FBT - Week 11: cell culture and stem cell LO

Fundamentals of Cell Culture

• Definition of Cell Culture: The process involve growing cells outside of a living organism under strictly controlled laboratory conditions.

• Target Organisms: This term usually refers to the culturing of animal cells.

• Historical Context:

  • First successfully performed by Ross Harrison in 1907.

  • The original experiment utilized frog nerve cells.

• Primary Utility: Allows for the study of specific cells in a controlled environment without the influence or complexity of the whole organism.

• Key Advantages:

  • Availability of controlled experimental conditions.

  • Produces highly reproducible results.

  • Facilitates the easier observation of complex cellular processes.

  • Reduces the necessity for animal experimentation.

Primary Cell Culture

• Definition: These are cells taken directly from animal tissue and placed into an appropriate culture medium.

• General Characteristics:

  • They closely resemble cells in the body (known as in vivo).

  • They provide results that are physiologically relevant.

  • These cells have a limited lifespan.

  • They eventually undergo senescence, which is the point at which they stop dividing.

• Standard Growth Requirements:

  • Temperature: $37^{\circ}C$.

  • Atmosphere: $5\% \, CO_2$.

  • Nutrients: An appropriate nutrient-rich culture medium is required for survival and growth.

Establishing Primary Cultures

• Explant Culture Method:

  • Small tissue fragments are attached directly to a culture vessel.

  • Process Steps:

    1. Tissue pieces are attached to a glass or plastic surface.

    2. Culture medium is added to the vessel.

    3. Cells migrate outward from the tissue.

    4. Cells begin to divide and proliferate.

• Enzymatic Dissociation Method:

  • Process Steps:

    1. Tissue is mechanically chopped into smaller sections.

    2. Proteolytic enzymes are added to the mixture. Specific enzymes used include Trypsin and Collagenase.

    3. The extracellular matrix (ECM) is digested by these enzymes.

    4. Individual cells are released from the tissue structure.

    5. Cells are then cultured separately.

• Selecting Specific Cell Types (Flow Cytometry/FACS):

  • Fluorescence-Activated Cell Sorting (FACS): Uses fluorescent antibodies to identify and isolate specific desired cells.

  • Applications:

    • Cell sorting procedures.

    • Stem cell isolation.

    • Immune cell analysis.

    • Use in research and clinical diagnostics.

Cell Lines and Strains

• Cell Line Definition: A primary culture becomes a cell line after the very first subculture (passage).

• Finite Cell Lines:

  • Possess a limited lifespan.

  • Typically undergo a range of $2$ to $100$ divisions.

  • They eventually reach senescence and stop dividing.

• Continuous Cell Lines:

  • Possess the ability to divide indefinitely.

  • Often derived from tumors or transformed cells.

  • Example: HeLa Cells:

    • Origin: Derived from the cervical cancer cells of Henrietta Lacks in 1951.

    • Importance: Vital for the development of the Polio vaccine, cancer research, virology, and general cell biology.

    • Ethical Issues: The cells were taken without informed consent, leading to significant discussions regarding research ethics and patient rights.

• Cell Strains:

  • Definition: A subpopulation selected from a cell line.

  • Obtainment: Acquired through cloning, genetic selection, or transfection.

  • Features: May possess unique characteristics and can differ genetically from the parent cell line.

Cell Culture Laboratory Equipment

Cell Morphology

• Lymphoblast-like Cells:

  • Grow in suspension (not attached to surfaces).

  • Shape: Round or spherical.

  • Non-adherent.

  • Example: Blood cells.

• Epithelial-like Cells:

  • Adherent (attach to surfaces).

  • Shape: Flattened and polygonal in appearance.

  • Example: Skin cells.

• Fibroblast-like Cells:

  • Adherent (attach to surfaces).

  • Shape: Elongated with a bipolar shape.

  • Example: Connective tissue cells.

Aseptic Technique and Contamination

• Purpose: To prevent the contamination of cultures.

• Vulnerability Reason: Culture media are warm, humid, and nutrient-rich, providing the perfect conditions for microbial growth.

• Types of Contamination:

  • Bacterial Contamination: Identified by a turbid (cloudy) medium, rapid changes in pH, and poor cell growth.

  • Fungal Contamination: Identified by visible fungal colonies, filaments (hyphae), and a cloudy medium.

  • Mycoplasma Contamination:

    • Extremely difficult to detect visually.

    • Requires PCR or enzymatic testing for detection.

    • Can seriously affect experimental results.

  • Cross-Contamination: Occurs when one cell line contaminates another. A major issue in research as many cell lines have historically been misidentified.

Growth Dynamics: Confluency and Passaging

• Confluency Definition: The percentage of the culture surface covered by cells.

