Cell Culture and Cell-ECM Interactions

Cell-Extracellular Matrix Interactions

  • Last lecture focused on extracellular matrix (ECM) components.
    • Basement membrane (secreted by epithelial cells).
    • Connective tissue (secreted by fibroblasts), includes collagens and elastins.
  • Cells physically attach to the ECM via integrins.

Integrins

  • Key proteins for cell-matrix attachment.
  • Heterodimers made of alpha and beta isoforms.
  • Transmembrane proteins with a single pass through the plasma membrane.
  • External side interacts with ECM proteins like fibronectin and laminin (particularly in the basement membrane).
  • Internal side attaches to the cytoskeleton.
    • Focal adhesions attach to the actin cytoskeleton.
    • Epithelial cells attach to basement membranes via intermediate filaments (keratin filaments).
  • Functions:
    • Positioning cells within tissues.
    • Transmitting signals from the external environment to the cell interior (changes in shape, pressure, growth factors).
    • Structural and signaling functions.

Focal Adhesions

  • Multi-protein complexes linking the cell to the ECM via integrins.
  • Clusters of integrin heterodimers.
  • Attach to ECM proteins (fibronectins, laminins).
  • Link to the actin cytoskeleton via a large multi-protein complex.
    • Key proteins: paxillin and focal adhesion kinase (FAK).
  • Serve as receptors that receive signals from the external environment.
  • Integrin activation changes protein shape, sending signals to the actin cytoskeleton, triggering changes in cell shape.
  • Changes in integrin structure promote binding and clustering of proteins, regulating interaction with the actin cytoskeleton.
  • Focal adhesions are mediated by integrins, interacting with the actin cytoskeleton and ECM proteins.
Cell Shape
  • Non-adherent cells (e.g., white blood cells) are round and do not attach to a matrix.
  • Adherent cells (e.g., fibroblasts, epithelial cells) need to attach to a matrix to receive signals for division or differentiation.
  • Trypsin is used to cleave proteins in focal adhesions, causing cells to detach from the ECM and round up.
  • Focal adhesion proteins (integrin, paxillin, FAK) are visible under a microscope via staining.
  • Actin is the key cytoskeletal protein linking cells to the ECM via integrins.
    • Strong actin fibers (stress fibers) provide cell strength and shape.
    • Focal adhesions are multiprotein complexes, with over 200 different proteins linking integrins to the ECM and actin cytoskeleton.
Mutations in Integrins
  • Mutations in focal adhesion proteins or integrins can lead to severe diseases.
  • Integrins have alpha and beta isoforms with multiple subtypes (around 24).
    • Different subtypes determine different functions within cells.
    • Example: alpha-five subunit with beta-one binds to fibronectin and is present in virtually all cells.
      • Mutation leads to non-viable embryos.
    • Mutation in integrins for muscle attachment (muscular dystrophy).
    • Mutation in integrins for epithelial cell attachment to the basement membrane (severe blistering).
Integrins and Extracellular Cues
  • Cell-matrix adhesions respond to external cues, such as stretching and mechanical pressure, sending signals via integrins to change cell shape and migration.
  • Integrins translate extracellular cues from growth factors, regulating cell behavior within the ECM.
  • Signals may tell a cell to detach and proliferate, migrate and invade, or inhibit apoptosis. (Metastatic cancer)
Cancer and ECM Interactions
  • Hyperproliferation of epithelial cells puts pressure on the basement membrane.
  • Fibroblasts secrete more collagen, inducing a fibrotic response.
  • Cancer-associated fibroblasts secrete collagen, forming tracks for cancer cells to migrate along. Cancer cells migrate along collagen tracks and enter blood vessels.
  • Collagen fibers promote tumor invasion.
Growth Factors
  • Localized growth factors accumulate within the ECM, signaling to surrounding cells to proliferate.
  • Epithelial cells must be attached to the ECM to respond to growth factors and divide.
Anoikis
  • Loss of interaction with ECM induces cell death (anoikis).
  • Anoikis is a protective mechanism preventing cells from dividing in the wrong place.
  • Cancer cells overcome anoikis, becoming anchorage-independent.
  • Anoikis is defined as apoptosis triggered by lack of proper cell adhesion to the extracellular matrix.
Cell-ECM Interactions Take Home
  • Interaction between cells and their external environment is vital for cell function and disruptions contribute to diseases.

Cell Culture

Key Concepts

  • Adherent vs. suspension cells.
  • Specific requirements for cell culture and key challenges.
  • Primary cultures, transformed cell lines, and stem cells.
  • Advantages and disadvantages of studying cells in culture vs. in their natural environment.
  • Applications of cell cultures.
  • Future of bioengineering and organ-on-a-chip assays.

