Lecture 2 animal cell culture


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Lecture 2 animal cell culture

  1. 1. Animal Cell Culture Animal Cell Culture Applications • Production of recombinant proteins • Production of MAb - Hybridoma • Study of cell biology Prof. S.T. Yang Dept. Chemical & Biomolecular Eng. • Tissue engineering - artificial organs The Ohio State University • in vitro cell toxicity and drug screening Cell culture vs. Animal Animal Cell Bioprocessing • Problems for large-scale animal cell cultures • Advantages (compared to microorganism) – Consistency and reproducibility results – Minimum inoculum size: ~105/ml or 1-5x104/cm2 – understanding the effects of a particular – low cell proliferation rate: tg = 12 - 48 h compound on a specific cell type (e.g., liver cells) – low productivity of target products – avoid contaminants – high medium costs (serum) • Disadvantages – low resistance to toxic metabolites (ammonia, – cell characteristics can change (growth and inhibit growth or lactic acid, change in pH) biochemical) – higher sensibility to outer stimuli, higher – cell may need to adapt to nutrients susceptibility to shear stress (only thin & soft cell membrane of lipid bilayer) 1
  2. 2. Products from Mammalian Cell Cultures Cell Types • Group I: cells as end • Group II: cell-derived products – Growth factors products • Nerve growth factor, Epidermal growth factor • Epithelial (skin) – Artificial skin – Proteases (Urokinase, etc.) – Artificial organs – Hormones • Fibroblast (most widely used, • Beta-islet cells (pancreas) • Hepatocytes (liver) • Human growth hormone, Insulin, Calcitonin, Parathyroid hormone bone cartilage and fibrous matrix – Bone marrow – Monoclonal antibodies for body) – Lymphocytes • single type of antibody binds to specific compounds selectively. – Gene/cell theraphy • Used in diagnostic and therapeutic agents • Muscle – Vaccines • Polio, Measles, Mumps, Rubella, Yellow fever, Rabies, Influenza • Neuron – Recombinant Glycoproteins (Cytokines) • Interferons, block virus replication • Blood and lymph (cells in Annals New York • Blood clotting factors (VIII, IX) Academy of Sciences, 356 • Glycoprotein hormone, EPO suspension) • Plasminogen activators 20 μm Animal Cells • Anchorage-independent Animal cells - characteristics – Blood cells • Unicellular organisms (microbes) – proliferate until – Cancer cells stop signal is detected – Hybridoma cells - Produce MAb (monoclonal • Multicellular organisms – proliferation is tightly antibody) regulated – Some cells never divide – nerve, muscle cells • Anchorage-dependent – Some cells are always dividing – stem cells – Need solid surface to support growth – Some cells don’t normally divide unless stimulated – – Primary cells fibroblasts – wound recovery – Chinese hamster ovary (CHO) cells - Produce – Loss of proper control cancer glycosylated recombinant proteins 2
  3. 3. Cellular Responses to Environmental Stimuli The Cell Cycle Growth Arrest Apoptosis M Growing Cell Differentiation G1 Cancer G2 Apoptosis Differentiation Cancer S Continue growth Tissue Cultures Transformed Cell Lines • Initiated by placing a piece of tissue in a glass dish that contains serum • Cells migrates out of the tissue along the surface of the glass and proliferate. They proliferate only if they are firmly attached to and spread out over an • Lost normal growth control adhesive substrate (anchorage dependence) • Proliferation ceases when the dish is covered with a single layer of cells. Once • Can be selected when tissue culture cell lines are the cells form a continuous sheet they stop dividing (contact inhibition) allowed to grow repeatedly to high densities • Transferring the contact inhibited primary cells at lower densities to new dishes that contain fresh medium induces them to resume proliferation. After • Produced by allowing cells to grow out of tumors about 50 divisions in tissue culture proliferation slows and cells begin to die (senescence) • Produced by treating primary cells or • Some of the cells in the culture accumulate genetic change that allow them to nontransformed cell lines with chemical carcinogens escape senescence. As long as they are transferred to new dishes periodically, these cells can divide indefinitely. These homogeneous populations are called or cancer-inducing viruses tissue culture cell lines. They resemble primary cells in their growth control. • When injected into animals without functional (non-transformed) immune system, transformed cell lines cause tumors. 3
