Week 11 - Cell Culture and Stem Cells summary

Definition: The process of growing cells outside a living organism (in vitro) under controlled conditions, primarily referring to animal-derived cells.
History: Successfully pioneered by Ross Harrison in 1907 to study frog nerve fiber development.
Applications:
- Basal Research: Studying cell biology, disease interactions, and aging.
- Medical Research: Toxicity testing for new drugs, virology (vaccine production), and cancer research.
- Bioengineering: Genetic engineering (protein production), gene therapy, and tissue engineering (artificial organs).

Core Equipment: Cell culture hood (laminar flow cabinet), incubator, water bath, centrifuge, refrigerator/freezer ( $-20^{\circ}C$ ), and cell counters (automated or hemacytometer).
Specialized Storage: Inverted microscope for visualization, liquid nitrogen for long-term storage (<-130^{\circ}C), and autoclaves for sterilization.

Basic Nutrients: Bulk ions ( $Na, K, Ca, Mg, Cl, P, Bicarb$ ), trace elements (iron, zinc, selenium), sugars (glucose), and 13 essential amino acids.
Complex Additives: Vitamins, choline, inositol, and antibiotics (to control bacterial/fungal contamination).
Serum (Fetal Bovine Serum - FBS): Provides growth-promoting factors, buffers toxic nutrients, neutralizes proteases, and contains essential hormones.
Incubation Parameters: Typically maintained at $37^{\circ}C$ with $5\%$ $CO_2$ to manage pH levels.

Primary Culture: Cells taken directly from animal tissue. They mimic the physiological state in vivo but have a finite lifespan (senescence).
Establishment Techniques:
- Explant Cultures: Small tissue fragments attached to a substrate; cells migrate out and divide.
- Enzymatic Dissociation: Mechanical disruption followed by treatment with proteases (trypsin and collagenase) to destroy the extracellular matrix.
Cell Separation: Purifying specific cell types using Flow Cytometry (FACS) or magnetic separation with specific antibodies.

Cell Line: Defined as the culture after the first subculture.
- Finite Cell Lines: Limit of $2-100$ divisions.
- Continuous Cell Lines: Transformed or tumor-derived cells that proliferate indefinitely (e.g., HeLa cells, derived from Henrietta Lacks in 1951).
Cell Strain: A subpopulation of a cell line selected via cloning or positive selection, often having specific genetic changes.
Morphological Types:
- Lymphoblast-like: Spherical, remain in suspension.
- Epithelial-like: Polygonal shape, attached to substrate.
- Fibroblast-like: Bipolar and elongated, attached to substrate.

Confluency: The percentage of the surface area covered by cells. Optimal subculturing occurs at $70-80\%$ confluency.
Subculturing: Uses Trypsin/EDTA to detach adherent cells. EDTA chelates calcium to inhibit trypsin and aid detachment.
Passage Number: The total number of times cells have been split and replated.
Hayflick’s Phenomenon: The observation that cells have a limited number of divisions correlated with aging.
Cryopreservation: Storage in liquid nitrogen ( $-195.79^{\circ}C$ ). Uses Dimethyl sulfoxide (DMSO) as a cryoprotectant to prevent lethal ice crystal formation, though DMSO is toxic and must be removed quickly upon thawing.

Characteristics: Immature, unspecialized cells capable of self-renewal and differentiation into specialized types.
Potency Levels:
- Totipotent: Can form all cell types (e.g., morula).
- Pluripotent: Can form many types (e.g., hESC, blastocyst inner cell mass).
- Multipotent: Tissue-specific (e.g., adult stem cells).
- Oligopotent: Progenitor cells.
- Unipotent: Fully differentiated.
Division Types: Can be symmetric (producing identical daughters) or asymmetric (producing one stem cell and one progenitor).

Mesenchymal Stem Cells (MSC): Multipotent stromal cells found in bone marrow/placenta. They can produce fat, bone, muscle, and cartilage, and possess anti-inflammatory properties.
Induced Pluripotent Stem Cells (iPSC): Adult somatic cells genetically reprogrammed to an embryonic stem cell-like state using pluripotency genes (e.g., Oct4, Sox2, Klf4, c-Myc).
- Clinical Utility: Drug screening, disease modeling (e.g., using patient-specific iPSCs), and therapeutic transplantation.

Methods:
- Microscopy: Observing cell morphology.
- RT-PCR: Analyzing gene expression (RNA collection).
- Immunocytochemistry: Using fluorescently labeled antibodies to identify protein locations and expression (e.g., Nestin and Sox1 in neural stem cells).