Seminar of Cell Culture Techniques

1. Seminar of Cell Culture Techniques Tapodi Antal Department of Biochemistry and Medicinal Chemistry, Faculty of Medicine, University of Pecs, Hungary

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

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

4. 1. PrimeryCultures • Tissue preparation 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. 2. Secondary cultures 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) • Hybridomas HeLa

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

7. Hybrid selection Metabolic pathways relevant to hybrid selection in medium containing hypoxanthine, aminopterin andthymidine (HAT medium). When the main synthetic pathways are blocked with the folic acid analogue aminopterin (*), 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.

8. Effect of HAT-medium Selection

9. 5-Amino Imidazole- 4-Carboxy Ribonucleotide * 5-Formido-Imidazole- 4-Carboxamine Ribo- nucleotide PRPP PP Hypoxanthine Inosine Monophosphate Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) Guanine Guanosine Monophosphate (GMP) PRPP PP Thymidine GDP dGDP Thymidine kinase RNA GTP dGTP dTMP dTDP d TTP DNA * Thymidylate Synthetase UDP dUTP dUMP dCTP dATP

10. Production of Polyclonal and Monoclonal antibodies

11. Neuro Hybryds • It works with adherent cells. • Cell fusion of HGPRT and TK-/-,no secreting neuoblastoma and neural cells. • Selection in HAT medium

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

13. Online Order of Cell Lines

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

15. 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.

16. 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.

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

18. Freezers

19. Centrifuges

20. Autoclaves

21. Vacuum Ovens

22. Microscopes

23. ELISA readers

24. FACS

25. 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)

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

27. Metabolic activity(MTT, viability assay) 4 • Seed the cells into 96-well plates at a starting density of 10 cell/well and culture overnight in humidified 5 % CO2 atmosphere at 37 °C. • Treat the cells modifying the their viability the following day. • Remove medium from the wells containing 0,5% water suluble mitocondrial 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 reder at 550 nm wavelength

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

29. 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

30. Apoptosis signalling

31. Activation and inhibition of Apoptosis

32. The Roll of mitochondria in apoptosis

33. Caspase Cascade

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

35. 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

36. DNA Laddering • To investigate the DNA fragmentation, the extracted DNA has to run on 1,5% agarose gel. • DNA fragments show ‘ladder-pattern’.

37. DNA Laddering

38. 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

39. 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

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

41. 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

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

44. Transfection I. pEGFP with NLS • Expression vector systems • pcDNA • pEGFP pEGFP without NLS

45. Transfection II. • RNAi • siRNA • stRNA or Dicer RNAi • shRNA Using vectors for RNAi analysis • siRNA cassette

46. Proposed mechanism for how siRNA works

47. stRNA or Dicer RNAi

48. Gene deletion (Demonstration)

49. Clinical Application of cultured Human Stem Cells • Not only can human embryonic stem cells be cultured in the laboratory. • But cells may be manipulated to produce cultures and Characteristics of particular tissue. • Possibility by damage and ageing (Parkinson’s disease, diabetes)

50. Epithelial Stem Cell identification and isolation • First methods involved in the separation of an epithelial cell type from other cells will be examined, followed by ways in which the proliferative capacity of such a cell type can be assessed. • Secondly, methods used for the maintenance of primery stem cells in culture and ways of caracterizing stem cells using immunocytochemistry will be described.