P01_Primary cell cultures : Isolation – Characterization – Propagation - Application

1. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application Cell culture techniques: Understanding how our cells function (Research) Looking for active compounds (Screening) Production of Biopharmaceuticals (Production)

2. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application • At the beginning: chicken embryo tissue culture • HeLa and Eagle: 50 years with permanent cell lines • Epigenetic factors: indispensable for cell differentiation • Primary cell cultures: how to get them from tissue • The social behavior of cells: Co-Cultivation and ECM • Movement: Migration and Invasion • From 2D to 3D: Emulation of the tissue • Gene expression and function: functional genomics • The future of cell culture: Good Cell Culture Practice

3. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application At the beginning: chicken embryo tissue culture • Benefits • Maintenance of histological structures • All cells present • cell-cell-interactions • cells remain attached to ECM • and disadvantages • Microscopic monitoring impossible • Uncontrolled environment • no quantification • heterogeneous cell population • of tissue cultures

4. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application HeLa and Eagle: 50 years with permanent cell lines Some major benefits of these technology • Dissociation of the tissue provides single cell suspensions • Cells grow adherent (most) on chemically modified surfaces • well defined cell culture medium inhibited bacterial growth • Actually more than 3.000 cell lines available • Freezing and storage in nitrogen • High throughput screening and bio production • cell culture technique established as a routine lab-work • reduction of experiments on animals • used in HTS and vaccine production

5. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application Starting modern flat cell culture systems 9. Februar 1951 George Otto Gey (1899-1970) Otto Gey and his wife 1950s they started the Tissue Culture Laboratory *Henrietta Lackst

6. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application HeLa and Eagle: 50 years with permanent cell lines The major drawbacks of these technology: • some (most?) of the cell lines proved to be genetically unstable • cross-contamination as a result of „bad handling“ • contamination with mycoplasms • loss of differentiation • cell culture media not really standardized • replacement of experiments using animals limited • “in vitro” fails to reflect “in vivo” • GCCP may diminish some of these limitations ECM : Extracellular macromolecules as matrix are necessary

7. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application Primary cell cultures: how to get them from tissue • Removal of organs or samples from surgery • sampling of tumour specimens • crude preparation of regions of interest • dissociation using enzymes • separation of cells according to surfaces molecules (Immunosorting) • cultivating on coated surfaces • cultivation in specific serum-free medium • selection of cells of „interest“ • controlling gene expression profile CAVE: Usually 2-6 subcultures are useable Actually a lot of primary cell cultures in low passages are available

8. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application Primary cell cultures: how to get them from tissue Some major problems and limitations using primary cell cultures: 1-Selection of cells of interest within a heterogeneous population 2-Damaging of the cells during enzymatic treatment 3-Loss of important cell populations 4-Rapid change in gene expression profile within hours 5-Limited material available 6-Ethical limitations 7-Time consuming and expensive 8-Standardization and quality control complex Only adult stem cells will start to divide and grow in vitro!

9. P01_Primary cell cultures: Isolation – Characterization – Propagation - Application P01.1: Primary Cell Cultures vs. permanent cell lines A classification • Primary Cell Cultures: • Cells grow out of the tissue tissue/organ • Cells have a limited lifespan / passaging potential • Cells are representative for the tissue of origin • Cells are differentiated or cells can be differentiated • Cell lines (permanent): • Spontaneous or immortalized • permanent – really? • Genetically modified • Genetically stable?

10. P01: Primary Cell Culture • P01.1 Cell lines • It all started with HeLa - but it was a happy accident! • largely from human tumor tissue • genetically heterogeneous • usually represent a cellular subtype • loss of differentiation a priori or over passaging • stability problems in long term cultivation • precise correlation in the absence of appropriate controls is often poorly • easy to augment  almost indefinite available • nearly no Batch-to-Batch-variations when using SOPs • immortalization not representative for normal tissue • therefore, HTS-compatible

11. P01: Primary Cell Culture • P01.1Primary Cell Culture • It all started with Roux (1885): chicken embryos in glass bottles! • And today? Why “back to the roots” of cell culture technologies? • regenerative medicine (SKIN) see PPP 12.09.2013 • replacement of animal experiments (ECVAM) • stem cell research (myocardium) • Searching for therapeutic targets (tumor stem cell) • molecular medicine • in vivo-like models in parallel to animal experiments • … and much more…

