Microcarrier Culture of Lepidopteran Cell Lines: Implications for Growth and Recombinant Protein Production

Abstract

Several microcarrier systems were screened with Sf‐9 and High‐Five cell lines as to their ability to support cell growth and recombinant (β‐galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex‐1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore‐1 and ‐2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra‐Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 × 106 (Sf‐9) and 2.7 × 106 (High‐Five) cells mL‐1 (8 × 106 and 5.5 × 106 cells per cm2 of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. β‐Galactosidase (β‐gal) production of Sf‐9 cells on Fibra‐Cel disks (infected at 3.3 × 106 cells mL‐1) was prolonged (192 h), and specific protein production was similar to that of high‐density free cell infection. Cultispher‐S microcarriers were found to be a very efficient system for the growth of High‐Five cells, whereas no growth of Sf‐9 cells took place for the same system. Concentrations of about 9 × 106 cells mL‐1 were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after β‐gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final β‐gal titers of 946, 1728, and 1484 U mL‐1 were obtained for infection densities of 3.4, 7.2, and 8.9 × 106 cells mL‐1, respectively.

Escherichia coli Biofilms Formed under Low-Shear Modeled Microgravity in a Ground-Based System

“…The results are representative of at least 10 beads from two independent experiments. (12,14,40), this approach has not, to our knowledge, been used to cultivate microbial biofilms. The primary obstacle has been providing bacteria with a surface for biofilm formation that could be maintained under LSMMG.…”

“…An overnight culture of the desired fluorescent strain [either AMS6(pAD123) or AMS150(pAD123)] was diluted in fresh Luria-Bertani broth (A 660 ϭ 0.1), and 5 g ml Ϫ1 sterile glass microcarrier beads (Sigma; 150 to 210 m diameter; 1.02 g/cm 3 ) were added to the diluted culture. These microcarrier beads are ideal for these investigations since they are smaller and less dense than those successfully used previously for the suspension culture of anchorage-dependent mammalian cells (12,14,40). The mixture of beads, inoculum, and medium was used to fill a pair of HARVs.…”

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

“…Sf-9 cells have been cultivated in suspension in various types of bioreactors such as stirred tank, airlift, spin-filter perfusion, and helical-ribbon impeller stirred tank (for review, see (Taticek et al 1995)). Besides, insect cells have been immobilized on various substrata such as microcarriers (Wickham and Nemerow 1993), Fibra-Cel disks (Ikonomou et al 2002) and glass beads (Chung et al 1993), and were maintained in suspension by fluid flow. The maximum cell densities achieved in these studies lie in the range of 3-14.5 Â 10 6 cells ml À1 .…”

Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside microcarriers in protein-free media

“…In particular, non-woven polyester fabrics, (NWPF), have been used successfully in fixed-bed reactor for the cultivation of some cell lines because their high surface-to-volume ratio allows the development of high cell densities. Fibra-cel Ò carriers, which are used in this paper, are commercially available NWPF products that have been used successfully with insect cell lines (Kompier et al 1991;Agathos 1996;Ikonomou et al 2002) and several mammalian cell lines, either anchorage-dependent (Racher et al 1995;Hu et al 2000;Kaufman et al 2000;Mertens et al 2001) or anchorage-independent (Wang et al 1992). In most of these studies serum-supplemented media were used, at least during the initial cell propagation phase.…”