Cell Culture & Stem Cells – Fundamental Biomedical Techniques

Cell Culture & Stem Cells

Cell Culture: Primary Culture and Cell Lines

• Cell culture is the process of growing cells outside a living organism under controlled conditions, typically referring to culturing cells derived from animal cells.
• The first successful cell culture was undertaken by Ross Harrison in 1907, using frog nerve fibers to study nerve fiber development.

Cell Culture Equipment:

• Cell culture hood (laminar flow cabinet)
• Incubator
• Water bath
• Centrifuge
• Refrigerator and freezer (-20°C)
• Cell counter (e.g., automated cell counter or hemacytometer)
• Inverted microscope
• Liquid nitrogen
• Autoclave

Applications of Cell Culture:

• Model systems for studying basic cell biology, interactions between disease-causing agents and cells, drug effects, aging processes, and nutritional studies.
• Toxicity testing: Studying the effects of new drugs.
• Virology: Cultivating viruses for vaccine production and studying their infectious cycles.
• Cancer research: Studying the function of chemicals, viruses, and radiation that convert normal cultured cells into cancerous cells.
• Genetic Engineering: Production of commercial proteins and large-scale production of viruses for vaccine production (e.g., polio, rabies, chickenpox, hepatitis B, measles).
• Gene therapy: Replacing cells with non-functional genes with cells having a functional gene.
• Tissue Engineering: Using cell and tissue culture to generate artificial tissues and organs.

Primary Cell Culture:

• Cells taken directly from animal tissue are added directly to a medium.
• Primary cells closely mimic the physiological state of cells in vivo, generating more relevant data representing living systems.
• Primary cell cultures have a finite lifespan.
  • Senescence: Stop dividing after a certain number of population doublings.
  • Temperature: 37°C, 5% CO_2
  • Media: pH-buffers, nutrients, growth factors

Media Components:

• Bulk ions: Na, K, Ca, Mg, Cl, P, Bicarbonate or CO_2 (buffers).

• Trace elements: iron, zinc, selenium.

• Sugars: glucose is most common.

• Amino acids: 13 essential.

• Vitamins

• Choline, inositol (cell structure and membrane integrity)

• Antibiotics: used to control bacterial and fungal contaminants.

• Serum: Fetal Bovine Serum (FBS) contains growth-promoting activities.

  • Buffers toxic nutrients by binding them.
  • Neutralizes proteases.
  • Has undefined effects on cell-substrate interaction.
  • Contains peptide hormones or hormone-like growth factors that promote healthy growth.

Methods for Establishing Primary Cultures:

• Explant cultures:

  • Small tissue pieces are attached to a culture vessel using plasma clots or fibrinogen and immersed in culture medium.
  • Individual cells move from the tissue explant onto the culture vessel surface, where they divide and grow.

• Enzymatic dissociation:

  • Tissues are broken up mechanically (scissors and forceps).
  • Fragmented tissue is treated with proteolytic enzymes like trypsin and collagenase to destroy the extracellular matrix and adhesion proteins.

• Cell separation:

  • Flow cytometry (immunoassay) uses EC-specific antibodies, a laser, nozzle tip, deflection plates, and fluorochrome to select a particular cell type.
  • Magnetic separation uses magnetic beads attached to antibodies to separate cells.

Cell Lines

• After the first subculture, the primary culture becomes a cell line.
  • Finite cell lines: Cell lines with a limited lifespan, typically 2-100 divisions.
  • Continuous cell lines: Cells transformed under laboratory conditions or in vitro culture, including tumor-derived cells.
    • Permanently established cell cultures proliferate indefinitely with fresh medium and space.
    • Considered single-cell derived and highly homogenous (though recent gene profile analyses challenge this).

Cell Strains

• A cell strain is a subpopulation of a cell line selected by cloning or other methods.
• Cell strains may undergo additional genetic changes since the initiation of the parent line.
• Individual cell strains may become more or less tumorigenic or be designated as a separate strain following transfection procedures.

Cell Morphology:

• Lymphoblast-like: Cells do not attach, remain in suspension, and are spherical.
• Epithelial-like: Attached to a substrate, appear flattened and polygonal.
• Fibroblast-like: Attached to a substrate, appear elongated and bipolar.

Cell Culture Contamination

• Aseptic Technique is crucial for minimizing microbial contamination. Work in a culture hood (laminar flow cabinet).
• Cell cultures are susceptible to contamination by bacteria or fungi due to the nutrient-rich media, humidity, and warmth.
  • Evident by a drop in pH (color change of media), turbidity of the medium, and presence of fungal colonies.
  • Mycoplasma contamination is difficult to determine, requiring PCR or enzymatic tests.
• Contamination by other cell lines is a significant issue.
  • 15-20% of cell biology experiments are conducted with misidentified or cross-contaminated cells.
  • Only ~50% of researchers regularly verify the identity/quality of their cell lines.

Cell Culture Confluency

• Confluency: How "covered" the growing surface appears upon visual inspection.
• Optimal confluence for moving cells to a new dish is 70-80%.
  • Too low, cells will be in lag phase and won’t proliferate.
  • Too high, cells will stop dividing or pile on one another, forming tumor-like formations.

