William R. Tolbert

Chapter 3 - Large-Scale Cell Culture Technology

Publisher Summary Large-scale growth of vertebrate cells in vitro offers a method for the production of biomolecules not available from other sources. This has focused the attention of researchers on the development of mass culture systems that offer opportunities for large-scale growth of such cells for production of desired biomolecules. This chapter describes a variety of mass culture systems that can be operated antibiotic-free and can be selected to meet the specific requirements for each cell/product combination. It reviews the data on the vertebrate culture methods currently available and in use for the production of biologically important molecules with a particular focus on large-scale cell culture technology. The chapter presents a review of present large-scale cell culture technology and describes the development in the laboratory of significant advances in a new, multifaceted approach to this area. It would be useful both for the establishment of large-scale cell culture facilities dedicated to the production of biomolecules for clinical or commercial application and for the research laboratory.

Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures

SummaryA system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 109 cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O2 and CO2 were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10-to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4-and 40-liter versions of this reactor have been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10×106 cells/ml with a total yield of 8.7×1011 cells from 360 liters of medium.

Cell aggregate suspension culture for large-scale production of biomolecules

SummaryA method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm2 flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1×1010 cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.

Rapid determination of cell volume density in mammalian cell suspensions

Abstract Cell volume density was shown to be a useful growth parameter to monitor both single cell and aggregate suspension cultures. A method is described to quickly and easily measure this parameter using the length of a cell pellet in a conical hematocrit tube. The tubes were prepared by sealing the small end of disposable micropipet tips, and for seven cell lines growth within the range from 0.1 to 5 ml/liter was reproducible with an average ± 5.6% SD. Good agreement was obtained for population doubling times determined from cell volume density, DNA concentration, and, where possible, cell number density.