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
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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
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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)
Types of cells
• On the basis of morphology
– Fibroblast like- cells attached to an substrate
Adherent cells in
monolayer culture
– Epithelial like-attached to a substrate
Suspension culture
– Lymphoblast like- cells do not attach
• On the basis of differentiation state
– Differentiated (chondrocytes, osteoblasts, hepatocyte,…)
– Undifferentited (stem cells)
Epithelial like
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)
What is a stem cell?
stem cell
SELF-RENEWAL
(copying)
stem cell
DIFFERENTIATION
(specializing)
specialized cell
e.g. muscle cell, nerve cell
Why self-renew AND differentiate?
1 stem cell
1 stem cell
Self renewal - maintains
the stem cell pool
4 specialized cells
Differentiation - replaces dead or damaged
cells throughout your life
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
Understanding tumorigenic potential
of pluripotent stem cells
(Cell Cycle. 2009;8(16): 2608-2612).
Hematopoietic stem cells
Human macrophages were derived from
hematopoietic stem cells in the bone marrow.
Mesenchymal stem cells
J Anat. Feb 2004; 204(2): 133–139.
Neural stem cell
Potential Uses of Stem Cells
• Basic research
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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)
Articular cartilage
Healthy Joint
Trumatic injury
Osteoarthritis
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Cartilage tissue engineering
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Scaffolds
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