In a perfusion cell culture process, productivitytiter can be influenced by differences in cell density or cellspecific perfusion rate cspr. Growing titers and viable cell densities, due to improved media and cell lines, and growing use of perfusion cell culture are making this downstream purification step more challenging. Piret1 1 biotechnology laboratory and department of chemical and bioresource engineering, university of british columbia, vancouver, bc, v6t 1z3 canada email. Eppendorf stainlesssteel spargers were used for cell retention.
Leelavatcharamas v 1997 growth, gamma interferon production and cell cycle in batch, continuous and perfusion culture of cho cells. Reducing variability in cellspecific productivity in. A cell density of over 30 million cellsml was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. An economic comparison of three cell culture techniques. Cell growth, viability, and titer highthroughput smallscale model in ambr 15 figure 3. Optimization of a mammalian cell perfusion culture in productivity and product quality.
The cell densities achieved in perfusion culture 30100 x 10 6 cellsml are typically higher than for fedbatch modes 525 x 10 6 cellsml. Pdf a practical guide to microfluidic perfusion culture. Pdf perfusion mammalian cell culture for recombinant protein. Furthermore, as culture productivity increased, and although it greatly benefited fedbatch processes, perfusion promised even greater output from a continuous culture. Two approaches for cell retention in perfusion culture systems.
While the culture media continuously perfuses through the system, the 3d aggregates of cells and scaffolds are retained in the cell culture chamber by a porous perfusion core. However, no similar activities related to perfusion cell culture in deepwell plates are known. Very high cell density perfusion of cho cells anchored. Flow perfusion effects on threedimensional culture and. Mammalian cell retention devices for stirred perfusion. Use of the centritech lab centrifuge for perfusion culture of hybridoma cells in proteinfree medium. Perfusion culture of t lymphocytes in the wave bioreactor. In vivomimicking microfluidic perfusion culture of. A suspensionbased perfusion culture with a working volume of 1. Culture of 3d cell aggregates in perfusion in a mini.
Mikosa,c,1 adepartment of chemical and biomolecular engineering, rice university, houston, tx 77005. A major challenge for perfusion reactor design and operation is. Onestep seed culture expansion from one vial of highdensity cell bank to 2000 l production bioreactor this application note describes how perfusion cell culturing can be used to reduce processing time, simplify operations, and maximize equipment utilization in seed culture expansion processes. Continuous perfusion microfluidic cell culture array for highthroughput cell based assays paul j.
A practical guide to microfluidic perfusion culture of. Process design and development of a mammalian cell perfusion. Fourteen days of perfusion cell culture were conducted with a working volume. These very high throughput techniques are very powerful for screening experiments either for cell line selection or media and feed development. Development of a microfluidic perfusion 3d cell culture system. In the study, development and implementation of a perfusionbased high cell density banking process, published in biotechnology. The tubular perfusion bioreactor system was used for perfusion culture, as described previously. Continuous perfusion microfluidic cell culture array for. How a high density cell culture medium can positively impact your upstream productivity and economics. The bioblu 5p vessel was inoculated at an initial cell density of 0. Using awardwinning alternating tangential flow atf technology, xcell atf cell retention devices deliver high cell concentration and process intensification during cell culture. Limited use of centritech lab ii centrifuge in perfusion. United states of america, animal cells have been immobilized on a ceramic matrix and medium is perfused through this matrix.
Continuous perfusion microfluidic cell culture array for highthroughput cellbased assays paul j. This perfusion process was then successfully scaled up to a 100 l single. Combined with an intensified perfusion bioreactor, continuous processing can greatly reduce cost, space requirements, and handling steps, while improving production efficiency. The reasons for using perfusion culture vary for these molecules. Easypperfusion for anchoragedependent cell culture in. Minusheet perfusion culture system is used for advanced cell culture experiments in combination with adherent cells and to generate specialized tissues in combination with selected biomaterials, special tissue carriers and compatible perfusion culture containers the technical development of the minusheet perfusion culture system was driven by the idea to create under in vitro conditions an. A practical guide to microfluidic perfusion culture of adherent mammalian cells. Recombinant chinese hamster ovary cho cells producing igg monoclonal antibody were cultivated in a novel perfusion culture system celltank, integrating. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. Our perfusion bioreactor systems allow for continuous feeding of the cell culture chamber from an external media bottle. Optimisation of cell culture parameters is an essential component of process development for the production of active biopharmaceuticals, vaccines, and monoclonal antibodies mabs. Because an hfpb cell culture on the ecs side of the fibers exists at concentrations. Process design and development of a mammalian cell.
In this study, we developed a stepwise procedure to use shake tubes st in combination with benchtop br bioreactors for the design of a mammalian cell perfusion culture at high productivity 23 pgcell. Perfusion cell culture using the 50 l hyperforma s. Within the spectrum of current applications for cell culture technologies, efficient largescale mammalian cell production processes are typically carried out in stirred fedbatch or perfusion bioreactors. Optimization and evaluation of perfusion medium in high cell density mammalian cell culture systems. Progress, the cell culture development group at biogen idec shared their expertise in using ge healthcares wave bioreactor to create a perfusionbased. System for culture of 3d cell aggregates in perfusion, the researchers inoculated four dasbox mini bioreactors with the human h157 cancer cell line and cultivated the cells as 3d aggregates in perfusion mode for up to 15 days. From the early 90s to today, different commercial molecules such as recombinant follicle stimulating hormone gonalf, interferon beta1a rebif and factorviii kogenatefs were launched using perfusion processes pollock et al. Continuous collection of stem cells from a human placenta perfusion coculture john j. Pdf high cell density perfusion culture has a maintained. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Perfusion mammalian cell culture for recombinant protein. Pdf use of the centritech lab centrifuge for perfusion.
