C. Schmatz

The use of macroporous gelatin carriers for the cultivation of mammalian cells in fluidised bed reactors

A fluidised bed system for the cultivation of mammalian cells on a new type of macroporous gelatin microcarrier is described. The volumetric cell densities achieved under controlled conditions for two ‘standard cell lines’ (VERO, CHO) were one order of magnitude higher compared to conventional techniques using spherical microcarriers. The system can be potentially used for both anchorage dependent and independent cells.

Influence of transfected SV40 early region on growth and differentiation of human endothelial cells

Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-termin vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extendedin vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years.Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 108 cells/ml a stable growth rate of 0.004 h−1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

Expression of SV40 tumour antigens enables human endothelial cells to grow independently from foetal calf serum and exogenous growth factors

Human endothelial cells were transfected with a plasmid containing the coding sequence of the large T protein of simian virus 40. Transfected cells were selected for their ability to grow in defined medium (DM). Several cell lines were derived and characterized in their response to endothelial cell growth supplement (ECGS), epidermal growth factor (EGF) and insulin (INS). In addition to cell lines that were dependent on these additives, others growing without any exogenous growth factor could be selected. No evidence of autocrine growth stimulation was found. For growth studies, a simple assay was used based on the acid phosphatase activity as a parameter for the cell number. Cell lines in defined medium showed less chromosome aberrations than those grown in serum-containing medium. Because of their long in vitro life span of about 100 generation doublings and defined medium requirements these cells represent valuable test material for all kinds of investigations on the vascular endothelium.

Long-Term Stability Of Continuously Perfused Animal Cells Immobilized On Novel Macroporous Microcarriers

The ultimate goal of optimizing biological production is the establishment of simple, safe, cheap, consistent, and easily manageable production systems. Various complicated techniques have been used for animal cell culture. In practice, however, only the simple ones are used for production purposes. A high cell density continuous perfusion in vitro system simulates a situation that most closely exists in the in vivo maintenance of differentiated cells arrested in the G1/G0-phase of the cell cycle. In order to make this a technological reality, a macroporous open structured carrier has been designed using high pressure polyethylene as a primary matrix material. During long-standing continuous perfusion of cultured mammalian cells such as Chinese hamster ovary (CHO) cells with a protein-free medium, the cells are maintained at high cell density (> 10 8 per ml) in a fully productive but nonpropagating state. Using the fluidized bed porous bead technology we do not expect any limitation in the scale-up of any technology using continuous mammalian cell lines.

Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.

PRODUCTION OF THE HIV-1 NEUTRALISING HUMAN MONOCLONAL ANTIBODY 2F5: STIRRED TANK VERSUS FLUIDIZED BED CULTURE

ABSTRACT The performance of a 6 litre stirred tank reactor and a fluidized bed reactor operated with 2 litre macroporous PolyporE beads have been compared. Under controlled perfusion conditions the productivity (anti-HIV-1 lgG3) of the fluidized bed culture system turned out to be 20-fold higher.

Monoclonal Antibody Production Using the Porous Glass Bead Immobilization Technique

The increase of cell density in bioreactors and thus immobilization techniques have received a substantial amount of attention.s2 The technique for mass production and maintenance culture of anchorage-dependent mammalian cells at high cell densities is established for the production of a wide range of biological^.^^^ There are a number of reasons claimed beneficial for using immobilization techniques for the propagation of hybridomas, for example, high cell density, decreased biomass separation cost, lower media costs, the use of protein-free media, and higher volumetric production rate^.^-^

Fluidized Bed Technology: Influence of Fluidization Velocity on Nutrient Consumption and Product Expression

Introduction Fluidized beds in combination with macroporous microcarriers are based on the perfusion technology. Perfusion technology was developed as it was recognised that cells in vivo are continuously supplied with blood, lymph, or other body fluids to keep them in a constant physiological environment. To exploit the high productivity potential per reactor volume of a fluidized bed system optimisation of fluidization velocities for anchorage dependent as well suspension cells immobilized in macroporous matrices are needed to guarantee a sufficient nutrient supply similar to in vivo conditions.