  4. 4. Requirements for cells used in cell culture Commonly used cell lines Property Examples Efficient expression of foreign genes Transcription and post -transcriptional process; Cell Line Cell Type Application Comment Protein synthesis; Secretion BHK (baby hamster kidney) Fibroblast Vaccine production Anchorage dependent, can be induced to suspension COS (African green monkey kidney) Fibroblast Transient expression of Contain a mutant of SV40 virus Post-translational modifications e.g. protein folding; glycosylation, phosphorylation recombinant genes Stability of transgene expression Genotypic (chromosomal stability) L (Mouse connective tissue) Fibroblast Tumour cell line Phenotypic (CpG methylation, chromosome structure alterations) 3T3 (Mouse connective tissue) Fibroblast Develop culture technique Vigorous growth in suspension; WI-38 (human embryonic lung) Fibroblast Human vaccine Finite life-span, “normal” cells Absence of adventitious agents Viruses; Mycoplasms Vero Fibroblast Human vaccine Established cell line capable of continuous growth but Cultivation requirements Serum Growth factors, stabilizing proteins with normal diploid charact. Fermentation properties Growth to high cell densities Resistance to shear forces CHO (chinese hamster ovary) Epithelial Genetic engineering Attach to surface if available, will grow in suspension HeLa (Human cervical carcinoma) Epithelial Fast growing human cancer cell isolated in the 1950's Aeration processes Production of toxic compounds (e.g. lactic acid) MPC-11 (Mouse myeloma) Lymphoblast immunoglobulin Derived from mouse tumor Production under fermentation High production at high cell density Namalwa (human lymphoma) Lymphoblast a-interferon Derived from burkitt's lymphoma patient conditions Reduced cell growth NB41A3 (Mouse Neuroblastoma) Neuronal Tumour cells have nerve cell characteristics including Protein requirements in medium response to nerve growth factor Cell Growth Fast growth at low cell density BW5147 (Murine thymus) Reduced growth at high cell density Mammalian Cell Biotechnology in Protein Production, H. Hauser, 1996 Cell Metabolism Culture Techniques • Culture containers - T-flask, multiwell, bioreactor • Culture media - BME, BMEM, DMEM, GMEM, Han’s F-12, CHO • CO2 incubator - 5-10%, pH 6.9-7.4, 37oC − CO2 + H 2O ↔ H 2CO3 ↔ H + + HCO3 basic acidic • Sterilization - Filtration • Liquid N2 storage 4
  5. 5. Media Supplements Extracellular matrix (ECM) proteins • Serum • Carbohydrate – proteins (immunoglobulins, albumin, Energy source transferin, fetuin, fibronectin) – growth factors glucose (4-5g/L), fructose – Insulin (glucose uptake) • Amino acid (0.1-0.2 mM) – steroids protein precursors – Trace minerals (Fe, Cu, Zn, Se) – Growth inhibitors glutamine (2-4mM) • Alternative to serum(serum free) • Salts (Buffer): K, Mg, Ca – cell type specific e.g. insulin, isotonic, osmolarity 300 mOsm/l transferrin, ethanolamine, sodium PBS, HEPES selenite • Vitamins (μM) and hormones • AntibioticsG (100 U/ml) inhibit G+ – Penicillin metabolic cofactors bacteria • Phenol red – streptomycin (50 mg/l) G+ & G- – amphotericin B (25mg/l) anti-fungi indicator, pH: 7.4 (red), – culture are easy to overgrow by bacteria 7.0 (orange), 6.5 (yellow) because of the difference in growth rates (typical doubling time: animal cell: 24 h, bacteria: 30min) Bioreactors for Culture in Bioreactor Systems Suspension or on Carriers for Animal Cell Cultures • Static flasks and roller bottles • Spinner flasks • Bioreactors (stirred, airlift, immobilized) Features for Wave Bioreactor: - Disposable Bioreactor Chamber. - Scalable to 500 liters. - Completely closed system. Operates without an incubator. - Easy to operate. 5
  6. 6. Microcarrier and Fibrous Support Matrix Porous microcarrier increases Non-woven fibers with large available surface areas for surface areas for cell anchorage-dependent cell attachment and growth growth 6
