibody concentration of 1250 μg/mL, a modified (multiple‐membrane) capsule resulted in intracapsular cell densities of about 6 × 107 cells/mL and monoclonal antibody concentrations of 5300 μg/mL. These significant improvements in cell density and monoclonal antibody concentration were attributed to a lower (22%) intracapsular alginate content as well as better retention of the cell product by the modified capsule membrane.

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Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33 degrees C followed by virus replication at 27 degrees C 3-4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27 degrees C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33 degrees C (6 degrees C above the optimal 27 degrees C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (14 1 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27 degrees C) to produce recombinant protein (beta-galactosidase). A cell density as high as 1 x 10(7) cells.ml-1, and a beta-gal concentration of up to 104,000 unit.ml-1 were achieved.

The desire to increase cell density and product concentration has been the primary driving force for the development of better animal cell culture processes. In the technique used in our laboratory-microencapsulation-insect cells (Spodoptera frugiperda), infected with a temperature-sensitive mutant of the Autographa californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules which had a controlled membrane molecular-weight cutoff and an intracapsular alginate concentration which was ca. 16% lower than that obtained in the commercially available single-membrane system. Cell culture experiments indicated that the intracapsular alginate concentration appears to be a key factor in achieving good cell growth. It was possible to obtain intracapsular cell densities of 8 x 10(7) cells/mL capsules and virus concentrations to 10(9) IFU/mL capsules. The virus litre in the supernatant was ca. 300 times lower, indicating that virtually all of the virus was retained within the capsules.

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