Cell Culture, Cytoskeleton and Cell Interactions

Cell Culture

• Lab next week is the following week - revise material beforehand.

• Requirements for cell proliferation and survival outside the body:

  • Temperature: Maintained at 37 degrees Celsius in an incubator.
  • Oxygen: Necessary for cell respiration, with 5% $CO_2$ to buffer media.
  • Humidity: Achieved via sterile water tray to prevent media evaporation and cell drying.

• Nutrients:

  • Minimal essential medium (MEM) was initial term.
  • Del Becco's modified MEM (DMEM) is commonly used due to increased nutrients.
  • Media needs changing every few days, even for non-proliferating but metabolically active cells, due to waste secretion and nutrient use.
  • Growth components: Sugar source (glucose), inorganic salts (magnesium, sodium, potassium), essential amino acids (13, including glutamine), and water-soluble vitamins.
  • Growth factors are added fresh, often sourced from fetal calf serum (FCS) or fetal bovine serum.
    • FCS has variability between lots and needs testing upon sourcing.
    • Usually added at 5-10% concentration.
    • Contains phenol red as a pH indicator (red at physiological pH, orange/yellow when acidic, purple when pH increases).
  • Antibiotics may be added due to bacterial growth preference in cell culture conditions; aseptic technique is essential (laminar flow hood or biological safety cabinet).
  • Specific components may be needed depending on cell type (e.g., nerve growth factor for nerve cells).

• Subculturing (Cell Splitting):

  • Required when cells proliferate and take over the flask, using nutrients, secreting wastes, and taking up space.
  • For non-adherent cells (suspension):
    • Gentle shake to create a homogeneous solution.
    • Remove a small amount and place into a new flask with fresh media.
    • Can be scaled up easily with agitation for gas exchange.
  • Subculturing dilutes cells (e.g., from $1 \times 10^6$ cells/mL to $1 \times 10^5$ cells/mL via 1 in 10 dilution).

• Adherent Cells:

  • Attach to the flask or extracellular matrix via focal adhesions or polylysine-coated plastic.
  • Require removal from the flask for subculturing.
    • Trypsin is used to degrade focal adhesions.
    • Remove old media, wash with phosphate-buffered saline (PBS), and add trypsin (3-5 minutes).
    • Inactivate trypsin by adding fresh media containing fetal calf serum (FCS).
    • Centrifuge to create a cell pellet, remove media, and resuspend in fresh media.
    • Count cells using a hemocytometer to determine the number of cells to seed into the next flask or for specific experiments.
  • Cell-cell contact inhibition occurs as cells become confluent, stopping proliferation.
    • Cancer cells may lose this inhibition and continue to grow, forming multiple layers or surviving in suspension.

• Bioengineering: 3D Cell Culture

  • Mimics natural tissue environment better than 2D culture.
    • Cells grow in 3D spheroids, embedded within an extracellular matrix.
    • Matrix: Hydrogels made of collagen, elastin, or fibronectin.
    • Allows cells to grow and differentiate into organoids.
    • More complex and finicky than 2D cultures but a better tissue model.
  • Advantages of 2D culture include simplicity, cost-effectiveness, speed, while disadvantages include a lack of realistic 3D interaction with the extracellular matrix and cells.
  • 3D systems enable the formation of organoids, with cells on the outside differentiating differently than cells in the middle due to oxygen and nutrient availability.
  • Modelling tumours in 3D:
    • Can model cells adapting to low oxygen and nutrients in the inner core.
    • Important for testing drug responses in tumor models.
  • Breast Cancer Cells Example:
    • Normal mammary epithelial cells form acini (mammary organoids) with cells around the outside differentiating and undergoing apoptosis on the inside.
    • Cancer cells or cells with oncogenes exhibit hyperproliferation without apoptosis, resembling a tumour.
    • Used to test drugs that inhibit proliferation, induce cell death, or inhibit migration using these 3D cultures.
  • Three-dimensional cultures offer the potential to generate organs in a dish and repair, maintain, or create new tissues.

Cytoskeleton

• Dynamic cytoskeleton regulates cell positioning within tissues.

• Components: microtubules, microfilaments, intermediate filaments.

• Learning targets:

  • General structure of microtubules and actin cytoskeleton.
  • Role of cytoskeleton dynamics.
  • How drugs affect the cytoskeleton.
  • Role of actin cytoskeleton in cell movement/migration and focal adhesions.

• Case Study: Helena, 45, diagnosed with breast cancer, prescribed paclitaxel.

