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The CD2 (T11) molecule belongs to a family of cell-surface glycoproteins that function as adhesion molecules in the immune system. Human CD2 is found exclusively on cells of the T lineage: peripheral T lymphocytes, NK cells, and thymocytes. CD2 binds specifically to the surface glycoprotein LFA-3. CD2/LFA-3 adhesion is the basis for the formation of rosettes between T cells and sheep erythrocytes (SRBC) which bear the sheep homologue of LFA-3. More importantly, CD2/LFA-3 adhesion functions in the immune system to augment T cell activation; it initiates conjugate formation between participating T cells and antigen-presenting cells (APC). We investigated the effects of soluble forms of CD2 (sCD2), produced in either baculovirus or CHO expression systems, on the rosetling of T cells with SRBC and on the activation of T cells by antigen plus major histocompatibility complex (MHC) molecules. Rosette formation between T cells and SRBC was completely inhibited by as little as 1 μ M sCD2. Furthermore. sCD2 effectively inhibited (at micromolar concentrations) the T cell proliferative response to recall antigens including rubella, tetanus toxoid, and herpes simplex virus (HSV-1), as well as alloantigens in a mixed lymphocyte culture. These findings are consistent with the notion that the CD2/LFA-3 interaction augments antigen-specific T cell functions. The use of a CD2 "decoy" molecule rather than anti-CD2 or anti-LFA-3 antibodies to block the CD2/LFA-3 interaction rules out secondary antibody effects, via the Fc portion, as the basis for inhibition of T cell activation and directly stresses the importance of this adhesion interaction in T cell responses.

Maintenance of the progenitor cells responsible for hematopoiesis has generally been accomplished using a feeder layer of stromal cells in stationary culture. Here, we compared the expansion of the total cell and progenitor cell populations using low-density mononuclear cells (LDMCs) obtained from human bone marrow in static culture (T-flasks) and in different cell culture bioreactors designed for the scale-up of mammalian cells. Static cultures were performed without the presence of a previously established stromal cell layer. Expansion of marrow in all cases was accomplished through the use of added cytokines such as IL-3, GM-CSF, and c-kit ligand. The results for the total cell expansion in static culture ranged from 4.4- to 32-fold. The cell number increase was affected by such factors as patient to patient variability, freeze-thawing, and the combination of cytokines used. Due to widespread use and the small amount of marrow needed, static cultures were used as a basis for comparison with other expansion systems. The cell culture systems used to evaluate the scale-up of marrow cultures included suspension, microcarrier, airlift, and hollow fiber bioreactors. Using identical media, cytokines, and feed schedules, LDMCs in the suspension bioreactor expanded to a value of 1.6 compared to a normalized value of 1.0 for static cultures for the two runs investigated. Expansion results for microcarrier cultures averaged 0.75 when compared to static cultures. A cell number increase in the airlift bioreactor resulted in an expansion which was 0.70 of the control static culture. Granulocyte-macrophage and erythroid progenitor assay data were also evaluated for the suspension, microcarrier, and airlift bioreactors.(ABSTRACT TRUNCATED AT 250 WORDS)

To compare modeling with experimental data of cell growth surrounding individual fibers, the growth profiles of hybridoma cells in the extracapillary space of single hollow fiber bioreactors were examined. Agarose was provided in the extracapillary space to provide support and minimize convection. By sacrificing bioreactors at various time intervals, the growth profiles of cells surrounding a single hollow fiber could be monitored with increasing time. Using photomicroscopy and viable staining, areas of viable and nonviable cell growth were examined at various stages of development ranging from initial seeding to stable growth conditions. Cells were found to act as nucleation sites for the growth of individual colonies within the agarose. Profiles at stable growth conditions resulted in a thick cell mass near the surface of the fiber wall followed by cell colonies of decreasing size with increasing radial distance. A simplified theoretical model for cell growth was developed using mass balance equations for substrate penetration into individual cell colonies as well as away from the wall of a single fiber. The resulting profiles derived from theory were compared with experiments and found to be in good agreement for entering oxygen concentrations of 5% and 20%.

A major problem in the use of plasmids as recombinant vectors is the problem of plasmid-free cell generation from plasmid shedding and subsequent growth. A common technique for controlling the population of plasmidfree cells is the use of selective media against these cells using an auxotrophic host and a plasmid that has the ability to produced the essential metabolite. A distributed model describing the growth of Saccharomyces cerevisiae containing a recombinant plasmid in selective media was developed. The model allows for growth and production of a metabolite by the plasmid-carrying strain and growth of the plasmid-free cells on resulting metabolite concentrations. Through a determination of system constants and numerical solution to the equations, experimental batch and continuous culture results for cell concentration transients could be simulated by the model. The results indicated that despite selective pressure, plasmid-free cell growth was significant.

In the design of bioreactors for mammalian cell culture, metabolic parameters such as oxygen uptake, acid output, and CO, generation are desirable. Knowledge of cellular oxygen uptake rates allows the design of systems which supply adequate oxygen for cell growth and product ex- pression. Knowledge of cellular acid output rates allows the formulation of media with sufficient buffering capac- ities to limit the pH change of cultures and provides a basis for the design of systems with pH control. Knowledge of cellular CO, and carbonate uptake or output allows the de- sign of systems which will supply or remove these species. The methods described in this study involve the use of a glass syringe for the culture of animal cells.’ The physical conditions under which the cells are exposed are similar to those of large scale cultures with respect to nutrient sup- ply, cell concentration, microcarrier concentration, etc. As such, the effects of various experimental conditions ob- tained from these techniques can be used for the design of large-scale processes. The oxygen uptake rate of mammalian cells in culture has been measured by various methods. Fleischaker and Sinskey’ measured the oxygen demand in human diploid foreskin cells in microcarrier culture using an oxygen bal- ance equation. The oxygen uptake rate (OUR) was deter- mined using the transport characteristics of the culture vessel by measuring the oxygen concentration transients. Frame and Hu3 measured the OUR of swine testicular cells in 50 mL flasks with no gas phase using an oxygen elec- trode. Additional methods for measuring the oxygen up- take of animal cells involve the measurement of oxygen tensions in completely filled tissue culture flasks with a polarographic electrode4 as well as measuring the disap- pearance of oxygen in a closed chamber by electron spin resonance techniques.5 Bicarbonate and CO, are important mainly in the control of pH as a result of the following equilibria: HC0