Basics of stem cell culture Dr Shafaei

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

Trypsinization

Growth curve Doubling time Population doubling

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)

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

Epithelial like

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

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

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

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

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

Embryonic and adult stem Cell

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

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

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

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

Neural stem cell

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

Potential Uses of Stem Cells

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, …)

Transfection

Recombinant DNA

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, …)

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

31 Articular cartilage Healthy Joint Trumatic injury Osteoarthritis

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33 Cartilage tissue engineering

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