Mammalian cell culture BME 262 S09

Transcription

1 Mammalian cell culture 1

2 Modern Uses of Mammalian Cell Culture Study of cell physiology and function Monoclonal antibody production (cell fusion) Clinical genetics Gene therapies Stem Cell therapies Tissue engineering 2

3 Tissue Engineering 3

4 Sources of cells for TE Autologous and Allogenic Bone Marrow (meshenchymal stem cells) Tissue Biopsy 4

5 Sources of cells for cell culture Tissue explant - primary cells Derivation from progenitor or stem cells (controlled differentiation) Continuous cell lines (man-made or tumor derived) - may retain basic features 5

6 Primary Tissue Explant 6

7 Blood as a Tool in Cell Culture Plasma is the cell free component of blood - it is dense in protein including the clotting factor fibrin The clotting components of plasma settle naturally under gravity. The remaining soup, is called serum it contains all the small, soluble proteins in the blood. Including the enzymes that cause plasma to clot The early scientist bathed tissue in serum w/embryo extract for nutrients and clotted plasma for affixing the tissue to the slide 7

8 Primary Tissue Explant Cells migrate out of tissue explant for obser vation 8

9 Mechanical Disaggregation Vigorous pipetting Pressurized passages through a series of (increasingly tighter) sieves Forcing tissue through syringe and needle Loose fitting dounce homogenizer Sonication 9

10 Chemical Disaggregation by trypsin Trypsin is a highly effective protease Crude and warm is more effective but more toxic to cells - pure and cold is more gentle Attacks any protein with an accessible Lys or Arg residue except when neighboring AA is proline- i.e. not specific 10

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13 Other Means of Chemical Disaggregation Collagenase is effective in many tissues Hyaluronidase attacks the backbone of glycosaminoglycans (ground substance in cartilage) Divalent cation chelators (EDTA, EGTA) gently disengage cadherins, and ocludin (tight junctions), and integrins from their substrates 13

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17 Cell Purification from Complex Harvests Differential Adhesion (different surfaces, different times) Differential Centrifugation (zonal separation: selecting cells based on size and shape; isopycnic - density based separation) Anitbody based methods - MACS, FACS, panning Selective Media Cloning 17

18 Cloning of primary cells Q. How do we nurture the growth of good cells in an environment whey they may grow more slowly than others? A. Generate a culture from a single cell or a small group of cells Problems: 1) will senescence happen before or soon after a clone is achieved 2) will phenotype drift undo your efforts anyway 3) what about the requirements for conditioned media 18

19 Cloning by Limiting Dilution Plate in multiwell dish < 1 cell/well check for colonies, trypsinize and transfer from individual wells 19

20 Cloning with cloning rings 20

21 Clonal Growth 21

22 Cell Culture Growth Adhesion-dependent Suspension 22

23 Culture growth kinetics 23

24 Subculture (passage) When cell density increases so that nearly all the surface of the plating vessels is covered, cells should be passaged Expands culture but also protects from nutrient depletion, and prevents contact inhibition Passage is typically done with trypsin/ EDTA 24

25 Trypsin/ EDTA 25

26 Subculture/Passage Before passage the cells are said to be 1, after this we count passage number The number of cell divisions matter - each division has the potential for genetic mutation - but the passage is easier to count A culture, nearly covering the surface of a plating vessel, is split 1:2 if the cells are divided over two more such vessels 26

27 Subculture/Passage Higher splits (1:6, 1:10) buy time between passages, but passage does not equal division number Diluted cultures may hurt because cells condition their media Propagating cells - if reasonably homogeneous in type - are classified as a line expected to have certain characteristics A line can be cryogenically frozen and revived 27

28 Simple culture growth model During expansion: N(t+dt) = N(t) +rn(t) dt N = the number of cells at time t r = per capita division rate r dt = fraction of cells dividing during interval dt This gives the classic population growth equation: dn/dt = rn 1 And the exponential solution: where N is the initial cell density rt N = N o e 28

29 Simple culture growth model Population growth cannot continue indefinitely Assume the division rate slows with increased cell density according to: 2 r(n) = r (1-N/k ) o where r is the rate of division when N = 0 and k is the carrying capacity of the culture r(n = k) = 0 29

30 Simple culture growth model this looks like... r ro 3 1 with 2 gives the logistic equation: dn/dt = r N(1-N/k) o k N 30

31 Simple culture growth model this looks like... Final solutions dn/dt k/2 k N N > k o N o > k/2 N o<< k 31

32 Culture Crisis 32

33 Differentiation/ Dedifferentiation Changes in phenotypes with successive passaging Dedifferentiation involves loss of specialized cell identity - ex. endothelial cells progressing backward to more fibroblast-like cells, markers - VE cadherin, ICAM, etc. Begin to disappear after passage 6,7 Differentiation is often desirable (ex. progenitor cells), inducible with culture conditions 33

34 Cell Senescence Primary cultures have a finite number of divisions Divisions rates in culture outpace those in the body divisions for mouse cells; cows; humans At senescence the bulk culture disintegrates and some cells may transform 34

35 Transformation 35

36 Characteristics of transformed cells They may: Grow in soft agar (suspension) Loss of contact inhibition Require less serum Some lines are immortal but do none of these (NIH 3T3 fibroblasts) 36

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38 A mitotic clock? 38

39 A mitotic clock? 39

40 How to live forever The lesson from senescence is that DNA damage accumulates over many divisions and eventually leads to non-proliferation signals Immortality therefore must must be changes that allow cells to ignore DNA damage 40