The chambers are interchangeable and specially designed to be compatible with 3d inserttm scaffolds of varying sizes ranging from 96well to 6well. Continuous collection of stem cells from a human placenta. Cell growth, viability, and titer benchscale bioreactors with minibiosep acoustic perfusion system figure 3. The specific aspects of each individual process that can be considered when determining the method of choice are presented. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and microassays independently. High titers and perfusion processes challenge cell. Pdf the enormous cost and time required for launching of a new drug on the market request a redesign of testing approaches and validation. Bioreactor systems facilitate threedimensional 3d cell culture by coping with limitations of static cultivation techniques. Microfluidic cell retention device for perfusion of.
Thus it is important to measure variability in productivity performance as cellspecific productivity qp, which incorporates all three factors. Results of perfusion culture of vero cells on cytodex 3 microcarriers in spinfilter impeller vessel. The working volume of the bioreactor was 3 l, and the in. The perfusion run of vero cells on cytodex 3 microcarriers was performed for 10 days. Scieprogetty images cell harvesting is a crucial step in biopharmaceutical manufacturing that can have significant impacts on product quality and the design of remaining downstream processes. Cell retention devices for suspendedcell perfusion cultures. An understanding of the constraints imposed at each stage of performing microfluidic perfusion culture e.
Two approaches for cell retention in perfusion culture systems zhaowei wang abstract a lack of efficient, economical, and reliable cell retention devices has limited the application of perfusion culture systems in the biopharmaceutical industry. Perfusion bioreactor culture of bone marrow stromal cells. Cells were sampled daily through the needleless sample port. Abstract the development of mammalian cell perfusion cultures is still laborious. Modelation of growth kinetics of mammalian cells in. The media also flows through an external silicone oxygenator via a. The small 10 cm high cell culture hydrocyclone has a maximum processspecific perfusion performance of 450720 liters of cell culture harvest per day while operating at a flow rate of 0. A perfusion cell culture process involves the constant feeding of fresh media and removal of spent media and product while retaining high numbers of viable cells figure 1.
High cell density perfusion culture has a maintained exoproteome and metabolome article pdf available in biotechnology journal 10. Leading perfusion device for mab and rprotein production. Use of cell cycle analysis to characterise growth and. Temperature was maintained at 37 c or shifted to 33 c with a heat jacket. To allow for the investigation of proper cultivation conditions and the reproducible generation of tissueengineered grafts, a bioreactor system, which comprises the control of crucial cultivation parameters in independentoperating parallel bioreactors, is beneficial. Perfusion is not exactly new for cell culture croughan et al. We characterized the key performance index of the cell retention device for longterm perfusion culture, in terms of cell retention efficiency, biocompatibility. Cellretention systems add a level of complexity to the process. Interest in hollowfiber perfusion bioreactors is growing.
Removing spent media while keeping cells in culture can be done using alternating tangentialflow atf and standard tangentialflow filtration tff. A streamlined singleuse solution for an intensified high. Most of these molecules are labile and their indication range is expressed in. Shake flasks were used since the beginning of suspended cell culture. Pdf the manufacturing of recombinant protein is traditionally divided in two main steps. In addition, you will find some recommendations for using human umbilical vein endothelial. Flow perfusion effects on threedimensional culture and drug sensitivity of ewing sarcoma marco santoroa,b, salaheddine lamhamedicherradib, brian a. Viable cell counts were performed using the standard trypan blue. Native pancreatic islets interact with neighboring cells by establishing threedimensional 3d structures, and are surrounded by perfusion at an interstitial flow level. Principles and approach to developing mammalian cell. H ighdensity 50 l perfusion cell culture using the xcell.
Product sieving challenges in tff perfusion cell culture. Optimization and evaluation of perfusion medium in high. Furthermore, modern highend cell culture processes aim to maintain the cells in a defined metabolic state in order to ensure stable product quality through. A brief history of perfusion biomanufacturing bioprocess. Br bioreactors for the design of a mammalian cell perfusion culture at. Onestep seed culture expansion from one vial of high. Microfluidic cell retention device for perfusion of mammalian. Todays renewed interest in perfusion culture is due to an increased awareness of its advantages, some general improvement in equipment reliability, and a broadening of operational skills in the biomanufacturing. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. A perfusion bioreactor system for cell seeding and oxygen. Mammalian cell retention devices for stirred perfusion bioreactors steven m. Pdf the optimization of bioprocesses for biopharmaceutical manufacturing by chinese hamster ovary cho cells can be a challenging. Labscale perfusion cultures 350 ml were used to demonstrate the.
The 3d perfusion bioreactor consists of multiple independent, autoclavable polycarbonate chambers. Schematic setup of an intensified cell culture using a perfusion cellbag bioreactor. Perfusion culture an overview sciencedirect topics. The basal medium for perfusion cultures was a laboratory formulation of serumfree medium, sfmmdm.
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