Extended cell bank

One consideration to bear in mind during the design of inoculums expansion is to demonstrate the genetic stability of the cell line beyond the expected number of generations required to operate at large-scale. This is usually accomplished by conducting measurements of product expression and genetic markers in cells from an extended cell bank (ECB). 

From the regulatory point of view, the characterization requirements for the cells will depend on the bulk culture process to be used. This will define the number of PDs (set within the limits discussed above) that the cells will undergo. An additional regulatory requirement is that the cells should be passaged beyond the anticipated PD level achieved at the end of each production run. This procedure should therefore establish the stability of cells well beyond the normal working limits. Therefore, after additional passaging, an extended cell bank is prepared for characterization procedures to be repeated. 

Human skin fibroblasts were generated from our cell bank, and vigorous growth ensured. Cells of 12 PDs and 31 PDs after isolation were used, together with fibroblasts transfected with a telomerase construct at 12 PDs, and shown to have extended lifespan after 44 PDs. Cells were applied to the surfaces, test and control, in known quantities (40 cells per surface), and allowed to grow under optimum conditions for 5 days. Surfaces were gently rinsed to remove non-adherant cells, and stained with propidium iodide (nuclear flourescent stain), and counted under flourescence microscopy. 

Fig. 1.3 Diagram of a simple batch (or extended-batch) process with suspension cells. Cells are obtained from a Master- or Working Cell bank (MCB/WCB) and inoculated into spinners for a defined subcultivation period (every 3—4 days, usually for up to 100 days or more). For maintenance purposes of the culture, the cells in the spinner are referred to as the seed train". Cells...

Currently, most RTFs and mAbs are expressed in rodent, bacterial or yeast cell cultures (see Part IV, Chapter 13). A Master Cell Bank (MCB) should be estabbshed and tested for authenticity and freedom from contamination [5, 8, 11] (see also Part IV, Chapter 4). A Working Cell Bank (WCB) is extremely useful to extend the lifetime of the MCB. 

Moreover hemopoietic stem cells-precursors derived from cord blood have higher potential of proliferation and expansion than their analogues from adult bone marrow. Extended cord blood use as a source of hemopoietic cells resulted in the necessity to establish cord blood banks where the samples could be stored in a frozen state at -196 C for a long time with no loss of their biological properties. 

The above results have obvious implications for the biosynthesis of cellulose mlcrofIbrlls. The parallel chain structure of cellulose I rules out any kind of regularly folded chain structure, and reveals the mlcrofibrils to be extended chain polymer single crystals, which leads to optimum tensile properties. Work by Brown and co-workers (22) on the mechanism of biosynthesis points to synthesis of arrays of cellulose chains from banks of enzyme complexes on the cell wall. These complexes produce a bundle of chains with the same sense, which crystallize almost immediately afterwards to form cellulose I mlcroflbrlls there is no opportunity to rearrange to form a more stable anti-parallel cellulose II structure. Electron microscopy by Hleta et al. (23) confirms the parallel sense of cellulose chains within the individual mlcroflbrlls stains reactive at the reducing end of the cellulose molecule stain only one end of the mlcroflbrll. 

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