Spinner culture

Bellco (Appendix 3) produce a 3 1 air lift bioreactor designed initially for use with hybridoma cells entrapped within alginate macro beads ( 3.6.4). In this system the rate of antibody production was over 10 times that achieved in a spinner culture (Familletti, 1987). 

Two types of polyvinylpyrrolidones (PVP-Bayer, Leverkusen, Germany) have been found beneficial to cell growth in spinner cultures, and a mixed molecular weight PVP has been found to protect hybridoma cells against shear injury in a bubble column reactor (Handa, 1986). 

We have found that cytoplasmic extracts from a number of cell types support nuclear import in vitro. The extracts are prepared essentially as described in Dignam et al. (1983) using cells grown on a large scale in spinner cultures or purchased from a commercial source as a frozen cell pellet. We routinely start with approximately 10 cells. The nuclei produced in the initial steps of the procedure can be used as a source of snRNPs. 

Grow mouse myelomas in static flasks or spinner culture and keep growing exponentially in DMEM containing 10% or 20% FCS (dilute to 2-3 x 10 ceUs/ml the day before fusion). 

The rat myeloma Y3 has to be grown in spinner culture to fuse weU. Seven to ten days before cells are required, about 5 x 10 cells stored frozen in liquid nitrogen as 1 ml aliquots in freezer medium are thawed quickly at 37 °C, diluted with 10 ml... 

Spinner culture is the simplest form of suspension culture available, relatively cheap to set up, and with reasonable scale-up potential (larger units and increasing the number of replicates). 

Culture procedures have been described from the simple flask and spinner cultures through increasing degrees of sophistication to the highly productive, but complex and expensive, perfusion bioreactors. The choice of culture system depends upon how much mAb is needed, the quality needed (i.e. for what purpose is it intended), and the expertise and cell culture facilities available. In order to choose on the basis of mAb yield the data in Table 4 summarizes each system described in this chapter with values for cell density and mAb production. Although somewhat hypothetical the values are based on an average... 

Preparation of HeLa cell-free extract (1) Obtain cells in log-phase spinner culture (5.2x 10 cells/ml). Spin cells in conical flasks at l,000rpm for 15 min. Resuspend cells in 10 ml of ice-cold PBS per liter of cells and transfer to a sterile 50-ml Falcon tube. Wash twice more, first with 50 ml/L cells, then with 5 ml/L cells (spin at 1,500 rpm for 10 min) and transfer to 15 ml Falcon tube before last spin. (2) Determine approximately the volume of packed cells and resnspend with same volume ice-cold hypotonic bnffer. Swell on ice for 10 min. 

Cells that survive exposure to a mutagen are fragile. If cells are exposed to mutagens in spinner cultures, the toxicity is markedly increased compared to exposure in stationary suspension culture. We have therefore found it convenient to expose the lymphoblasts to the test agent in stationary suspension culture. 

The adsorption and penetration of AAV containing H-thymidine-labeled DNA has been measured with KB cells in spinner culture (Rose and Koczox, 1972 Berns and Adler, unpublished data). Adsorption reached a maximum and plateaued one to two hours after infection. Approximately 16-30% of the added viral label was irreversibly associated with the cells by this time. Depending on the method of cell fractionation, 25-45% of the bound radioactivity was eventually (by 20 hours) found in the nuclear fraction, the majority within two hours. Uncoating, as measured by sensitivity of the labeled DNA to DNase, took place in the nucleus. Maximum uncoating occurred by I6 hours post infection and represented approximately 85% of the total DNA (Berns and Adler, unpublished data). 

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