Future bioreactor use considering protein expression experiments

With regards to high density cell cultures previous applications include recombinant antibody expression from hybridomas (Casablancas, et al., 2013; Jain, et al., 2011) and protein expression from CHO cells, mouse myeloma (NS0), baby hamster kidney (BHK), human embryo kidney (HEK-293) and human retinal cells (Kim, et al., 2012; Wurm, 2004). Although our drip-perfusion bioreactor was not cultured with hybridomas the MTT and fluorescent staining results of high density culture of mammalian fibroblasts indicated potential. For high density cell culturing using scaffold architecture similar to the FDM disc stacks would be advantageous and would allow a continuous culture for 3 to 4 weeks without passaging. Prolonging this process could involve a simple approach of aseptically removing the inferior most located disc approaching critical density and replacing it with a fresh disc on the stack’s superior most location. Complete removal and renewal of medium every two to three days would allow for purification of expressed proteins within the collected medium. This situation would expect the cultured cells to be capable of synthesising and utilising a signal sequence for the N-terminal of the target protein and eventual protein secretion into the medium for purification (Blobel & Dobberstein, 1975; Hegde & Bernstein, 2006). To efficiently isolate protein from the medium, preceding and proceeding protocols to drip-perfusion culturing are proposed and can involve, respectively, engineering an affinity tag into a specific location in the target protein sequence and isolation of recombinant protein by exploiting the binding efficiency of the affinity tag via a chromatography technique (Gräslund, et al., 2008). Various affinity tags and their

corresponding benefits and disadvantages have been reviewed ranging from the smaller 0.8 kDa polyhistidine and polyarginine tags to the larger 20.0 kDa tags of glutathione S- transferase and could provide initial points of research for the unfamiliar investigator (Gräslund, et al., 2008; Terpe, 2003).

Protein production in the drip-perfusion bioreactor is anticipated to experience difficulties when aiming to produce ECM related proteins. Although current commercial sources of ECM proteins such as collagen I, IV, fibronectin and vitronectin are derived from mammalian sources including rat tail tendons, human placenta, and human plasma instead of being recombinantly produced, due to the complexity of interplay between genes, enzymes, proteins and cofactors involved in their respective syntheses and PTMs (Canty & Kadler, 2005; Myllyharju & Kivirikko, 2004). Recent advances in molecular engineering have provided an approach to recombinant collagen production using plant cells (Shoseyov, et al., 2013) and may be provide translatable techniques for use with the drip-perfusion bioreactor. The design of the drip-perfusion bioreactor utilised a scaffold to support adherent cell culture which relied on their deposition of ECM proteins to bind to the functionalised scaffold. The type of functionalization and amount of scaffold surface treated requires consideration as literature reported increased migration impingement proportional to surface area functionalised (Mann, et al., 1999; Mann & West, 2002). This impinged migration would impact on the uniformity of scaffold cell culture and possibly hinder cell proliferation rates due to dense cell population in confined regions. Examining different parameters of scaffold surface functionalization with respect to a selected cell type for ECM protein production would be necessary when investigating this procedure in the drip- perfusion bioreactor.

The second consideration with ECM protein production is the method of extracting secreted ECM protein from within an adherent cell culture. A possible approach could involve the use of magnetic particles/beads, diameter of 1-4 μm, coated with an affinity ligand capable of binding the affinity tag engineered into the target protein. Although literature has shown success purifying peptidases, polysaccharide hydrolases, albumin and peptides with the magnetic separation technique when applied to cell lysates (Safarik & Safarikova, 2004) and novel optimisations have been performed to increase purification yields (Xu, F., et al., 2011) this magnetic separation technique would require adaptation and optimisation when used for purifying ECM proteins. This magnetic technique could be applied to this drip-perfusion system by injecting magnetic particles into the media circulation loop (Fig. 3-8 B, 2i) prior to the air trap via an integrated 3-way stopcock, the circulating medium would carry the particles to the drip feed outlets and drip-perfuse them through the cell culture. The affinity

ligand coated magnetic particles would be expected to bind affinity tagged protein as the particles were transported throughout the scaffold by the medium (Fig. 5-1). The medium containing the protein bound particles could then be extracted from the reservoir with the manual hand pump and a magnetic separator applied to the harvested medium yielding the protein bound particles. These particles could be treated with an enzyme cleaving the affinity tag and isolating the desired protein. This procedure could be performed to coincide with medium exchange intervals reducing an accumulation of magnetic particles in the cell culture.

Figure 5-1 Schematic of drip-perfused medium carrying magnetic particles coated with affinity ligands

The magnetic particles would be carried through the scaffold by the perfusion of medium and collected after medium extraction using a magnetic separator. Particles trapped due to dense cell culture would involve sacrificing the cultured scaffold, lysing the cell culture and separating the magnetic particles via magnetic attraction.

If the density of the cell culture had approached critical cell density, the FDM disc stack may need to be sacrificed for protein purification. Magnetic particles lodged within the cultured scaffold could be used advantageously after lysing the adherent cell culture with a chemical approach for example RIPA (radio-immunoprecipitation assay) buffer, M-PER (Mammalian Protein Extraction Reagent) solution and T-PER (Tissue Protein Extraction Reagent) solution (Thermo Fisher Scientifc Inc., 2014); post cell lysis the magnetic particles binding target

Adherent cell culture

Medium flow

Magnetic particles coated with affinity ligands

protein, could be separated from the lysate using the magnetic separator as reported in the literature (Safarik & Safarikova, 2004). Although these methods are proposed optimisation would be anticipated for each protocol during the endeavour of investigating high yields of produced protein.