Perfusion culture systems

Third type of culture is stroma-non contact . In this system primitive progenitor cells are sustained when cells are co-cultured with irradiated allogeneic stroma but separated from it by the 0,4 micron membrane in transwell inserts (Costar, Cambridge, MA). These cultures are maintained by daily supplementation of stromal feeder conditioned media (Roller et al. 1998, Verfaillie, 2001) successfiilly expanded umbilical cord blood cells in a novel automated perfusion culture system. Development these approaches followed in the studies of investigators who incorporated the stromal components into the expansion culture. Recently published trials by McNiece et al. 2000 are more encouraging where cells were expanded in static culture for 10 days in Teflon bags (American Fluoroseal, USA). 

Roller M., Manchel L, Maher R., Goltry K., Armstrong R., Smith A. (1998) Clinical scale human umbilical cord blood cell expansion in a novel automated perfusion culture system,. Bone marrow transplant. 21(7), 653-665. 

A great deal of effort has gone into the development of high-density immobilized perfusion culture systems, which can be operated continuously. The limitation with many of these systems (e.g. hollow fibre) is that, whilst they achieve very high unit cell density (typically 10 mH), they do not scale up well volumetrically. This limitation has been overcome by the use of porous carrier immobilization techniques, where high unit cell density can be combined with good volumetric scale-up potential and long-term continuous operation. 

Perfusion Culture Systems as a New High Density Culture... 

The perfusion culture systems are classified into two types by static and dynamic methods as shown in Fig. 12. 

To eliminate the use of a membrane and a filter, we have also tried to make a perfusion culture system using a sedimentation column,... 

High Density Culture Using a Perfusion Culture System with Sedimentation Column... 

Fig. 1 Perfusion culture of the cells in a gel-free 3D microfluidic cell culture system (3D-nFCCS). (a) Representation of a one-pass perfusion culture system, (b) Optimization of perfusion culture flow rate for maximum cell viability [26]...

Dong, H., Tang, Y. J., Ohashi, R., Hamel, J. F. P. (2005). A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells. Biotechnology Progress, 21, 140—147. 

Rudolph, A., Eickner, T., Seidhtz, A., et til. Investigation of in vitro drug release behaviour of drug-eluhng stents using a perfusion culture system. Biomed. Eng. Biomed. Tech. (2013)... 

Xie Y, Hardouin P, Zhu Z, Tang T, Dai K, Lu J. 2006. Three-dimensional flow perfusion culture system for stem cell proliferation inside the critical-size beta-tricalcium phosphate scaffold. Tissue Eng 12(12) 3535-43. 

Fig. 5. A suspension perfusion culture process having ceU recycle. System employed at Amgen using CHO cells for production of a recombinant protein.

The procedure for the estimation of qs and qp is identical to the one presented for fed-batch and continuous cultures. The only difference is in the estimation of the specific growth rate (p). Since perfusion cultures behave as batch cultures as far as the biomass is concerned, p can be obtained as described earlier for batch systems. Namely p is obtained as the slope in the plot of / Xv(t,) versus t,. 

Continuous panel processes, 20 107 Continuous perfusion cell culture systems, 5 350, 354-355 pros and cons of, 5 35 It Continuous polymerization (CP) process, 19 749, 842... 

Figure 27. Human osteoblast-like MG 63 cells in cultures on porous (A) or fibrous (B) poly(L-lactide-co-glycolide) scaffolds. A A summarizing picture of horizontal optical sections. The depth of cell ingrowth into the pores (average pore diameter of 400-600 mm) is indicated by spectral colors (blue 0-60 mm, green 80-160 mm, yellow 180-220 mm, orange 240-300 mm, red 320-400 mm, violet 420-480 mm). Day 14 after seeding, cells stained with propidium iodide. B cells grown for 4 days in static culture followed by 2 days in dynamic perfusion cell culture system. Cell membrane stained with Texas Red C2-maleimide and the nuclei counterstained with Hoechst 33342. Leica TCS SP2 confocal microscope, objective 5x (A) or lOx (B) [37].