Cell Culture Terminology

• Trypsin/EDTA: An enzyme used to detach cells from a culture dish.
  • Trypsin cleaves peptide bonds (LYS or ARG) in fibronectin of the extracellular matrix.
  • EDTA chelates calcium ions in the media that would normally inhibit trypsin and also affect cell adhesion molecules.
• Passage number: The number of times the cells have been removed (or “split”) from the plate and re-plated.
• Hayflick’s Phenomenon: Cells will continue to grow and divide normally for a limited number of passages, after which they stop dividing and eventually die.
  • There is a correlation between the maximal number of passages and aging; the number of passages decreases when cells are harvested from older individuals.

Cell Cryopreservation

• Most mammalian cells can be stored at temperatures below -130°C for many years. Liquid nitrogen > -195.79°C.
• Ice crystals that form during freezing can puncture the plasma membrane, leading to cell death.
• Dimethyl sulfoxide (DMSO) protects cells by:
  • Partially solubilizing the membrane to reduce puncture risk.
  • Interrupting the ice lattice to reduce crystal formation.
• DMSO is toxic, so cells should be thawed quickly and removed from DMSO as soon as possible.

Stem Cells

Stem Cell Characteristics:

1. Immature, unspecialized cells that do not perform any specific function.
2. Reproduce themselves.
3. Can differentiate/mature into many different specialized cell types.
   • Example: A stem cell can produce itself and a liver cell, skin cell, nerve cell, etc.

Types of Stem Cells:

1. Embryonic stem cells: totipotent (all) or pluripotent (many).
   • Human embryonic stem cells (hESC)
2. Adult stem cells: multipotent (few) tissue-specific stem cells.
3. Adult differentiated cells genetically reprogrammed to an embryonic stem cell-like state.
   • Induced pluripotent stem cells (iPSCs)
     The potency of a cell is the number of different cell fates open to that cell.

Stem Cell Properties

• Self-renewal: When a stem cell divides, it produces:
  • One daughter cell that remains a stem cell.
  • One daughter cell that becomes a specialized cell.
• Proliferation: Stem cells proliferate throughout life for tissue repair.
• Differentiation potential: With the correct factor (e.g., hormone), a stem cell can produce a specialized cell of any type, regardless of the tissue of origin.

Stem Cell Potency

• Totipotent (e.g., morula)
• Pluripotent (e.g., inner cell mass of the blastocyst)
• Multipotent (most adult stem cells)
• Oligopotent (progenitor cells)
• Unipotent (differentiated cells)

Stem Cell Division

• Symmetric cell division
  • Stem cell to stem cell
  • Progenitor cell to progenitor cell
  • Differentiated cell to differentiated cell
• Asymmetric cell division
  • Stem cell divides into stem cell and progenitor cell.
• Terminal differentiation
  • Progenitor cell differentiates into differentiated cell.

Stem Cell Applications (Embryonic)

• Differentiation of pluripotent stem cells into:
  • Nerve cells for Parkinson's & Alzheimer's
  • Heart muscles for heart disease
  • Blood cells for leukemia

Stem Cell Applications (Adult)

• Wound healing, third-degree burns treatment.
• Autologous skin grafts (patient’s own unaffected skin).
  • Keratinocytes are isolated from a biopsy of unaffected skin.
  • Keratinocyte stem cells are cultured in vitro for 2-3 weeks to form sheets of epidermal cells.
  • These epidermal sheets are grafted onto the patient’s burnt skin.
  • The grafted skin is well-differentiated and similar to non-grafted skin except for the lack of hair follicles and sebaceous glands.
• Multipotent stromal cells (e.g., in bone marrow, placenta)
  • Produce cells for fat, muscle, bone, and cartilage.
  • Can also differentiate into nerve cells or fibroblasts.
  • Have anti-inflammatory and immune-suppressing properties.
  • Of particular interest in research.

Mesenchymal Stem Cell Differentiation

• Growth and differentiation factors like Noggin, BMP-2, 4, 5, Insulin, Wnt-5b, FGF basic, TGF-Beta affect differentiation into:
  • Adipogenesis (white and brown adipocytes)
  • Myogenesis (Skeletal, Smooth, Cardiac muscle)
  • Osteogenesis (Mature Osteoblasts)
  • Chondrogenesis (Chondrocytes)

Stem Cell Applications (iPSC)

• Introduction/Expression of pluripotency genes reprograms differentiated somatic cells into induced pluripotent stem cells (iPSCs).

• Screen affected cell type by differentiating patient specific iPS cells in vitro after skin biopsy:

  • Treatment with disease-specific drugs
  • Screening for therapeutic compounds

• Use gene targeting to repair disease-causing mutations to get repaired iPS cells:

  • Transplantation of genetically matched healthy cells

Stem Cell Analysis:

• Cell Morphology – Microscopy
• Genetic Analysis: Gene Expression - RT-PCR
• Protein analysis: Immunocytochemistry

Immunocytochemistry

• Uses the immune system to generate antibodies against a foreign substance (antigen).
• Antibodies are applied to sectioned tissue or fixed cells.
• A label attached to the antibody or secondary antibody (fluorescent or enzyme).
• The location of the protein is identified under a fluorescence microscope or colored substrate deposition.

Stem Cell Common Applications:

• Study mechanisms of pluripotency
• Study mechanisms of human development
• Cells for transplantation (e.g., spinal cord injury)
• Cells for biotechnology
• Cells for basic/translational research
• New human disease models
• Toxicity testing
• Drug metabolism studies
• Identify new drug targets
• Cell biology studies