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Cell Culture Techniques

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  1. Cell Culture Techniques Balazs Veres

  2. I. Cell Types II. Introduction to Cell Culture Lab III. Techniques Contents

  3. Primary cultures Continuous cultures Normal Immortalized Spontaneous Transformation Transfection Somatic Cell Fusion(Hybridomas, Hybrids) Cell lines Adherent Suspension Cells from ATCC and ETCC I. Cell Types

  4. PrimaryCulture • Tissue preparationdirectly from young animal, or isolation of cells from blood, intraperitoneal fluid, etc. • Tissue dissociation • Dissection then Homogenization with Knife or Blender • Enzymatic Digestion(collagenase, papain, trypsine)/cleaving of DNA of damaged cell with DNase • Dissociation of cells in medium and selection of organiccell types CO2 Incubator Knife Blender

  5. Secondary culture, extended culture • Secondary culture:From primary cultures, after 1 passage. • Extended culture (multipassage culture, cell strain):From primary cultures after several passages. Restricted lifespan (40-100 passages), limited growth potential.

  6. Continuous culture Cell line: genetically homogeneous, everlasting lifespan H9c2 • Normal cell lines • They were spontaneously immortalized.(e.g.: Cardio-myocytes from rat) • Immortalized • Transfected with some sort of oncogene; SV40 (Simian virus) Large T antigen (T IDBL) • Tumor cells (e.g.: Human cervix carcinomas: HeLa 1952) • Hybridomas HeLa

  7. Growth-curve of cultured cells

  8. Hybridomas • Cell fusion of HGPRT and TK-/- myeloma and B-cells from immunized animal • Selection of hybridomas in HAT (Hypoxanthine, Aminopterine and Thymidine) medium

  9. Hybrid selection Metabolic pathways relevant to hybrid selection in medium containing hypoxanthine, aminopterine andthymidine (HAT medium). When the main synthetic pathways are blocked with the folic acid analogue aminopterine (*), the cell must depend on the “salvage” enzymes HGPRT and TK (thymidine kinase). HGPRT (-) cells cannot grow in HAT medium unless they are fused with HGPRT (+) cells.

  10. Effect of HAT-medium Selection

  11. Ribose-5-P PRPP IMP 1) adeniloszukcinát xantilát (XMP) adenilát (AMP) guanilát (GMP) ADP GDP ATP GTP Purine and pirimidine nucleotide biosynthesis 2) HCO3- karbamil-P + Asp karbamil-aszpartát uridilát (UMP) UDP UTP CTP 3) PRPP adenin hipoxantin (uracil) guanin adenin-(uracil) foszforibozil- HGPRT transzferáz AMP IMP (UMP) PPi GMP 4) kinázok uridin UMP citidin CMP adenozin AMP guanozin GMP timidin TMP ATP ADP

  12. Production of Polyclonal and Monoclonal antibodies

  13. Cell lines • Adherent (WRL-68, HepG2, HeLa etc.) • Suspension (Jurkat) • Cells from ATCC and ETCC WRL-68 Jurkat HeLa HepG2

  14. Online Order of Cell Lines

  15. Cells in culture • Medium: salt, glucose, essencial amino acids, vitamines, buffer, phenol red indicator, serum, antibiotics • Environment: • Temperature: 37°C • High humidity • 5% CO2

  16. CO2-thermostats Airflow Solutions Dishes Freezers Liquid nitrogen Centrifuges Autoclave Vacuum ovens Cryotubes Microscopes ELISA-readers II. Introduction of Cell Culture Lab(Equipment)

  17. CO2 Incubators • Water Jacketed CO2 incubator • 3 Gas/CO2 Incubator with RH Control • Precise control of Oxygen levels combined with CO2, N2 and RH ensure accurate conditions for applications such as, hypoxic cell studies and cancer research.

  18. Laminar Flow Box • HEPA filter rated at 99.99% efficient for 0.3 micron particulates. The HEPA filtered air is then directed vertically across the work surface.

  19. Dishes • Dishes • Multiwell plates • Flasks • Flasks on slide

  20. Freezers

  21. Centrifuges

  22. Autoclaves

  23. Vacuum Ovens

  24. Microscopes Cell growth Contamination Cell counting Dye Transfection efficiency

  25. ELISA readers

  26. FACS

  27. II. Introduction of Cell Culture Lab(Culture) • Growth of the cells in adequate media with serum(FCS/FBS) and antibiotics and antimycotics (chemically defined serum-free media) • Environment: • Temperature: 37°C (34 °C, 41 °C) • Highhumidity • 5% CO2 • Split: Trypsin-EDTA • Count of Cells (Thrypan Blue)

