The heterogeneous nature of polyethylenimine-DNA complex formation affects transient gene expression.

Polyethylenimine has been used widely in transient gene expression with mammalian cells. To further understand its mediation of gene transfer, the transfection of HEK 293-F cells with dynamically prepared PEI/DNA complexes was studied with the help of fluorescent labeling. The efficiency of complex endocytosis/phagocytosis was found to correlate with the average sizes of complexes applied and complexes greater than 1 µm in diameter were likely excluded by the cells. Coupled with complex growth in size, the degree of association between PEI and DNA increased with the time of complex formation in the presence of competing ions. The blocking of transcription by complex formation necessitated complex dissociation in the nuclear environment for transcription to happen. Intracellularly, the fates of PEI complexed DNA therefore may be mostly determined by the degree of association. Results also suggested that the uptake of PEI/DNA complexes and subsequent protein expression were independent of the cell cycle stages of HEK 293-F cells.

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media.

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca(2+) and Mg(2+) for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl(2) and MgCl(2) concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca(2+)/Mg(2+) feeding to 117 h with Ca(2+)/Mg(2+) feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 x 10(6) and 9.12 x 10(6) cells ml(-1), respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.

A High-throughput End-point Assay for Viable Mammalian Cell Estimation.

A single wavelength colorimetric microplate-based assay was developed using non-cytotoxic dye resazurin for the estimation of viable cell concentrations of Chinese hamster ovary (CHO) and hybridoma cells. Experimental results showed variations in pH and temperature caused by cell cultivation and assay operations were well tolerated. Cell concentrations can be effectively determined in the range of 10(5)-10(7) cells ml(-1) using a microplate reader at the wavelength of 605 nm. This assay can be performed in a high-throughput manner such that a large number of cell culture samples can be screened within a relatively short time frame. When used together with a cell culture system of high-throughput format, it may have potential utilities in applications such as cell culture medium formulation and optimization.