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Basics of stem cell culture Dr Shafaei
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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.
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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)
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Trypsinization
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Growth curve Doubling time Population doubling
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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)
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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) Types of cells Adherent cells in monolayer culture Suspension culture
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Epithelial like
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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) Types of cells
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stem cell What is a stem cell? stem cell SELF-RENEWAL (copying) specialized cell e.g. muscle cell, nerve cell DIFFERENTIATION (specializing)
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1 stem cell Self renewal - maintains the stem cell pool 4 specialized cells Differentiation - replaces dead or damaged cells throughout your life Why self-renew AND differentiate? 1 stem cell
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Self Renewal (Regeneration) Stem cells are capable of dividing & renewing themselves for long periods
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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
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Embryonic and adult stem Cell
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Embryonic vs Adult 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
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(Cell Cycle. 2009;8(16): 2608-2612). Understanding tumorigenic potential of pluripotent stem cells
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Human macrophages were derived from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells
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Mesenchymal stem cells J Anat. Feb 2004; 204(2): 133–139.
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Neural stem cell
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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
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Potential Uses of Stem Cells
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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, …)
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Transfection
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Recombinant DNA
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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, …)
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Mechanical shear stress Red – F-Actin (Phalloidin-Alexa 633) Green – VE-Cadherin (VE-Cadherin (D87F2) XP) Blue – Cell nuclei (DAPI)
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31 Articular cartilage Healthy Joint Trumatic injury Osteoarthritis
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33 Cartilage tissue engineering
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Scaffolds 34
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