Fundamental Biomed Week 11 - Cell Culture & Stem Cells

Cell Culture

  • Cell culture is the process of growing cells outside of a living organism under controlled conditions, typically referring to animal cells.
  • First successfully undertaken by Ross Harrison in 1907, using frog nerve fibers.

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 cell biology, interactions between disease-causing agents and cells, drug effects, aging, and nutritional studies.
  • Toxicity testing: Studying effects of new drugs.
  • Virology: Cultivating viruses for vaccine production and studying infectious cycles.
  • Cancer research: Studying chemicals, viruses, and radiation effects on converting normal cells to cancerous cells.
  • Genetic Engineering: Production of commercial proteins, large-scale virus production for vaccines (e.g., polio, rabies, chickenpox, hepatitis B, measles).
  • Gene therapy: Replacing cells with non-functional genes with cells having functional genes.
  • Tissue engineering: Generating 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.
  • Primary cell cultures have a finite lifespan.
    • Senescence: Stop dividing after a certain number of population doublings.
    • Temperature: 37°C, 5% CO2CO_2
    • Media: pH buffers, nutrients, growth factors.

Media Components

  • Bulk ions: Na, K, Ca, Mg, Cl, P, Bicarb or CO2CO_2 (buffers).
  • Trace elements: Iron, zinc, selenium.
  • Sugars: Glucose (most common).
  • Amino acids: 13 essential amino acids.
  • Vitamins
  • Choline, inositol: Cell structure and membrane integrity.
  • Antibiotics: Control bacterial and fungal contaminants.
  • Serum: Fetal Bovine Serum (FBS).
    • Contains growth-promoting activities, buffers toxic nutrients, neutralizes proteases, affects cell-substrate interaction, and contains peptide hormones or hormone-like growth factors for healthy growth.

Methods for Establishing Primary Cultures

  • Explant cultures
    • Small pieces of tissue are attached to a culture vessel 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 mechanically broken up.
    • Fragmented tissue is treated with proteolytic enzymes like trypsin and collagenase to destroy the extracellular matrix and adhesion proteins.
  • Selecting a particular cell type for culture
    • Flow cytometry (immunoassay)
    • Magnetic separation

Cell Lines and Cell Strains

  • Primary culture → Sub-culture → Cell Line.
  • Cell Line: After the first subculture, the primary culture becomes a cell line.
  • Finite cell lines: Cell lines with a limited lifespan (2-100 divisions).
  • Continuous cell lines: Cells transformed under laboratory conditions or in vitro culture.
    • Permanently established cell cultures that proliferate indefinitely with fresh medium and space. Considered single-cell derived and highly homogenous.
  • HeLa Cells:
    • Derived from Henrietta Lacks in 1951 without her knowledge.
    • Used in the development of the Polio vaccine in 1953.
    • Showed that the human papilloma virus causes cancer in 1989.
  • Cell Strain: Is a subpopulation of a cell line that has been positively selected from the culture by cloning or another method.
    • Undergo additional genetic changes since the initiation of the parent line.

Cell Morphology

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

Contamination

  • Aseptic Technique: Work in a culture hood (laminar flow cabinet) to minimize microbial contamination.
  • Cell cultures are susceptible to contamination by bacteria or fungi.
    • Evident by a drop in pH (change of media color), turbidity of medium, and the presence of fungal colonies.
    • Mycoplasma contamination is difficult to determine; require PCR or enzymatic tests.
  • 15-20% of cell biology experiments are conducted with misidentified or cross-contaminated cell lines.

Cell Culture Maintenance

  • Confluency: How covered the growing surface appears by visual inspection.
  • Optimal confluence for moving cells to a new dish is 70-80%.
    • Too low: Cells will be in the lag phase and won’t proliferate.
    • Too high: Cells will stop dividing
  • Trypsin/EDTA: Enzyme used to detach cells from a culture dish.
    • Trypsin cleaves peptide bonds in fibronectin of the extracellular matrix.
    • EDTA chelates calcium ions, which inhibit trypsin and affect cell adhesion molecules.
  • Passage number: The number of times the cells have been removed and replated.
  • Hayflick's Phenomenon: Cells will grow and divide normally for a limited number of passages before stopping.

Cell Preservation

  • Most mammalian cells can be stored at temperatures below -130°C for many years.
  • Liquid nitrogen > -195.79°C.
  • As the suspension of cells freezes, ice crystals form, leading to cell death.
  • Dimethyl sulfoxide (DMSO) protects the cells by:
    • Partially solubilizing the membrane so that it is less prone to puncture.
    • Interrupting the lattice of the ice, so that fewer crystals form.

Stem Cells

  • Immature, unspecialized cells that reproduce themselves and can differentiate into many different specialized cell types.
  • A stem cell can produce itself and specialized cells like liver, skin, or nerve cells.

Types of Stem Cells

  • Embryonic stem cells: Totipotent (all) or pluripotent (many).
  • Adult stem cells: Multipotent (few) tissue-specific stem cells.
  • Adult differentiated cells: Genetically reprogrammed to an embryonic stem cell-like state [Induced pluripotent stem cells (iPSCs)].

Potency of Cells

  • Potency: The number of different cell fates open to that cell.
  • Totipotent: Can differentiate into all cell types (e.g., morula).
  • Pluripotent: Can differentiate into many cell types (e.g., inner cell mass of the blastocyst).
  • Multipotent: Can differentiate into a limited number of cell types (most adult stem cells).
  • Oligopotent: Can differentiate into a few cell types (progenitor cells).
  • Unipotent: Can differentiate into only one cell type (differentiated cells).

Stem Cell Division

  • Symmetric cell division: Produces two identical stem cells or two identical progenitor cells.
  • Asymmetric division: Produces one stem cell and one progenitor cell.
  • Terminal differentiation: Progenitor cell differentiates into a specialized cell.

Stem Cell Applications (Embryonic)

  • Pluripotent stem cells can differentiate into nerve cells (Parkinson's, Alzheimer's), heart muscle (heart disease), and blood cells (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 and cultured in vitro to form sheets of epidermal cells.
    • Epidermal sheets are grafted onto the patient’s burnt skin.
  • Mesenchymal stem cells:
    • Found in bone marrow, placenta, etc.
    • Produce cells for fat, muscle, bone, and cartilage.
    • Differentiate into nerve cells or fibroblasts.
    • Anti-inflammatory and immune-suppressing properties.
Driving Mesenchymal Stem Cell Differentiation
  • Growth and differentiation factors (e.g., BMP-2, 4, Insulin, Wnt-5b, FGF basic, TGF-Beta, Noggin)
  • Induction media for adipogenesis, myogenesis, chondrogenesis, and osteogenesis

Stem Cell Applications (iPSC)

  • Induced Pluripotent Stem Cells: Differentiated somatic cells are reprogrammed by introducing pluripotency genes.
  • Disease-specific drug screening: Affected cell types are differentiated in vitro and treated with drugs.
  • Patient-specific iPS cells: Used for transplantation of genetically matched healthy cells.
    • Gene targeting repairs disease-causing mutations.

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 is attached to the antibody or secondary antibody (fluorescent or enzyme).
  • The location of protein is identified under a fluorescence microscope.

General Stem Cell Applications

  • Study mechanisms of pluripotency, human development, gene function, and cell biology.
  • New human disease models, toxicity testing, drug metabolism studies.
  • Identify new drug targets and novel therapeutics.