Basics of stem cell culture

In name of Allah

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 celle.g. muscle cell, nerve cell

DIFFERENTIATION(specializing)

1 stem cell

Self renewal - maintains the stem cell pool

4 specialized cells

Differentiation - replaces dead or damagedcells 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): 2608-2612.)

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)

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Articular cartilage

Healthy Joint Healthy Joint Trumatic injury Trumatic injury

OsteoarthritisOsteoarthritis

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Cartilage tissue engineering

Scaffolds

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