In a fedbatch reactor, fresh media is continuous or sometimes periodically added but there is no continuous removal. The fermenter is emptied or partially emptied when reactor is full or fermentation is finished. It is possible to achieve high productivities due to the fact that controlling the flow rate of the feed entering the reactor can optimize the growth rate of the cells.
Bioreactors for animal cell culture Cell and tissue engineering Professor Claudia Lobalto da Silva Claudia Bartolucci Claudia Siverino 72240 72322 What is a bioreactor? • An apparatus for growing organisms (yeast, bacteria, or animal cells) under controlled conditions • Used in industrial processes to produce pharmaceuticals, vaccines, or antibodies • Also used to convert raw materials into useful byproducts such as in the bioconversion of corn into ethanol • Bioreactors supply a homogeneous (same throughout) environment by constantly stirring the contents • Bioreactors give the cells a controlled environment by ensuring the same temperature, pH, and oxygen levels Required properties of bioreactors • Simplicity of design • Large number of organisms per unit volume • Uniform distribution of micro-organisms • Simple and effective oxygen supply • Low energy requirement • Uniform distribution of energy • providing information about the formation of 3D tissue Types of bioreactors You can classify bioreactors based on three different parameters Sterility of the container : Sterile : used for the production of antibiotics or vitamins Not sterile : used for example in conventional fermentations such as in the production of beer, or more modern as the treatment of water Conditions imposed by the bioprocess Organisms growing in bioreactors may be: • Suspended • Immobilized A simple method, where cells are immobilized, is a Petri dish with agar gel Large scale immobilized cell bioreactors are: • moving media, also known as Moving Bed Biofilm Reactor (MBBR); • Packed bed; • Fibrous bed; • Membrane Methods of cultivation of micro-organisms BATCH culture A typical batch reactor consists of a tank with an agitator and integral heating/cooling system These vessels may vary in size from less than liter to more than 15,000 liters The advantages of the batch reactor lie with its versatility A single vessel can carry out a sequence of different operations without the need to break containment FED-BATCH culture In a fed-batch reactor, fresh media is continuous or sometimes periodically added but there is no continuous removal The fermenter is emptied or partially emptied when reactor is full or fermentation is finished It is possible to achieve high productivities due to the fact that controlling the flow rate of the feed entering the reactor can optimize the growth rate of the cells Continuous PERFUSION culture Perfusion bioreactors involve continuous culture, feeding, and withdrawal (harvesting) of spent media for long periods Perfusion systems accumulate no waste products Once established, bioprocessing with perfusion bioreactors can in many cases be simpler and experience fewer failures CONTINUOUS-FLOW (chemostat) culture A chemostat (from Chemical environment is static) is a bioreactor in which fresh medium is continuously added, while culture liquid is continuously removed to keep the culture volume constant By changing the rate with which medium is added to the bioreactor the growth rate of the microorganism can be easily controlled Others Bioreactors - Spinner flask Used in tissue ingeneering bioprocessing, in particular for cartilage grown in static medium, even if it is still too thin for clinical use - Rotating wall bioreactor The wall of the vessel rotates, providing an upward hydrodynamic drag force that balances with the downward gravitational force, resulting in the scaffold remaining suspended in the media As tissue grows in the bioreactor, the rotational speed must be increased - Rotary Perfusion bioreactors System allows a continuous feeding of the cell chamber from external media bottle; cells are retained in the cell chamber by molecular weight cutoff membrane - Compression Bioreactor It provides a controllable mechanical and physiological environment for simulating in vivo conditions in vitro This class of bioreactor is generally used in cartilage engineering and can be designed so that both static and dynamic loading can be applied Case study Rotating double-chamber bioreactor design The device allows seeding and culturing on both surfaces of a tubular matrix and includes rotatory movement of the scaffold around its longitudinal axis providing oxygenation to the 3D structure and improves mass transport between the culture media and the adhering cells Rotating double-chamber bioreactor design T= 37 °C Pp= 5% CO2 main components: • culture chamber, • motion • control units o polymeric culture chamber biologic sample and the medium o cylindrical scaffold holders working ends 10-25 mm in diameter o central portion expose the luminal surface of the matrix for seeding and culturing o chamber closed by a Petri-like cover permit oxygenation and sterility of the culture environment o system can be autoclaved reduction of contamination risks Bone marrow stem cell (BMSC) culture Mesenchymal BMSCs were isolated and cultured Isolation Proliferation Differentiation Chondrocytes The multi-lineage differentiation