Cell Culture

  • Cell culture is a key tool to study protein function, elucidate molecular mechanisms of disease, and provide models for drug development.
Background
  • Cell culture has been around for over 100 years.
  • First reported cell culture was in 1906-1907 with frog cells.
  • Developments in cell culture media in the 1940s and 1950s led to Eagles medium.
  • HeLa cells were the first human cell line able to continuously divide in culture.
Applications
  • Making recombinant proteins for therapeutics, such as insulin.
  • Making antibodies, monoclonal antibodies, for therapeutics/lab tools.
  • Testing drugs and understanding cell behavior.
  • Inducing differentiated cells back into a stem cell-like phenotype (induced pluripotent stem cells; iPSCs).
Cell Examples
  • Fibroblasts (migratory, attach to the flask bottom).
  • Muscle cells (align together, can be induced to contract).
  • Nerve cells (large cell bodies, long extensions, can induce action potentials).
  • Epithelial cells (form sheets of cells, like to be next to each other). They form contacts with each other, as well as contacts with the extracellular matrix
  • Suspension cells (little round circles or spheres). Are able to survive and proliferate unattached
  • Cell cultures are used to look at proliferation, differentiation, migration, cell death, etcetera.

Advantages of cell cultures

  • Enables you to study the specific molecular nature of that individual cell away from all of the other cell types that might be there.
  • Study the molecular nature of individual cells.
  • Clone from one individual cell.
  • Manipulate the conditions in culture easily (add or remove growth factors, drugs).
  • Visualize cells down the microscope
  • Reduces the number of animals in experiment.

Disadvantages of cell cultures

  • Consider the biological relevance to the original tissue environment.
  • Primary culture is grown on a plastic dish, not within the context of their whole tissue
  • Continuous cultures are able to continuously proliferate
  • The more a cell divides, the more cells proliferate, the more chance they have of developing changes, developing mutations to their DNA, and also of getting epigenetic changes.

Types of Cell Cultures

Primary Cell Cultures
  • Cells taken directly from an animal (human or lab animal).
  • Isolate the specific cell type we're interested in (e.g. nerves).
  • Can grow these for a little while
  • Terminally differentiated cells like a nerve, they're not going to proliferate
  • Cells isolated using a protease (trypsin) or collagenase.
  • Used for fibroblasts, hepatocytes, chromaffin cells, neuronal cells.
  • Issue: they're not going to grow indefinitely
  • Fibroblasts can undergo 50-100 generations from embryos.
  • They reach a defined lifespan and stop dividing.
  • Closely related to the original site that they came from.
  • Very expensive to do experiments on because you have to keep going back to the primary source to be able to get those cells
Continuous or Transformed Cell Lines
  • Capable of indefinite growth in culture, when we give them the right environment and the right nutrients.
  • Derived from cancerous tumors or transformed cells (e.g. fibroblasts).
  • Treated with a DNA tumor virus that induces oncogenes to be expressed and inhibits tumor suppressors so that those cells can continue to divide and pass that senescent phase or that crisis phase.
  • Genetic mutation enables them to divide continuously and avoid programmed cell death and also senescence.
  • Undifferentiated or immature cells.
  • First immortal human cell line are HeLa cells that were originally derived from a 31 year old woman in America, Henrietta Lacks, and she had terminal cervical cancer.
  • Informed consent is required.
  • Easy to multiply in a lab setting, used world wide.
  • HeLa cells were used to test polio vaccine, isolate HIV.
Stem Cells
  • They're able to divide but also to differentiate.
  • Stem cells can be a really powerful model in the laboratory because we can have cells that have the ability to continue to proliferate.
  • Can be quite expensive to grow because the types of growth factors specifically to keep them alive
  • HEK293 cells were derived from human embryonic kidney cells in the 1970's
  • Embryonic stem cells can be controversial. So isolating embryonic stem cells would usually be from embryos the are left from IVF that are no longer required.
Induced Pluripotent Stem Cells (iPSCs)
  • Taking differentiated cells from an individual and give them the right transcription factors to switch on genes that tells it to behave like a stem cell.
  • Can use that same process to study cloning.
  • Identified just four genee that needed to be switched back on to reprogram those cells back into an embryonic stem cell.
Importance of iPSCs
  • Important for being able to study cell behavior and being able to study differentiation, the process of differentiation.
  • Really important for precision medicine. So, testing different drugs on that individual patients cells.
  • Can induce them to proliferate into a cell type the patient might need more of.