Design and application of NMR-compatible bioreactor circuits for extended perfusion of high-density mammalian cell cultures.

MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.

Iteration of hybridoma growth and productivity in hollow fiber bioreactors using 31P NMR.

Applications of nuclear magnetic resonance (NMR) spectroscopy to isolated or cultured mammalian cells have been limited because of technical difficulties in maintaining cultures at the extremely high densities required by NMR. Among the well-engineered systems available for such analyses, hollow fiber bioreactors (HFBRs) can maintain the greatest cell density. This attribute of HFBRs makes them ideal for application to NMR-based studies. These systems are currently being applied in biotechnology, where they are used for the production of mammalian cell-derived products, such as monoclonal antibodies. In this paper, the application of a HFBR system designed especially for NMR-based investigations is described. Performance of this system is monitored by NMR and the resulting stability and density of hybridoma cultures are reported. The resulting signal-to-noise per unit time is the highest seen to date for a mammalian cell system.

Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles.

The influence of electric surface charges on the polar headgroups and the hydrocarbon region of phospholipid membranes was studied by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with charged amphiphiles. A positive surface charge was generated with dialkyldimethylammonium salts and a negative surface charge with dialkyl phosphates. The POPC:amphiphile ratio and hence the surface charge density could be varied over a large range since stable liquid-crystalline bilayers were obtained even for the pure amphiphiles in water. POPC was selectively deuterated at both methylene segments of the choline moiety and at the cis double bond of the oleic acyl chain. Additional experiments were carried out with 1,2-dipalmitoyl-rac-glycero-3-phosphocholine labeled at the C-2 position of the glycerol backbone. Deuterium, phosphorus, and nitrogen-14 nuclear magnetic resonance (NMR) spectra were recorded for liquid-crystalline bilayers with varying concentrations of amphiphiles. Although the hydrocarbon region and the glycerol backbone were not significantly influenced by the addition of amphiphiles, very large perturbations of the phosphocholine headgroup were observed. Qualitatively, these results were similar to those observed previously with other cationic and anionic molecules and suggest that the electric surface charge is the essential driving force in changing the phospholipid headgroup orientation and conformation. While the P-N dipole is approximately parallel to the membrane surface in the pure phospholipid membrane, the addition of a positively charged amphiphile or the binding of cationic molecules moves the N+ end of the dipole toward the water phase, changing the orientation of the phosphate segment by more than 30 degrees at the highest amphiphile concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and dynamics of the phosphatidylcholine and the phosphatidylethanolamine head group in L-M fibroblasts as studied by deuterium nuclear magnetic resonance.

Mouse fibroblast L-M cells were grown in tissue culture medium containing selectively deuterated choline or ethanolamine. Both compounds were incorporated into the corresponding phospholipids at levels greater than 50% thus leading to a selective deuteration of these phospholipid head groups. Choline and ethanolamine were labeled at either the alpha- or the beta-carbon atom and well-resolved deuterium and phosphorus n.m.r. spectra were obtained from intact cells, crude plasma membranes and lipid extracts, leading to the following conclusions. (i) A large fraction, if not all, of the phospholipids in the intact L-M cell membranes were organized in a liquid crystalline bilayer. (ii) The phosphoethanolamine and the phosphocholine head group conformation were found to be remarkably similar in pure lipid bilayers and in intact L-M cell membranes with the head group dipoles being oriented parallel to the membrane surface. (iii) The deuterium T1 spin lattice relaxation times fell in the range of 7-25 ms and were similar in intact L-M cells and in pure lipid model membranes, suggesting that the two head groups are not involved in strong interactions with membrane proteins. The rotational diffusion rate of the two head groups was reduced by at least a factor of 10 compared to molecules of the same size in aqueous solution. (iv) The phosphocholine head group was sensitive to the size and sign of membrane surface charges as verified in mixing experiments with charged lipids. In L-M cell membranes the phosphocholine appeared to sense an electrically neutral environment in spite of the fact that L-M cell membranes contain 10-20% negatively charged lipids.