Avinoam Kadouri

Modified CelliGen-packed bed bioreactors for hybridoma cell cultures.

This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG1 monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen™ bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 108 cells per cm3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1 %) of serum as well as serum-free media.These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time.The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.

Comparison of retinoic acid effects on anchorage-dependent growth, anchorage-independent growth and fibrinolytic activity of neoplastic cells.

Abstract We have compared the abilities of retinoic acid to modify the fibrinolytic activity, the anchorage-dependent growth and the anchorage-independent growth of various cultured tumor cells. Treatment with 10 μM retinoic acid for 3 or 6 days increased 2- to 4-fold the level of cell-associated plasminogen activator in S91-C2 and B16 (cell lines F1, F10, F10-B2, BL6 and LR6) mouse melanoma cells and in a human osteosarcoma (Hs781). In a few of the B16 sublines there was a similar increase in fibrinolytic activity secreted into harvest fluids. In contrast, there was no significant effect of retinoic acid on the fibrinolytic activity of five human melanomas (A375, Hs294, Hs852, Hs939 and SH4), two human carcinomas (HeLa-S3 and A431), two human osteosarcomas (Te85 and Hs791) and a murine fibrosarcoma (UV-2237P). Both the anchorage-dependent growth and the anchorage-independent growth were inhibited by retinoic acid to varying degrees in most of the cells employed (A375, Hs939, the B16 sublines, S91-C2, HeLa-S3, Te85 and Hs781), whereas only the anchorage-independent growth of the Hs852, the SH4 and the UV2237P cells was inhibited. The anchorage-dependent growth of the Hs294 cells was stimulated, whereas their anchorage-independent growth was not altered by retinoic acid. These results show that there is no correlation between the effects of retinoic acid on plasminogen activator and on cell growth. The data also indicate that retinoic acid can inhibit the anchorage-independent growth of cells which are resistant to retinoic acid under anchorage-dependent conditions.

Cultivation of anchorage-dependent mammalian cells and production of various metabolites

Abstract Cell culture is an important tool for the production of various biologically active products such as pharmaceuticals, viral vaccines, viral insecticides, monoclonal antibodies, polypeptides and growth factors. These products are produced by normal, transformed and genetically engineered cells. The majority of these cells are anchorage dependent and require a solid surface to attach to in order to proliferate, metabolize nutrients and produce biomolecules. The need for cell culture was intensified with the enhanced development of mammalian genetic engineering, which raised a new demand for advanced cell culture technologies. Intensive studies have been undertaken by scientists to develop new technologies for large-scale cell culture. A major effort was invested to develop cell culture systems with high ratios of surface area to volume. At present there is a wide variety of systems, each with its own advantages and limitations. In this short review, the various large-scale cell culture systems are described. Cell culture technology in general, and in particular the substrates to which the cells adhere, play important roles in the product formation of a given macromolecule; therefore the system should be chosen very carefully and adapted to the biological process. The production process of tissue plasminogen activator from normal human fibroblasts and a new cell carrier, “Fibra-cel”, which is made of non-woven polyester fabric, are presented as examples in order to demonstrate the state of the art in this field.

Restrictin‐P: The First Member of a Putative Family of Novel Inhibitors

MBA-2.1 cells produce an activity, designated restrictin-P, which is specifically inhibitory to the growth of plasmacytomas and mature B cell lymphomas. We examined whether the activity of this stromally derived glycoprotein could be attributed to a well-characterized growth factor. Restrictin-P-producing cells were therefore screened for the expression of transcripts of a variety of growth suppressors. With the exception of TGF-beta 1, none was produced in detectable amounts by these cells. Furthermore, recombinant forms of the inhibitory molecules tested did not exert a biological effect similar to that of restrictin-P. Restrictin-P was shown to elicit a G0/G1 arrest in the cell cycle of its target cells, as soon as 24 h after their exposure to the inhibitor. This effect could not be mimicked by TGF-beta 1. We suggest that restrictin-P is part of a novel family of inhibitors which are required for the maintenance of cell-type specificities in the hematopoietic microenvironment.

CONTINUOUS PRODUCTION OF MONOCLONAL ANTIBODIES IN CELLIGEN PACKED BED REACTOR USING FIBRA-CEL CARRIER

This study demonstrates the use of a CelliGen packed bed bioreactor for the growth of hybridoma cells and for production of Mab. The bioreactor is packed with disc carriers made of polyester non-woven fabric (Fibra-cel carrier, Sterilin England). Hybridoma cells (mouse-mouse ATCC HB-57, produces IgGl against human IgM) were seeded and grew on the carrier. Then a continuous production of Mab was maintained under controlled conditions over 46 days with a Mab concentration up to 400 μg/ml. The high productivity of Mab obtained in this system suggests that the culture conditions achieved in this packed bed bioreactor are excellent for the continuous propagation of hybridomas.

Polystyrene substratum for bulk culture of anchorage dependent cells

Twisted ribbons made of polystyrene were used as a packing material for the cultivation of anchorage dependent cells. Normal human fibroblast cells grown on this support in a laboratory scale reactor reached densities of about 5–7×105 cells/ml. The cells adhered strongly to the carrier and no cell detachment was observed upon transfer to serum free medium. The properties of this packing material and its potential use are discussed.

Design and Performance of a Perfusion Based Packed Bed Bioreactor for the Production of Secreted Protein

A bioreactor employing a packed-bed system for the production of secreted proteins has been developed. The bioreactor is able to achieve volumetric productivities, with most cell lines, that are five to ten times higher than standard stirred tank perfusion systems. The stationary basket uses polyester fiber disks (Fibra-Cel™- Sterilin UK & New Brunswick USA) as a support matrix for immobilized cell propagation and is capable of cultivating either anchorage dependent or anchorage independent cells. Typical cell densities achieved are about 108 cells per cm3 of Fibra-Cel in serum or serum-free medium. A big advantage of this system is the fact that it can be scaled linearly. Similar productivities were obtained in the benchtop bioreactor and in the larger pilot scale system.