• Optimal Confluency: Usually between $70\%$ and $80\%$.

• Confluency Problems:

  • Too Low (<70\%): Cells may remain in the lag phase, resulting in slow growth.

  • Too High (>80-90\%): Growth slows down, contact inhibition occurs, and cells may begin to pile up on top of each other.

• Passaging Cells (Subculturing):

  • The process of transferring cells into fresh culture vessels.

  • Trypsin/EDTA Role:

    • Trypsin: A protease enzyme that cleaves proteins involved in cell attachment.

    • EDTA: A chelator that binds to calcium ions ($Ca^{2+}$), weakening cell adhesion.

    • Together, they work to detach adherent cells from the flask surface.

• Passage Number:

  • Refers to the number of times cells have been subcultured.

  • Higher passage numbers may alter cell behavior, which is a critical factor for experimental reproducibility.

• Hayflick's Phenomenon:

  • Normal cells can only divide a limited number of times.

  • Eventually, they stop dividing, enter senescence, and die.

  • This is linked to the process of aging; older donors produce cells with fewer possible divisions.

Cryopreservation

• Purpose: Long-term storage of cells to preserve them for future use.

• Storage Conditions:

  • Must be kept below $-130^{\circ}C$.

  • Typically stored in a liquid nitrogen tank, which is approximately $-196^{\circ}C$.

• Freezing Challenges: Ice crystal formation can damage cell membranes and kill the cells.

• DMSO (Dimethyl Sulfoxide):

  • Functions: Protects membranes and reduces the formation of ice crystals.

  • Limitation: DMSO is toxic to cells and must be removed quickly after the cells are thawed.

Applications of Cell Culture

1. Basic Cell Biology Research: Studying growth, cell signaling, and cell-cell interactions.

2. Drug Testing & Toxicology: Testing new drugs for efficacy and assessing toxicity/safety before animal or human trials.

3. Virology:

   • Used to grow viruses and study replicate cycles.

   • Essential for vaccine development (e.g., Polio, Rabies, Measles, Hepatitis B).

4. Cancer Research: Investigating tumor development, cancer-causing agents, and testing anti-cancer therapies.

5. Genetic Engineering: Production of recombinant proteins, large-scale virus production, and biotechnology applications.

6. Gene Therapy: Aimed at replacing defective genes with functional ones for inherited diseases.

7. Tissue Engineering: Creating artificial tissues and replacement organs for regenerative medicine.

Stem Cells

• Definition: Stem cells are characterized by three primary traits:

  1. Unspecialized Cells: They have no specific function initially.

  2. Self-Renewing Cells: They can produce more stem cells.

  3. Differentiating Cells: They can become specialized cells (e.g., liver cells, skin cells, neurons, muscle cells).

• Key Properties:

  • Self-Renewal: When a stem cell divides, it produces one stem cell and one specialized/progenitor cell.

  • Proliferation: Can continue dividing for long periods, essential for growth and tissue repair.

  • Differentiation: The process of becoming specialized cells when exposed to signals like hormones or growth factors.

Stem Cell Classification and Potency

• Types of Stem Cells:

  1. Embryonic Stem Cells (ESCs): Derived from embryos. They are totipotent or pluripotent and can generate many cell types, but present ethical concerns.

  2. Adult Stem Cells: Tissue-specific and multipotent. Examples include bone marrow and skin stem cells. Their function is tissue maintenance and repair.

  3. Induced Pluripotent Stem Cells (iPSCs): Adult differentiated cells that are genetically reprogrammed into stem-cell-like cells. These avoid ethical concerns and allow for patient-specific therapies.

• Potency Levels:

  • Totipotent: Ability to form all cell types, including extraembryonic tissues (Example: Morula).

  • Pluripotent: Ability to form almost all body cell types (Examples: Inner cell mass of blastocyst, Embryonic stem cells).

  • Multipotent: Ability to form several related cell types (Example: Adult stem cells).

  • Oligopotent: Ability to form a few cell types (Example: Progenitor cells).

  • Unipotent: Ability to form only one cell type (Example: Differentiated cells).

Differentiation Pathways and Division

• General Pathway: Stem Cell $\rightarrow$ Progenitor Cell $\rightarrow$ Differentiated Cell.

• Symmetric Division: Produces two stem cells OR two progenitor cells.

• Asymmetric Division: Produces one stem cell and one progenitor cell.

• Terminal Differentiation: The production of fully specialized cells.

Medical Applications of Stem Cells

• Leukemia: Treated via bone marrow stem cell transplantation.

• Parkinson's Disease: Potential replacement of damaged neurons.

• Alzheimer's Disease: Research into replacing lost neurons.

• Heart Disease: Generation of new cardiac muscle cells.

• Burn Treatment: Use of skin regeneration and autologous skin grafts.