We currently established cultural system (amphycultural diffusion capsules) that allowed for conditions favorable for stem cell expansion in vitro. Many cell types and culture protocols and their combination with cytokines, growth factors, feeder layers can be implemented with ADC. Capsules are characterized by high perfusion rates that ensure that allow dilution of inhibitory autocrine factors and support long-term cell expansion. We have shown that ADC in vitro provides optimal cellular microenvironment that supports long term hematopoiesis (Bilko et al. 2005). 

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

The DynacelT culture system (Millipore corp Appendix 3) allows medium constituents to diffuse through a 0.6/xm membrane into a thin cell growth compartment particuarly suitable for growth of suspension cells. Several units can be coupled together to run from the same perfusing bottle. 

Due to the increased application of MoAbs in diagnostics and therapeutics, considerable effort has been made to develop technology for the large-scale production of MoAbs. Examples of the currently employed culture systems are hollow-fiber systems, suspensions, solid-phase cell immobilization, perfusion reactor, and encapsulation... 

New cell culture techniques, which may improve the applicability of renal epithelial cultures, are also required. Currenfly there exist two commercially available cell culture perfusion systems, which allow the continuous perfusion of culture media and optimized oxygenation [243]. These systems allow stable longterm culture of quiescent adherent cells [244]. Continuous medium perfusion furthermore may lead to the re-expression of lost functions in continuous cell hues and the maintenance of differentiated properties in primary cells. Recently our laboratory has demonstrated that LLC-PKj cells maintained in a newly developed perfusion system (EpiFlow ) changed from a glycolytic to a more oxidative phenotype [72]. Evidence is also available from experiments in our laboratory that this mode of cultivation helps to prolong the lifetime of primary cultures of proximal tubular cells. Combining perfusion culture with co-culture of a cell type that is an anatomical neighbour in vivo (e.g. epithelial with endothelial, interstitial or immune cells) may improve the state of differentiation of both partner cells and increase the complexity of autocrine and paracrine interaction [73]. 

Felder E, Jennings P, SeppIT, and PfallerW. LLC-PK(l) cells maintained In a new perfusion cell culture system exhibit an improved oxidative metabolism. Cell Physiol Biochem 12 153-162,2002. 

There are several commercially available products that use this principle. The device is commonly a plastic ring with a membrane attached, which can be placed into a multiwell plate or dish. Cells can be grown on one side of the membrane surface, on both sides of the membrane (transmembrane co-culture) or on both the membrane and plate well bottom for co-culture. This membrane device is commonly used in a static system (e.g. multiwell plate or culture dish) but it may also be mounted in an appropriate chamber for continuous perfusion culture. Examples of these products are the Falcon Cell Insert (Becton Dickinson Labware, NJ, USA), Millicell (MiUipore, MA, USA) and Transwell (Costar, MA, USA). 

Typical commercial cell culture systems include batch or fed-batch suspension reactors and perfused immobilized-cell reactors. However, the transient nature of batch culture causes difficulties in studying the effects of external stimuli on growth, metabolism and product formation. Due to metabolite concentration gradients, and the difficulty of obtaining representative cell samples, immobilized-cell reactors are also poorly suited for the analysis of cell growth and metabolism. As a result it is desirable to use well-defined model systems. Continuous-flow suspension reactors allow metabolic parameters to be measured at steady state, after cells have adapted to new (and possibly inhibitory) conditions. Perfusion reactors (with cells immobilized on suspended or stationary supports) extend these benefits to anchorage-dependent cells, and provide model systems for cell responses in vivo. However, while it is instructive to study the behaviour of cells under well-defined conditions, the results obtained must be verified in the culture system selected for commercial production. 

Continuous perfusion culture differs from chemostat in that all nutrients are kept in excess and cells are retained within the bioreactor. The most productive culture system gives t5 ical yields of 10 mg H day for 50-100 days (see section 5.9)... 

Bellco-Corbeil Culture System (Bellco). A roller bottle is packed with a cluster of small glass tubes (arranged in parallel and separated by silicone spacer rings). Models are available in lOOO-cm, 10 OOO-cm and 15 000-cm sizes. Medium is perfused through the vessel and the bottles rotate 360° in alternate directions to avoid tube twisting and the need for special caps. 

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