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Culture and cryopreservation of melanoma cells for PALS measurements – biomedical study

Culture and cryopreservation of melanoma cells for PALS measurements – biomedical study. Kraków, 13 September 2018 Agnieszka Kamińska, MSc. Eng. Faculty of Physics, Astronomy and Applied Computer Science Jagiellonian University 3 rd Symposium on Positron Emission Tomography, Kraków 2018.

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Culture and cryopreservation of melanoma cells for PALS measurements – biomedical study

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  1. Culture and cryopreservation of melanoma cells for PALS measurements – biomedical study Kraków, 13 September 2018 Agnieszka Kamińska, MSc. Eng. Faculty of Physics, Astronomy and Applied Computer Science Jagiellonian University 3rd Symposium on Positron Emission Tomography, Kraków 2018

  2. Characteristic of melanoma tumor Stages: Stage 0: the canceris in the outermostlayer of skin Stage 1: the canceris less than 1 mm thick Stage 2: the canceris 1-4 mm thick Stage 3: the cancerhasspread to lymphnodes Stage 4: the cancerhasspread to distantlymphnodesororgans https://www.medicalnewstoday.com/articles/154322.php https://www.verywellhealth.com/melanoma-staging-what-it-means-and-reveals-3010755

  3. Culture of melanocytes and melanoma cells • Culture conditions: • 5% CO2 • 37 °C HEMa-LP (melanocytes) WM-115 (primary melanoma) WM-266-4 (metastatic) • medium 254 supplemented • with HMGS (Human Melanocyte • Growth Supplement) • 0.5% FBS • Penicyllin/Streptomycin • RPMI (with L-glutamine) • 10% FBS • RPMI (with L-glutamine) • 10% FBS

  4. MELANOCYTES MELANOMA CELL LINE WM-115 Images taken by E. Kubicz

  5. Melanoma cells vs. melanocytes • irregularshape • reducedamount of cytoplasm • bigger/multipliednuclei • higherlevel of cortactin • VEGF • E-cadherin • N-cadherin • differentialexpression of miRNA a) b) Melanocytes (a) and melanoma cells (b), images taken by E. Kubicz

  6. Storage of the cells DEWAR

  7. Freezing of the cells – damagingfactors • Thermal shock • Formation of ice crystals • Dehydration • Increased salt concentration • Osmotic shock

  8. Cryopreservation – the technique of freezing cells and tissues • at very low temperatures (-196 °C) which requires the use of a • cryoprotective agent. • biological material retains genetically stable • stopping the biological activity • minimaizing ice crystals formation

  9. Cryoprotectants Dimethyl sulfoxide Trehalose 1,2 - Propanediol

  10. Lyophilization Liquid Solid Gas https://courses.lumenlearning.com/cheminter/chapter/phase-diagram-for-water/

  11. Lyiophilization Sample preparation Primary drying Secondary drying Final product Freezing

  12. Cryopreservation • Freezing media: • PBS (Ca2+, Mg2+free) • PBS (Ca2+, Mg2+free) + 10% DMSO • RPMI+20% FBS+10% DMSO • 1.5M PROH+0.5M Trehalose • 0.25M Trehalose http://www.mcrh.com/treatments success/ivf/cryopreservation

  13. Cryoprotectant - Trehalose • Disaccharide of glucose (342 Da) • α, α-1, 1-glycosidic bond • Bioprotectiveaction of trehalose - hypothesis: • Waterreplacement • Rearrengement of watermolecules • Transition to glassystate

  14. Raw image from LUNA II Automated Cell Counter LUNA II, image taken by B. Leszczyński Tagged image from LUNA II

  15. MELANOMA WM-266-4 after revitalizaton Taken by E. Kubicz

  16. Conclusions • The preservation of cellsisanextremelyimportantaspect of cellculture • To ensure high viability of cells (>90%) itisveryimportant to choose • appropriatecryoprotectantand handlingtechniques. • Supplementation of standard cryoprotective medium with trehalose • improving post-thawcellviability and metabolism. • Lyophilization of cultured cells allowsto performingPositron • Annihilation Lifetime Spectroscopy (PALS) measurements for several • hours without cell damage.

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