12. P01: Primary Cell Culture P01.1 Primary Cell Cultures – how it all began Skin Grafting for Full-Thickness Burn InjuryMaribeth Wooldridge, Judith A. SurveyerNov., 1980 The Use of "Artificial Skin" for Burns T Jaksic, and ­ J F Burke Vol. 38: 107-117 (Volume publication date February 1987)

13. P01: Primary Cell Culture • P01.1 Primary Cell Culture • targeted isolation of single, differentiated cell subpopulations • propagation in vitro • maintenance of function and differentiation • combination with other cells (co-culture, TE) • autograft transfer • immortalization • storage

14. P01: Primary Cell Culture • P01.1 Primary Cell Culture • Some important (even ethical) aspects: • Where does the tissue come from? animal / human • healthy tissue or malignant tumor? • „Informed consent“ from donator/patient • positive vote of the der ethics commission • access to tissue banks (institutions/private/commercial) • purchase from commercial suppliers of Primary Cell Cultures • control of the der Batch-to-Batch variability

15. P01: Primary Cell Culture • P01.1 Primary Cell Culture • Some important (even ethical) aspects : • tissue from donators rather unproblematic • agreement and hygienic controls (HIV) • some tissue are always problematic: hepatocytes! • variability can be very large: age + gender • fetuses: the material from which dreams/nightmares are made • do stem cells really solve the problem?

16. P01: Primary Cell Culture • P01.1 Primary Cell Culture • Some important aspects: non-critical material • donation skin after plastic surgery (breast, bottom, belly) • foreskin of boys (post partem until a few years) • endothelium from umbilical cord • adipocytes from donation tissue • hematopoietic cells from blood, bone marrow, cord blood • urothelial cells from bladder irrigations • alveolar cells from lung grafts

17. P01: Primary Cell Culture • P01.1 Primary Cell Culture • Some important aspects: critical material • hepatocytes from liver tissue • intestinal epithelium • myocardial tissue • differentiated tissue of internal organs • nerve tissue • tumor biopsies – smaller and smaller….Biobanking can help to propagate Fetal tissue (abortion material) after vote of the ethics commission Adult / neonatal stem cells?

18. P01: Primary Cell Culture • P01.1 Primary Cell Culture • Crucial question: Why? AIM and GOAL • fundamental research: TUMOR BIOLOGY • target screening • drug control • regenerative medicine • commercial/non-commercial difficult to distinguish • genetic reprogramming could solve the quandary!

19. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Before you start: Biopsy, transport, storage: SOP • What tissue type? • Accessibility? • Who characterizes/carries out the biopsy? • Environment (contamination)? • Wherein should the tissue be stored/removed? • Tissue stability (autolysis)? • Organization of the transport? • How long can the tissue temporarily be stored?

20. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Biopsy, transport, storage: SOP • What tissue type? • the better characterized, the more easily the Primary Cell Culture! • the more complicated the composition, the more difficult the selection • the fresher the tissue, the more successful the Primary Cell Culture

21. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Biopsy, transport, storage: SOP • Accessibility? • Who characterizes/carries out the biopsy? • good knowledge of the objective is advantageous • short routes • pathologist/surgeon/veterinarian as contact person • what are the quantities required? • the better the primary isolation, the more successful the cultivation!

22. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Biopsy, transport, storage: SOP • Environment (contamination)? • Wherein should the tissue be stored/removed? • sterile environment is required! • maybe problems in the animal facility • even set up a sterile laboratory in the pathology! • surgical sterility is not identical to cell culture sterility • sterility controls obligatory • add 2-4 x antibiotics to the medium for transport • no fungicide

23. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Biopsy, transport, storage: SOP • Tissue stability (autolysis)? • controls required (e.g. RNA-stability) • keep cool and dry • no general predication possible • fibroblasts cultures can be established until 48 h post mortem • endothelial cells from umbilical cord can be established until 96 h after birth • standardization required (see transport)

24. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Biopsy, transport, storage: SOP • Organization of the transport? • docket required • use suitable transport vessels! • log temperature and time • the faster the better: 30 min after biopsy on the benchtop! • responsibility, preparation of the laboratory • overnight-express possible • observe hygiene regulations!

25. P01: Primary Cell Culture • P01.2 Primary Cell Culture • Biopsy, transport, storage: SOP • How long can the tissue temporarily be stored in our lab? • according to tissue type 30 min up to 4 days • watch the pH-stability of the medium (+HEPES) • full-medium required • maybe pre-preparation of the tissue (-fat tissue, -erythrocytes etc.) • photo-documentation • take sterility controls • SOP for everything!

