Bioreactors 2024 Student Copy PDF

Dr Yogambha Ramaswamy Senior Lecturer, School of Biomedical Engineering, University of Sydney  Topic-specific Learning Objectives Students should understand the importance of bioreactors and the parameters that can be modulated to control cell growth in bioreactors Student...

Dr Yogambha Ramaswamy Senior Lecturer, School of Biomedical Engineering, University of Sydney  Topic-specific Learning Objectives Students should understand the importance of bioreactors and the parameters that can be modulated to control cell growth in bioreactors Student should understand various design criteria that must be considered in bioreactors. Student should understand the significance of empirical equations in using bioreactors Student should understand the broad applications of bioreactors in the context of biomedical engineering https://pharsol.com/knowledge-hub/blog/bioreactor-history NJ Dominy, Proc Natl Acad Sci U S A. 2015  Factory for cells: Bioreactors Devices/apparatus that facilitates the growth of cells/microbes under controlled conditions JI Betts et.al, Microbial cell factories, 2006  Simplest bioreactor: Static cell culture (2D) Tissue culture plastics + Culture medium + incubator (Gas, Optimal temp) https://www.cbt.sci.edu.vn/single-post/2018/01/20/animal-cell-culture-a-historyAnimal cell culture history Food source for cells often come in the form of culture media Amino acids Building blocks for proteins Sugars Most often glucose Need for metabolism Other proteins/growth factors/ vitamins for cell growth and proliferation Media often supplemented with serum (e.g. fetal bovine serum 10v/v%) There are tons of different media for different purposes Culture media also supports local aqueous environment Inorganic salts (Na, K, Ca, etc) For osmotic ionic balance Trace elements (Zn, Cu etc) For specific protein interactions Bicarbonate buffer (pH 7.2-7.4) + Phenol red pH indicator Useful to quickly determine contamination Orange/red when normal Yellow when acidic Bright pink when basic  Incubator – controlling the environment Temperature CO2 for pH, not directly for cells Media often contain bicarbonate buffer We can control [CO2] to control the reaction as necessary Often [CO2] at 5 v/v% in air is sufficient to keep pH at physiological levels Tissue culture plastic material Gas permeable bags (Adherent cells) (Non-Adherent cells) Plasma-treated polystyrene No surface for cells to adhere Helps adsorption of protein Permeable to gases (i.e. O2 and CO2) layer Fibronectin, vitronectin Helps cells attach to surface  Dimensionality: From 2D to 3D and 4D 2D monolayer cell culture models 3D invivo cell microenvironment 4D biomimetic materials with time-modulated properties  Scaling up: Instrumented bioreactor Each of the labels play an important function in the basic running of a typical bioreactor JI Betts, Microbial Cell Factories 2006  Bioreactor design Cell culture parameter considerations: Type of cells, appropriate media, cell density, substrate consumption, pH, oxygen, temperature, GMP Bioreactor design: – Design of bioreactor and material types and properties used to make the reactors – Apply equations of continuity and diffusion to model performance – Even cellular distribution – Shear stress regulation – Perform experiments to verify model Bioreactor design considerations  Spinner flask bioreactors Impeller mixes culture media and cells Media can be filtered from cells if cells are bound to macroporous particles Increased cell density V. Drapal et.al J Biomed Mater Res. 2022  Rotating wall bioreactors Low shear force (and consequently low turbulence) high mass transfer of nutrients 3D cell culture applications http://www.cellon.lu/rccs-synthecon.html  Hollow fibre perfusion bioreactor Consistent and physiologic culture environment Automated cell harvesting and inoculation High yield High density cultures Reduced cell stress due to negligible shear Economical and sustainable D. Bartis et.al, Three dimensional tissue cultures and tissue engineering, 2011 https://www.kdbio.com/fibercell-hollow-fibre-technology/ https://www.fibercellsystems.com/advantage/ Bioreactors and clinical translation: Insulin production https://www.nlm.nih.gov/exhibition/fromdnatobeer/exhibition- interactive/recombinant-DNA/recombinant-dna-technology- alternative.html  Principles of Tissue Engineering Bone/cartilage Cardiac tissue Skin Ocular Nerve tissue Materials tissue Repair of damaged tissue Cells Biomolecules Bioreactors: Applications in biomedical engineering  Bioreactors design for tissue engineering Bone Tissue engineering Cells that can make bone (osteoblasts) or cells that can eventually turn into cells that can make bone (mesenchymal stem cells) Environment Hydrodynamic shear stress (i.e. flow) Slightly basic pH (~7.8-8.4) Ca2+ ions (and other trace ions Si, Zn, Mg, Sr) Mechanical loading  Increased expression of related proteins Cells or Growth factors (e.g for bone. ALP, OC, BSP, Col I) cell proliferation Substrate 3D, porous, degradable (e.g. PLGA, collagen, CaP)  Bioreactors design for bone tissue engineering Grow bone grafts to repair a large bone defect Role of bioreactor: Optimized design parameters Relevant materials Controlled channels to provide nutrients Controlled hydrodynamic shear Desired interstitial flow velocity