potential of BMSCs was assessed by examining their osteogenic, adipogenic and chondrogenic capacities Anthony Hollander (Bristol) group found a new and sure method to allow the differentiation of mesenchymal in chondrocytes Respiratory epithelial cells culture Respiratory epithelial cells were isolated from airways and trachea and cultured Biopsy Culture Differentiation Human tracheal decellularization Scaffold = support structure that allows the cell growth, in order to create a tissue with some specific biologic, functional and morphologic characteristics Decellularization of the biological scaffold: A cm tracheal segment was retrieved from a transplant donor, having removed all loose connective tissue After 25 cycles of decellularization, epithelial and glandular cells were completely removed from the tracheal matrix, while only a few chondrocytes were still visible Matrix inside the bioreactor during seeding Cell seeding was performed inside the bioreactor, avoiding construct manipulation and thus limiting the risk of cell construct contamination Bioreactor cultivation of the trachea construct Ethical permission was obtained from the Spanish Transplantation Authority and the Ethics Committee of the Hospital Clinic, Barcelona At the end of the culture period, the bioreactor rotation was turned off, both chambers were emptied and completely refilled with fresh media and the bioreactor was delivered to the operating room The graft was then cut to shape and implanted into the patient as a replacement for her left main bronchus Bioreactor cultivation of the trachea construct Re-cellularization Matrix after seeding Results At two months, graft biopsy showed vigorous angiogenesis and remodeling gradients of oxygen and nutrients exist in engineered tissue, due to the balance between transport and rates of cellular consumption Immunofluorescence histology confirmed the presence of angiogenesis and showed reconstitution of epithelium, the continued presence of viable chondrocytes, and a reappearance of the mucosal lymphoid cells that typically densely populate normal tracheal mucosa Advantages vs Disadvantages low level of automation of the system : an automatic medium conditioning and exchange system is desirable in order to minimize contamination risks and protect homeostasis, permitting intermittent, sterile evaluation of pH, nutrient or waste concentration a system controller for tissue-engineering process : monitoring the data provided by Sensors, will allow more control and, thereby, reproducibility of expansion, differentiation and migration of cells within the scaffold re-personalization of a donor trachea and successfully airway transplantation without the need of any immunosuppressive therapies no sign of rejection at one year post-implantation vigorous angiogenesis viable chondrocytes, and a layer of viable epithelial cells at two months surgery However, the epithelial layer, was microscopically discontinuous Future • improve epithelial cell coverage of the internal surface of the graft pre-operatively • application of flow stimuli to the internal compartment of the bioreactor will encourage appropriate alignment and function of cilia prior to implantation, thereby initiating appropriate clearance of mucus from the first post-operative day • refinements to permit scale-up and full clinical trials, as well to explore hypothetical ways of improving graft production, such as encouraging angiogenesis and orientated ciliary function References • “NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure” , Xiao-hua Lei1., Li-na Ning1., Yu-jing Cao1, Shuang Liu1,2, Shou-bing Zhang1,2, Zhi-fang Qiu1, Hui-min, Hu1,2, Hui-shan Zhang1,2, Shu Liu1,2, En-kui Duan1* • “A novel bioreactor to simulate urinary bladder mechanical properties and compliance for bladder functional tissue engineering”, WEI Xin, LI Dao-bing, XU Feng, WANG Yan, ZHU Yu-chun, LI Hong and WANG Kun-jie, Chin Med J 2011;124(4):568-573 • “A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: From concept to clinical trial” , M Adelaide Asnaghi a,*, Philipp Jungebluth b, Manuela T Raimondi c,d, Sally C Dickinson e, Louisa E.N Rees f, Tetsuhiko Go b, Tristan A Cogan f, Amanda Dodson f, Pier Paolo Parnigotto g, Anthony P Hollander e, Martin A Birchall h, Maria Teresa Conconi g, Paolo Macchiarini b, Sara Mantero Biomaterials 30 (2009) 5260–5269 “Clinical transplantation of a tissue-engineered airway”, Paolo Macchiarini, Philipp Jungebluth, Tetsuhiko Go, M Adelaide Asnaghi, Louisa E Rees, Tristan A Cogan, Amanda Dodson, Jaume Martorell, Silvia Bellini, Pier Paolo Parnigotto, Sally C Dickinson, Anthony P Hollander, Sara Mantero, Maria Teresa Conconi, Martin A Birchall, Lancet 2008; 372: 2023– 30 Obrigada pela sua atenção ... four parts of the culture chambers P1 P2 The assembled culture chambers Cell culture Human bladder smooth muscle cells (SMC) and urothelium cells (UC) were cultured under cell culture condition... suspension culture environment and enhance cell- cell interactions Several researches showed that RCCS contribute to cellular aggregation, intercellular adhesion and formation of 3D cell clumps... Introduction • Rotating cell culture system (RCCS) is a cell culture device made by NASA to simulate microgravity condition It is also a 3D dynamic culture system for cell growth • The rotational