Efficient strategies for the production of recombinant proteins are gaining increasing importance, as more applications that require high amounts of high-quality proteins reach the market. Higher production efficiencies and, consequently, lower costs of the final product are needed for obtaining a commercially viable process. In this chapter, common problems in recombinant protein production are reviewed and strategies for their solution are discussed. Such strategies include molecular biology techniques, as well as manipulation of the culture environment. Finally, specific problems relevant to different hosts are discussed.

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Production of recombinant proteins: challenges and solutions.

We introduce the first lab-on-a-chip platform for complete mammalian cell culture. The new method is powered by digital microfluidics (DMF), a technique in which nanolitre-sized droplets are manipulated on an open surface of an array of electrodes. This is the first application of DMF to adherent cell culture and analysis, and more importantly, represents the first microfluidic platform capable of implementing all of the steps required for mammalian cell culture—cell seeding, growth, detachment, and re-seeding on a fresh surface. Three key innovations were required to implement complete cell culture on a microfluidic device: (1) a technique for growing cells on patterned islands (or “adhesion pads”) positioned on an array of DMF actuation electrodes; (2) a method for rapidly and efficiently exchanging media and other reagents on cells grown on adhesion pads; and (3) a system capable of detachment and collection of cells from an (old) origin site and delivery to a (new) destination site for subculture. The new technique was applied to cells from several different lines which were seeded and repeatedly subcultured for weeks at a time in 150 nL droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. To illustrate an application for this system, a microfluidic method was developed to implement transient transfection—we propose that the combination of this technique with multigenerational culture allows for “on-demand” generation of transiently transfected cells. Broadly, we anticipate that the automated cell microculture technique presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

A microfluidic platform for complete mammalian cell culture

Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer.

Large-scale transfection of mammalian cells for the fast production of recombinant protein.

https://www.cysticfibrosisjournal.com/article/S1569-1993(04)00094-3/pdf

Established cell lines used in cystic fibrosis research.

Background: The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results: Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions: In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.

/pdf/comparative-transfection-of-dna-into-primary-and-transformed-4d8brdg2lx.pdf

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

https://www.future-science.com/doi/pdf/10.2144/00292rv01

A. R. Holmes

Papers

Transfer and Expression of Foreign Genes in Mammalian Cells