Bhumirathna et.al, Sci Trans Med, 2016  Bioreactors for tendon tissue engineering Cells that reside in the tendon and maintain biological function (tenocytes) Cells that can differentiate into tenocytes (tendon stem cells, mesenchymal stem cells*, embryonic stem cells) Environment conventional media (ions?) Mechanical dynamic loading Growth factors such as Tendon related transforming growth proteins factor, growth (e.g. Col I, Col III, elastin, differentiation factor 5 TNC, TNM) (GDF5) cell proliferation Substrate Aligned fibres, degradable (e.g. silk, collagen)  Bioreactors for tendon tissue engineering NA Dyment et.al, J Ortho Res, 2020 GH Altmen et.al, Transactions of the ASME, 2002  Bioreactors for cardiac tissue engineering Cells that can make the cardiac muscle (cardiomyocytes) or cells that can eventually turn into cardiomyocytes (mesenchymal stem cells, iPSC) Electromagnetic field Environment Hydrodynamic shear stress (i.e. flow) Electrical stimulation Mechanical loading Heart related proteins (e.g. cardiac troponin, Growth factors or cells actinin, myosin, connexin) cell proliferation Substrate 3D, porous, degradable, compliant  Bioreactors for cardiac tissue engineering Multimodal modular bioreactor system and experimental design bioartificial cardiac tissue Effect of mechanical stretch Mechanical stimulation and direct real-time contraction force measurement Perfusion chamber for continues media exchange Increased expression of cardiomyocyte markers in the stimulated samples G Kensah et.al, Tissue Engg, 2021  Bioreactors for vascular grafts Bioreactor system enhanced cellular proliferation, alignment, and maturation of vascular smooth muscle cells Bioreactor preconditioning accelerates the formation of a significant muscular layer on decellularized scaffolds SK Yazdani et.al, Tissue Engg, 2009 Lab-on-a-chip: the new ‘it’ in bioreactors Miniaturized bioreactors Can become “organ-on-a-chip” if cells are incorporated Series of channels and chambers where reagents/cells/ biomolecules flow and interact Precise control of reagent flow in/out in pico- litre levels I.e. fluidic connections Multiple inputs/outputs + precise control of flow in each Also have electrical connections and integrated circuits Lab-on-a-chip: the new ‘it’ in bioreactors How do we get signals from cells and biomolecules? Stages of cell signalling Cell signalling in miniaturized factories  How is lab-on-a-chip made? Generally speaking: A tough and flexible polymeric material Should be either castable (on molds) or etchable Translucency often desired PDMS is a popular choice (also cheap and easy to handle) Engrave/print (either on the material itself or its mold) the required channels, chambers, plugs Add and connect the necessary electronics and pipes “Install” cells and appropriate fluid + solutes through the chip Current and potential applications for lab-on-a-chip devices Bioreactors: Significance of mathematical equations How fast does a cell multiply? - Cell growth rate µ = change in number of cells over change in time dN/dt = µ(S). N and N is Number of cells after a given time N = exp(µt) x No How fast do cells take up (or produce) metabolites? - change in mass over change in time at a particular instant (uptake rate U = dm/dt) If we know the number of cells, then Uptake rate per cell (“specific uptake rate”) = U/N = Us (empirical) Total cumulative uptake m by N number of cells over a period of time = integral of U.dt = integral of UsN.dt Now, because N is also a function of time, we can’t simply multiply Us by N and change in time to find cumulative consumption over a period of time Cumulative consumption m = [NoUs/µ]x[eµt – 1] Bioreactors: Significance of mathematical equations How fast do cells grow/multiply in a “limited supply” environment? Growth rate of cells is actually dependent on substrate supply There is also a substrate concentration where the growth rate is half the maximum growth rate (let’s say give it a constant K) If we know μm and K, we can estimate the growth rate μ for any substrate concentration S, using Monod’s equation/model Bioreactors: Significance of mathematical equations How fast do cells grow/multiply in a “limited supply” environment? μ = μm (S/S+K) Subsequently, we can control the growth rate of cells by controlling the substrate supply We can also stop growth (μ = 0) when there is no substrate Bioreactors: Significance of mathematical equations Typical growth curve of microbes/yeast, fixed ‘food’ Bioreactors: Significance of mathematical equations How fast do molecules diffuse from one place to another? Molecules move from high concentration to low concentration The rate of diffusion depends how “strong” this difference is (“i.e. a concentration gradient”) Each molecule has its own diffusion characteristics, defined by its diffusion coefficient Diffusive “flux” is the volume displacement per unit time (i.e. rate of volume movement J = -D(dc/dx)  Summary Bioreactors are special chambers whereby biological reactions can be carried out at a large scale These are designed with an ‘end-goal’ in mind Often the production of a specific product for medical or industrial use Controlling the environment, input and output of these chambers is critical in the function of these cells/organisms Bioreactor Environment Input Cell Output Substrate