• Cell Cytoskeleton:

  • Dynamic, not static.
  • Receives signals from outside and forces changes.
  • Provides shape, structure, robustness, polarity.
  • Involved in a range of functions, particularly cell division.

• Key components:

  • Microtubules: Made of tubulin dimer (alpha/beta tubulin).
    • Determine positions of organelles.
    • Intracellular transport.
    • Essential for mitotic spindle.
  • Microfilaments: Composed of actin protein.
    • Determine shape of cell surface.
    • Cell locomotion/migration.
    • Cytokinesis.
  • Intermediate Filaments: Composed of different proteins, mainly keratin.
    • Provide mechanical strength to cells within tissues.
  • These three components interact and regulate each other.

• Actin and Microtubules during Interphase and Mitosis:

  • Interphase:
    • Lots of actin microfilaments around the cortex of the cell and actin stress fibres going along the length of the cell into filipodia.
  • Mitosis:
    • The microtubules form kinetochore microtubules.
    • Actin is primarily around the periphery of the cell.
    • A contractile ring forms around the two daughter cells during cytokinesis.

• Microtubule Structure:

  • Formed by the addition of heterodimers composed of an alpha and a beta tubulin molecule.
  • 13 protofilaments around the circle.
  • Dynamic due to ability of alpha beta tubulin dimers to attach and fall off.

• Dynamic Instability:

  • Growth of microtubule always goes in the positive direction (plus end).
  • Back end is attached to the centrosome (minus end).

• Regulation by polymerization:

  • Addition of tubulin dimers and depolymerization.
  • Controlled by GTP.
  • On beta tubulin protein, there will be a GTP molecule bound.
  • When the dimer attaches to the end of the growing microtubule, that GTP will eventually be hydrolysed to GDP.
  • Once that's hydrolysed to GDP, then the microtubule is less stable, and it shorten.
  • The more dimers that you have adding and the more quickly you have dimers adding and you have still got GTP there before it gets hydrolysed, the microtubule will be growing.
  • If you only have low concentrations of dimers adding, then the GTP cap will be hydrolysed and the microtuber will start shrinking.
  • Catastrophe: Rapid shrinkage due to GTP hydrolysis.
  • Rescue: Regrowth when more dimers are added again.
  • If attached to the centrosome, growth and retraction occur at the plus end.
  • Stabilized by microtubule-associated proteins (MAPs).
  • Tau stabilizes microtubules; its phosphorylation is involved in Alzheimer's disease.

• Centrosome/Microtubule Organizing Center:

  • Anchors growing microtubules.
  • Composed of microtubules with gamma tubulin.
  • Important for cell division/mitosis; centrosome duplication occurs during the cell cycle.

• Mitosis:

  • Centrosomes duplicate during interphase.
  • Spindles separate sister chromatids during anaphase.
  • Microtubule dynamics are essential for all parts of mitosis; blocking polymerization or depolymerization can halt cell division and lead to cell death.

• Taxol (Paclitaxel):

  • Drug that binds to beta tubulin and stabilizes microtubules, blocking depolymerization and disrupting cell division.
  • Blocks cancer cells in mitosis, leading to cell death.
  • Effective for highly proliferative cancers but causes side effects by killing other dividing cells (e.g., epithelial stem cells, hair follicle cells).

• Actin Cytoskeleton:

  • Like tubulin, actin forms polymeric structures made of monomers that wrap around each other in an alpha helix.
  • Formed by G actin (globular actin).
  • Adds to the plus end of the microfilament.
  • Controls shape of the periphery of the cell.
  • Actin microfilaments/stress fibres are dispersed but concentrated under the plasma membrane and organized into lamellipodia and filipodia.

• Highly dynamic:

  • Constantly growing and shortening: growth at plus end, depolymerization at minus end.
  • Regulated by ATP: ATP-bound actin adds to the plus end, and ATP is hydrolyzed to ADP over time, leading to microfilament instability and loss of actin at the minus end.

• Natural Compounds Targeting Actin:

  • Death cap mushroom: Contains phalloidin, which binds to filamentous actin and prevents depolymerization, leading to liver failure and organ failure.
  • Cytochalazins: Derived from molds, bind to plus end and prevent polymerization.
  • Used in labs to study dynamics of the actin cytoskeleton rather than as therapeutics.

• Cell Migration:

  • Helena's breast cancer spread to the liver and bones.
  • Cancer cells can migrate and invade, entering the circulatory system and lodging elsewhere.
  • Migratory Phenotype: Cancer, fibroblasts, neutrophils.