26. P01: Primary Cell Culture Tumor Umbilical vein Lung cancer

27. P01: Primary Cell Culture • P01.3 Strategy for the preparation of a single cell suspension: • Cells are united in the tissue • cell-matrix-connection through hydrophilic interactions (integrine) • cell-cell-interaction through homophile interactions (cadherine) • mechanical fragmentation with scalpel • mechanical fragmentation with scissors • mechanical fragmentation through shear force (soft tissue) • +/- enzymatic cell disruption (perfusion)

28. P01: Primary Cell Culture Cell-CellandCell-Matrix-Interactions

29. P01: Primary Cell Culture Cell Isolation out of a tissue: Whereismycellofinterest?

30. P01: Primary Cell Culture Preparation Enzymatic digestion Preparation of single cell suspension Cell adherence

31. P01: Primary Cell Culture • P01.3: Strategy for the preparation of a single cell suspension • mechanical fragmentation with scalpel • Previously, cut tissue samples into small pieces with (if necessary) • optical control with magnifying glass • removing of macroscopic/microscopic different tissue • cut small pieces while avoiding severe trauma • tissue samples may not dry out • use HEPES-buffered medium (pH-stability) • compromise between small samples (low diffusion) and trauma • cutting tissue with scissors leads to the crushing of the tissue

32. P01: Primary Cell Culture • P01.3: Strategy for the preparation of a single cell suspension: • Enzymatic dissociation of cells from the tissue (1) • no highly specific enzymes known • trypsin is an unspecific protease, effective but toxic • collagenases specifically digest some matrix proteins • dispase is dissolving cell-matrix-interactions, but not the homophilc cell-cell- adherence • elastase-pre-treatment • variability in concentration, time and temperature • pre-treatment with cold solutions could reduce the toxic actions • total digestion of matrix proteins generally not recommended • enyzmes should be tested before (Lot-variability)

33. P01: Primary Cell Culture Enzymes usedfortissuedigestion: Pronases: Neutral metalloprotetasederivedfromStreptomycesgriseus. P. Cleaves predominantly peptid-bonds neighboredto hydrophobe aminoacids Elastases: Cleaves peptid-bonds on thecarboy-end ofsamllhydrophobicaalike e.g. Glycin, Alanin, Valin....efficacyratherunspecificwhichcausesdamageofthecells. Collagenases: Collagenasesareendopeptidasesthatdigest native collagen in thetriplehelixregion. Collagensarethemajorfibrouscomponentofanimalextracellularconnectivetissue. Trypsin: Summary of a classofendopeptidaseswhichcleaveproteinsatdefindedsequences e.g. nearserine: Unspecificwhichmarkedunwantedsideeffectsandcelldamage!

34. P01: Primary Cell Culture • P01.3: Strategy for the preparation of a single cell suspension: • Enzymatic dissociation of cells from the tissue(2) • resuspend suspension with pipettes (big notch) after digestion • staining cell suspension with cell-strainer of different pore size • rinse sieve with medium (inactivation of the enzyme solution) • microscopic control of the yield in the flow-through • treat the remaining tissue samples with strainer • alternatively, rinse and use as a particle culture • count and seed cells, probably further purification by MACS/FACS

35. P01: Primary Cell Culture • P01.3: Strategy for the preparation of a single cell suspension: • Enzymatic dissociation of cells from the tissue(3) • PERFUSION • perfusion for the preparation of hepatocytes, endothelial cells, SMC`s • enzymes according to regulations, low concentration and short incubation time • note diffusion gradient (hepatocytes) • reduced temperature when treating vessels • cell straining generally not necessary • mechanical support can be helpful (massage) • generally good yield and pure cell population

36. P01: Primary Cell Culture • P01.3: Strategy for the preparation of a single cell suspension: • Enzymatic dissociation of cells from the tissue(4) • Examples • Perfusion • endothelial cells from umbilical cord • SMC`s from umbilical cord • Mechanical fragmentation and diffusion • keratinocytes from skin • fibroblasts from skin

37. P01: Primary Cell Culture P01.3: Strategy for the preparation of a single cell suspension: Enzymatic dissociation of cells from the tissue(5) Examples – overview of the enzymes see additional supplement

38. P01: Primary Cell Culture P01.4: Strategies for Selection - a challenging work • Ambition: Enrichment of the desired cell population • macroscopic/microscopic fractionation • different speed for detachment of the cells (endothelial cells/SMC) • different stability concerning the used enzymes • different speed for adherence (macrophages) • different preference of the surface (coating) • different density (Ficoll, centrifugal elutriation) • different surface marker (FACS, MACS) • different cell culture media • isolation of single clones (“cloning rings”) • isolation of single clones (micro dissection) • selective trypsinization • other selection procedures

