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Basics of stem cell culture

Basics of stem cell culture. Dr S hafaei. In name of Allah. Definition of cell culture. Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. . Terminology . Organ culture vs. cell culture

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Basics of stem cell culture

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  1. Basics of stem cell culture DrShafaei In name of Allah

  2. Definition of cell culture Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.

  3. Terminology • Organ culture vs. cell culture • Primary Culture (heterogeneous population of cells) • Confluency • Sub culture • Passage number • Clone • Cell strain • Cell Line (Finite vs. Continuous Cell Line)

  4. Trypsinization

  5. Growth curve • Doubling time • Population doubling

  6. Terminology • Organ culture vs. cell culture • Primary Culture (heterogeneous population of cells) • Confluency • Sub culture • Passage number • Clone • Cell strain • Cell Line (Finite vs. Continuous Cell Line)

  7. Types of cells • On the basis of morphology • Fibroblast like- cells attached to an substrate • Epithelial like-attached to a substrate • Lymphoblast like- cells do not attach • On the basis of differentiation state • Differentiated (chondrocytes, osteoblasts, hepatocyte,…) • Undifferentited (stem cells) Adherent cells in monolayer culture Suspension culture

  8. Epithelial like

  9. Types of cells • On the basis of morphology • Fibroblast like- cells attached to an substrate • Epithelial like-attached to a substrate • Lymphoblast like- cells do not attach • On the basis of differentiation state • Differentiated (chondrocytes, osteoblasts, hepatocyte,…) • Undifferentited (stem cells)

  10. What is a stem cell? stem cell SELF-RENEWAL (copying) DIFFERENTIATION (specializing) specialized cell e.g. muscle cell, nerve cell stem cell

  11. Why self-renew AND differentiate? 1 stem cell 4 specialized cells 1 stem cell Differentiation - replaces dead or damaged cells throughout your life Self renewal - maintains the stem cell pool

  12. Self Renewal (Regeneration) • Stem cells are capable of dividing & renewing themselves for long periods

  13. Two types of stem cells • Embryonic Stem Cells (ESC): received from: • Embryos created in vitro fertilization • Aborted embryos • Adult Stem Cells (ASC): can be received from: • Limited tissues (bone marrow, adipose tissue, muscle, dental pulp, amnion, synovial membrane) • Placental cord • Dental pulp

  14. Embryonic and adult stem Cell

  15. EmbryonicvsAdult Stem Cells • Totipotent • Differentiation into ANY cell type • Large numbers can be harvested from embryos (Legal issue ) • May cause immune rejection • Rejection of ES cells by recipient has not been shown yet • Teratoma • Multi or pluripotent • Differentiation into some cell types, limited outcomes • Limited numbers, more difficult to isolate • Less likely to cause immune rejection, since the patient’s own cells can be used

  16. Understanding tumorigenic potential of pluripotent stem cells (Cell Cycle. 2009;8(16): 2608-2612).

  17. Hematopoietic stem cells Human macrophages were derived from hematopoietic stem cells in the bone marrow.

  18. Mesenchymal stem cells J Anat. Feb 2004; 204(2): 133–139.

  19. Neural stem cell

  20. Potential Uses of Stem Cells • Basic research • Human development • molecular basis of cancer • Molecular mechanisms for gene control • Role of signals in gene expression & differentiation of the stem cell • Signaling pathways • Biotechnology • drug discovery • Cell therapy

  21. Potential Uses of Stem Cells

  22. Cell biology methods • Transfection • GFP • siRNA • Live cell imaging •Chemotaxis •Angiogenesis •Migration / Wound healing •Cell Culture Under Flow • Real time RT PCR • Cytological methods ( H&E, …)

  23. Transfection

  24. Recombinant DNA

  25. Cell biology methods • Transfection • GFP • siRNA • Live cell imaging •Chemotaxis •Angiogenesis •Migration / Wound healing •Cell Culture Under Flow • Real time RT PCR • Cytological methods ( H&E, …)

  26. Mechanical shear stress Red – F-Actin (Phalloidin-Alexa 633) Green – VE-Cadherin (VE-Cadherin (D87F2) XP) Blue – Cell nuclei (DAPI)

  27. Trumatic injury Healthy Joint Articular cartilage Osteoarthritis

  28. Cartilage tissue engineering

  29. Scaffolds

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