• Cell Migration Steps:

  • Remodelling of focal adhesions at the leading edge.
  • Cells extend protrusions (lamellipodia and filipodia) in the direction of movement.
  • Formation of new focal adhesions.
  • Contraction of actin cytoskeleton.
  • Removal of focal adhesions at the back end of the cell using protease, calpane.
  • Actin cytoskeleton is the main component driving cell migration, but microtubules and intermediate filaments are involved as well.

• Cancer metastasis:

  • After getting into lymphatic/blood system, cancer cells need to extubate into a new tissue to form a new tumor.
  • Cancer cells lose migratory capacity, regain epithelial cell properties, and proliferate to form another tumour.
  • Targeting migration and invasion of cancer cells is one avenue for therapy.
  • Simultaneous addition of actin monomers at the plus end and removal at the minus end is called treadmilling.
  • Filopodia are thin, finger-like projections composed of bundled actin microfilaments involved in sensing the direction of movement.
  • Focal adhesions anchor the cells to the extracellular matrix for traction.

Cell Interactions

• Cells interact with each other, forming networks with importance for functions and cell polarity.

• Learning targets:

  • Cell adhesion definition.
  • Main types of cell junctions/adhesions and how they link to the cytoskeleton.
  • Importance of adherens junctions, tight junctions, desmosomes.
  • Functional significance of cell adhesions in defining cell polarity.
  • What can go wrong when cell adhesion/polarity is dysregulated, especially in cancer.

• Cell Adhesions:

  • Not just interactions with extracellular matrix (focal adhesions, hemidesmosomes).
  • Lateral cell to cell interactions are important.
  • Extremely important for development and maintenance of tissues.
  • Human cancers disrupt cell adhesions to proliferate and metastasize.

• Three main types of cell adhesions, from apical to basal:

  • Tight junctions: Most apical; forms a tight seal and controls what can and cannot move between cells.
  • Adherens junctions: Connection of cadherin proteins to the actin cytoskeleton; important for cell structure, response to cell signals, and coordinated movement.
  • Desmosomes: Composed of cadherin protein, attaches to intermediate filaments (e.g., keratins); structural support across a sheet of cells.
  • Gap junctions: Involved in movement of ions and signaling molecules between cells.

• Adherens and desmosomes are driven by cadherins rather than integrins like extracellular matrix adhesions.

• Tight Junctions:

  • Tight junctions stop pathogens and large macromolecules from getting in between cells.
  • Important controls proteins on the apical vs basolateral membranes.
  • Made up of claudens and occludens that interact with a zona occludins to the cytoskeleton.
  • Desmosomes: most basal cell cell junction, involved in adhering cells.

• Connect cell contact sites to that intermediate filament cytoskeleton made of keratin filaments to provide a lot of mechanical strength for the cell.

• Desmoglein and descolin are on the external side of cell and heterodimers are going to interact with same from adjacent cell that will form that desmosome, or adhesion.

• They can disassemble and reassemble, provides inter-cellular adhesion, important for strength in tissue that resists large stress, heart, skin.

• Mutations can lead to severe phenotype: Skin blistering and thickening because of too much fibrosis, they are very fragile blisters.

  • Cardiomyopathy also.

• Adherence Functions:

  • Slightly more apical from the desmosome.
  • Composed of proteins called coherence, but these are classified as classical coherence or e coherence.

• Cell Polarity:

  • Loss of polarity: all cancers.
  • Loss of cell cell adhesion contributes to cancer development.
  • Loss/ reduced expression of e cadherin, tight junction protein, zone 1, or example: then those cells becomes tumors and more migratory, they becomes meta- static.
  • Can promote tissue disorganization, which also leads too more cell division mitosis and, so faster growing cancers.

• Important component:

  • Loss of expression of E cadherin those cells are able to break loose of the tissue.
  • Can become migratory.

• Epithelial To Mesenchymal Transition TRANSITION:

  • That enables them to get into the blood vessels or the lymphatics.

• Matrix matalloproteasas: important to note key to breakdown tissue for growth.

• With normal EMT WOUND healing process is really and epithelial important cells for to process connect heal well via EMT like wound for epithelial process transition also.

• THE ABILITY of fibroblasts move a also with tissue damage EMT also via.

• What's the PRIMARY for characterized EMT for properties acquiring ADHESION to CELL lose epithelial and important. also for wound AND healing IMPORTANT development with METASTASES Cancer