39. P01: Primary Cell Culture P01.4: Strategies for Selection • Ambition: Enrichment of the desired cell population • macroscopic/microscopic fractionation • The mechanical pretreatment is usually the first and the most important • step in the selection

40. P01: Primary Cell Culture P01.4: Strategies for Selection • Ambition : Enrichment of the desired cell population • different speed for detachment of the cells (endothelial cells/SMC) • Preferred selection through perfusion. The penetration of the enzyme can be controlled by variation of the incubation time. Optimal time/concentration • temperature-combination has to be determined. • Good and pure separation is possible, if the different cell types are stratified • (skin, vessels)

41. P01: Primary Cell Culture P01.4: Strategies for Selection • Ambition: Enrichment of the desired cell population • different sensitivity concerning the used enzymes • The combination of different enzymes may preferably promote the selective isolation of single cell types. • In fact, this principle is an enrichment, the contamination with undesired cell • types is likely. • Preferably for heterogeneous tissue (lung, tumors).

42. P01: Primary Cell Culture P01.4: Strategies for Selection • Ambition: Enrichment of the desired cell population • different speed for adherence • Successful strategy, especially in combination with coating • Combination with antibodies also successfully tested (panning) • Can be used as positive or negative selection as well • Is also described for the removal of macrophages from lung tissue • Time must be determined empirically • Selection markers must be present

43. P01: Primary Cell Culture P01.4: Strategies for Selection • Ambition: Enrichment of the desired cell population • different preference of the surface (coating) • Surface is defined in terms of charge and matrix • Cells have got a different Zeta-potential • Cells differ in their integrin configuration • No all-or-nothing principle • combination with time factor to favor

44. P01: Primary Cell Culture - Coating HeLa and Eagle: 50 years with permanent cell lines The major drawbacks of these technology: • some (most?) of the cell lines proved to be genetically unstable • cross-contamination as a result of „bad handling“ • contamination with mycoplasms • loss of differentiation • cell culture media not really standardized • replacement of experiments using animals limited • “in vitro” fails to reflect “in vivo” • GCCP may diminish some of these limitations ECM : Extracellular macromolecules as matrix are necessary

45. P01: Primary Cell Culture Signaltransduction from ECM to the genome by integrins Actin Actin Actin

46. P01: Primary Cell Culture ECM as Epigenetic factors: indispensable for cell differentiation Integrins trigger Differentiation Apoptosis and Proliferation Ligands for Integrins: Collagen Laminin Fibronectin vWF Vitronectin Fibrinogen Fibronectin RGD`s ICAM VCAN-1 ……

47. P01: Primary Cell Culture ECM as Epigenetic factors: indispensable for cell differentiation Some major benefits of using ECM-coating • more standardized than „self-coating“ by FCS • surface adapted to a specific cell type • inhibition of apoptosis • growth rates are adapted to in vivo values • improved differentiation • improved adhesion • reversion to a biological response

48. P01: Primary Cell Culture ECM as Epigenetic factors: indispensable for cell differentiation Some common coating techniques: • Matrigel ™ to mimic basal membranes containing collagen IV • Proteoglycanes and Glycoproteins • Collagen I as the major component of the connective tissue • Fibronectin as adhesion molecules for fibroblasts • Laminin enables adherence of epithelial cells by “RGD motifs” Matrigel™ and Collagen I can be used as thin or thick (gel) coating Influence on cellular behaviour depends on the ECM-concentration Problems: How can we standardise the protocols i.e. concentration, thickness, stability?

49. P01: Primary Cell Culture ECM as Epigenetic factors: indispensable for cell differentiation Some common features of ECM-coating • Matrigel ™ induces cell specific gene expression (PSA in prostate) • Matrigel ™ reduce proliferation in primary cell cultures • Matrigel ™ allows tube formation in thick gels • Matrigel ™ can be used as a barrier in invasion assays • Matrigel ™ modulates signal transduction • Matrigel ™ influences shape and orientation Important: Even permanent cell lines respond to ECM-coating Thus coating may be a helpful parameter in HTS

50. P01: Primary Cell Culture ECM as Epigenetic factors: indispensable for cell differentiation The future of ECM-coating Modification of cell culture surfaces by Microstructuring and Nanomodification Coated with Si3N4