  28. III. Techniques • Metabolic activity (MTT) • Detection of Apoptosis and Necrosis • Western blot from cells • Transfection • Gene deletions • Clinical Application of cultured Human Stem Cells • Flow Cytometric Methods • FISH-probes • DNA Array

  29. Metabolic activity(MTT, viability assay) 4 • Seed the cells into 96-well plates at a starting density of 10 cells/well and culture overnight in humidified 5 % CO2 atmosphere at 37 °C. • Treat the cells modifying their viability the following day. • Replace mediumto medium containing 0.5% water soluble mitochondrial dye, (3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT+). • Incubate 3 hours and solubilize the water insoluble blue formasan dye by 10 % SDS in 10mM HCl. • Determine the optical density by an ELISA reader at 550 nm wavelength.

  30. Effect of HO-3089 (Novel PARP-inhibitor) on WRL-68 in Oxidative Stress

  31. Detection of ROS • Count cells • Treatment (eg: H2O2, PARP inhibitor) • C-400 dye binds to ROS: fluorescence signal • ELISA reader

  32. Detection of Apoptosis and Necrosis • Activity of Caspase3 and Caspase 8 • Release of Cytochrome c and AIF • Fluorescence dyes • Hoechst 33342 • Annexin V • Propidium iodide • Rhodamine • DNA Laddering • Induction and protection • PARP

  33. Apoptosis signalling via the Fas receptor

  34. Activation and inhibition of Apoptosis

  35. The Role of mitochondria in apoptosis

  36. Caspase Cascade

  37. Ca2+ CsA FIGURE 3 AIF P - + - + - - + + AIF S EndoG P S EndoG P cyt-c S cyt-c Protein release from mitochondria

  38. DNA Laddering • To investigate the DNA fragmentation, the extracted DNA has to run on 1.5% agarose gel. • DNA fragments show ‘ladder-pattern’.

  39. DNA Laddering

  40. Fluorescent dyes I. • Hoechst 33342:blue • Selective nuclear dye • Chromatin condensation, fragmentation • Rhodamine 110: green • Bis-L-asparic acide amide (substrate by caspase 3): green • TMRE: polarization of mitochondria: red

  41. Fluorescent dyes II. • Propidium iodide: Late-stage apoptotic and necrotic cells: red • YO-PRO-1: Viable cell nuclei green • Annexin V: early-stage apoptotic cells: green

  42. Induction and Protection of Apoptosis • Induction: • Hydrogen peroxide • Etoposide • Death domains: TNF, FAS, TRAIL • BAD • Protection: • BCL-2 family • IAP • Inhibition of PARP • HSP27, 70, 90

  43. PARPpoly-(ADP-rybose)-polymerase • Nuclear enzyme • Structure of PARP • 1st activator of PARP are ssDNA-breaks • The role of PARP in necrosis and apoptosis or repair-mechanism • The role of poly(ADP-ribose) glycohydrolase (PARG)

  44. Ad Nic-R-P-P-R (NAD+) Ad Ad R-P-P-R-R-P-P-R Reaction catalyzed by PARP Ad Ad Ad N + -R-P-P-R-R-P-P-R-R-P-P-R-R-P-P-R PARP Glu CONH 2 Nic

  45. Gene transfer • Calcium phosphate: DNA-calcium phosphate precipitatum complex • Alkali complex: DNA-protein complex • Liposome: DNA in lipid membrane-bounded bodies • Electroporation • Magnetofection: magnetic particles-DNA complex • Transient (transfected DNA does not incorporate to the genomic DNA); stable transfection (selection; G418-neomycin). • Viral: Adeno, retro, lenti and adeno-associated viruses; „new” cell-line, incorporated DNA.

  46. Transfection pEGFP with NLS • Plasmide: double stranded circular DNA • 3-20 kb., hundreds of copies • Heavy metal, antibiotic resistency • MCS: Multiple Cloning Site • Gene of interest pEGFP without NLS

  47. RNA interferency • RNAi: RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementarynucleotide sequences. • DNA→ transcription→ mRNA→ translation→ protein • siRNA • Dicer RNA (RNase III.) • siRNA cassette

  48. Mechanism for how siRNA works

  49. Therapeutic approaches • Chromosoma methylation • Diabetes • Viral infection • HIV • Tumor therapy: • β-cathenine: increased cell growth (tumors) • Supressed β-cathenine: tumor inhibition • SIDE EFFECTS!!!

  50. Clinical Application of cultured Human Stem Cells • Not only human embryonic stem cells can be cultured in the laboratory. • But cells could be manipulated to produce cultures and characteristics of particular tissue. • Possibile solution for damage and ageing (Parkinson’s disease, diabetes)