  7. 7. Considerations in Bioprocess Design • Media composition selection • DO (dissolved oxygen) • Accumulation of toxic metabolites • Shear stress to cell when oxygen or mixing is needed • Other factors: Temp, pH, osmotic pressure, etc. Optimization and control Minimizing Mechanical Stresses of culturing conditions • Precise pH control • Design agitation system • Precise DO control • Thermal environment must be uniform • Accurate temperature control • No disturbing the system during adding • Reliable monitoring of important cell culture parameters (cell density, conc. of glucose, nutrients and removing wastes lactate, ammonia) • Adequate oxygen delivery without • Dependable nutrient feed and harvest rate excessive foaming or shear damage controls • Automatic impeller acceleration control for 20- 225 rpm 7
  8. 8. Industrial Scale Cell Culture Typical Oxygen Transfer Coefficients • serum free media (chemical defined, easy separation) for Cell Growth in Various Systems • up to 20,000L • oxygen System KLa (h-1) • bubble: direct sparging • bubble-free: surface aeration or flow through cage Animal cells, 2 x 106 cells/ml (0.5 g/L) 1 -25 • perfusion culture to remove toxic metabolites Bacterial cells (10 - 20 g/L) 100 - 1000 • reduce physical stress Yeast cells (10 - 30 g/L) 100 - 1000 • pH, osmolality controlled by buffer, PBS, HEPES (pH~7) • development of anchorage-independent cell line Culturing Mode • Batch – CSTR or PFR – maximum cell density 106 cell/ml • depletion of an essential nutrient • accumulation of an inhibitor • complete cover of available growth surface (space) • Fed-Batch • Continuous – Chemostat vs. perfusion • Cell Immobilization – surface attachment vs. entrapment 8
  9. 9. Bioreactor as Cell Factory Hematopoiesis Hole, Cells Tissues Organs 1999;165:181-189. Kaufman et al, Proc. Natl. Acad. Sci. 2001;98:10716-10721 Tissue Engineering Embryonic Stem Cells Embryonic Stem Cells (Inner Cell Mass) Ectoderm Mesoderm Endoderm Heart Hematopoietic Epidermis Muscle Intestines stem cell (HSC) Nervous system Kidney Liver (Neural stem cell) Vascular system Pancreas Mesenchymal Lung stem cell Blood cells 1.Muscle cells are seeded on a tube of biodegradable polymer matrix. 2.It is placed in a bioreactor. Bone Cartilage Adipocyte Skeletal Muscle 3.Two month later, smooth muscle is generated. 4.Endothelial cells are added to line the tube. The blood vessel can be used for instance in heart surgery 9
  10. 10. Artificial Liver Bioreactor for Drug Screening Chorionic villous Intervillous space Circulating The liver-on-chip Water Capillary bioreactor from Syncytiotrophoblast Hepatometrix. Media Media Media Media Fibrous matrix Circulating Water • Human placenta trophoblast cells • Human colon cancer cells EGFP production under CMV promoter can be Microbioreactor for Gene/Cell Therapy used as S-phase marker for drug screening S- S-phase Three critical issues in implant devices – Tissue – Immune rejection – Device design GFP vs Cells 8.0E+05 A B C 7.0E+05 6.0E+05 y = 0.657x + 22068 A. Cells expressing GFP 5.0E+05 CHO GFP B. Cells stained with BrdU Fluorescence (CPS) 4.0E+05 C. Superimposed image 3.0E+05 2.0E+05 1.0E+05 0.0E+00 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 GDNF: A novel treatment for Parkinson’s Disease Parkinson’ Cell Num ber Glial cell line Derived Neurotrophic Factor Cell Number vs. GFP Fluorescence 10
  11. 11. Fermentation & Cell Culture Go Micro-bioreactor High Throughput • Functional genomics call for high throughput in cell cultivation • Reported working volumes: 250 μL - 2 mL • Miniaturized and automated pH, temperature and dissolved oxygen measurements and/or reproducible gas delivery • Combined with printed circuit board, integrated circuit sensors and electrochemical gas generation system Michel et al (2004), Biotechnol Bioeng, 85,376-381 BioProcessors Corp. Microscale cell culture analog - Microfluidic Bioreactor Array Animal on a chip HepG2/C3A in the liver chamber L2 cells in the 3T3-L1 in the fat lung chamber chamber Cell-based high throughput screening for drug discovery 11