41 Exposed DNA ends look like damaged DNA 41

42 The molecular basis of immortality The answer appears to be mutations occur in Rb, p53 (or their regulators) These proteins inactivate cell proliferation signals in response to DNA damage (and other reasons) 42

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45 Making continuous cell lines (making good from cancer) DNA viruses naturally lead to cancers Human papillomavirus -> warts, and cervical cancer SV40- virus, a monkey virus used intentionally to transform cells 45

46 DNA virus produces proteins that block cell arrest, others that keep things under control Control may be lost if viral DNA incorporates into host genome 46

47 To block cell arrest, papillomavirus produces two proteins: E6 to sequester host cell p53 and E7 to sequester host cell Rb SV40 produces a single large protein ( large T-antigen ) that does both jobs 47

48 Culture Media Until 1950 s people used tissue extracts Attempts at defined media by analysis of body fluids and tissue A variety of minimal media were defined but required a small supplement of serum 48

49 Types of Media Minimal Media -> Balanced salt solutions (BSS) - Earle s, Hanks - maintain cell viability only temporarily (i.e. cells don t pop or shrivel up) Complete media (Essential) -> Minimal Media plus essentials for cell life but not stimulatory Serum supplemented complete media involves uncertainties In Defined media everything is known = control 49

50 General composition of complete media Osmolality ~ 260 mosm/kg <-> 320 mosm/kg - once established maintain at ± 10 mosm/kg Amino Acids - the body s 8 essential plus tryosine and cysteine - many cells require an abundance of glutamine (anaerobic respiration) Glucose - energy source probably also glutamine Dissolved Oxygen - less than standard atmospheric is required 50

51 General composition of complete media Salts: Na+, K+, Cl-, Mg++, Ca++, SO4, PO - and HCO 3 CO 2 : an elevated pressure is required (2-10%) when HCO - is present 3 ph typically and this is maintained by teamwork between by CO and 2 bicarbonate: H 0 + CO <-> H CO <-> H HCO <-> H NaHCO Na

52 Cell quiescence Nutrient depletion or contact inhibition causes (non-transformed) cells to withdraw from the cell cycle into G = quiescence 0 In a quiescent cell much protein synthesis is halted including the production of cell cycle regulators (the cyclins and Cdks) while slow degradation continues 52

53 General composition of complete media Organic supplements like pyruvate, and fatty acids in many complete media Growth factors and hormones come from serum: along with a whole bunch of other things... 53

54 What is in serum? proteins - fibronectin (cell matrix), immunoglobulins, albumin (lipid and IG carriers), lipids, alpha-2-macroglobulin (a trypsin inhibitor) cytokines hormones lipids vitamins and minerals (ZN, Cu) And many others growth factors 54

55 Disadvantages of Serum Lot-to-lot variability Poor definition Availability Contamination Mytogenic and cytostatic compounds 55

56 Cell adhesion requirements Solid tissue cells require adhesion and spreading to survive and proliferate (some transformed versions do not) Cells wet surfaces in a fashion similar to water - i.e. charges are good Glass works when clean - + SiOH <-> SiO + H 56

57 Cell adhesion requirements Plastic - polystryene (PS), polyvinylchloride (PVC), polytetrafluorethylnene (PTFE) etc. - all cheap, optically clear and hydrophobic Must be treated - ion discharged, gamma irradiation Cells will adhere and spread over either positively or negatively coated surfaces 57

58 Cell matrix synthesis most cells can polymerize fibronectin but fibroblasts are the professional synthesizers fibroblasts and some epithelial cells can synthesize collagens epithelial cells synthesize laminin 58

59 Advantages of serumfree media Selective media - the correct combination of growth factors can help us select for one cell type in a complex Regulation of proliferation and differentiation - amplify culture in one growth factor and switch to another to induce differentiation Enhanced production of biomolecules 59

60 Disadvantages of serumfree media Multiplicity of media Undesired selectivity - greater chance for a sublineage that is not characteristic of the original tissue Slower cell proliferation - typically true Availability 60

61 How to replace serum Begin with complete media w/ 10% serum Add cell specific growth factors PDGF, TGF-beta etc. see table 9.3 in Freshney and try to reduce serum Repeat to find required media supplements Time consuming - today start with good guesses - existing recipes and knowledge that some factors (ex. transferrin, and insulin) are required by all cells 61

62 Other factors may need to add fibronectin or precoat with collagen, laminin, polylysine trypsin inhibitors - and changed protocols for passaging 62

63 Feeder layers Some cells fail to grow at the low cell densities required for cloning Feeder layers are homogeneous or heterologous cells that cannot divide but continue to secrete factors that support cell growth 63

64 Characterization/ conformation of cell type confidence in cell type is clearly an issue because 1) complex origins and 2) genetic instability must keep records of origin and handling - provenance records of cell growth rates, and morphology to monitor culture gold standard: check for specific cell markers/ activity 64

65 Morphology bovine aortic endothelial cells fibroblasts astrocytes 65

66 Characterization/ Conformation The goal is a good model for your purposes Any functional assay important to your cell type or your study should be part of the provenance Unlikely to retain all in vivo characteristics Must follow markers with passage because of dedifferentiation 66

67 Contamination Bacteria, yeasts, fungi, mold, mycoplasma all appear as contaminants in tissue culture Sorces: non-sterile technique, improper maintenance of hoods and incubators, importation of infected cell lines Antibiotics in media fight bacteria Fungicide in incubator water fight fungi and molds 67

68 Some are obvious, some are not bacteria mycoplasma yeast 68

69 Tissue Culture in Tissue Engineering Constructs 69

70 Engineering Vasculature 70