Biology 2382: Tutorial 1 - Why We Study Cells and Cell Culture Techniques

Introduction to Cell Biology Foundations

• Tutorial Series Introduction: Dr. Joshua Pemberton introduces the Biology 2382 tutorial series, a guided tour of cell biology topics including why cells are studied, the history of cell theory, and modern cell culture techniques.

• Primary Inquiry: The tutorial seeks to answer why cells are considered such a powerful model for understanding biological systems.

Causation and Complexity in Biological Systems

• Concept of Multilevel Representation: Prominent physiologist Dennis Noble introduced the idea of multilevel representation of causation in biology.

• Hierarchical Levels of Organization: Biological complexity builds from the most basic units up to environmental interactions:

  • Genes

  • Proteins

  • Subcellular networks

  • Cells

  • Tissues

  • Organs

  • Organ systems

  • Organism

  • Environment

• Experimental Approaches in Biology:

  • Reductionist Causal Chain: This approach attempts to understand the most fundamental building blocks first. It moves from characterizing individual genes to understanding proteins, how they form networks, and how those networks regulate cell and tissue function.

  • Downward Causal Chain: This approach attempts to understand higher-order systems first. By characterizing systemic behavior, researchers identify key components at lower levels of complexity.

• Centrality of Cells: Cells occupy a unique position in the middle of biological complexity, making them excellent models for both reductionist and downward causal chain research.

Evolution and Historical Foundations of Cell Theory

• Evolutionary Ancestry: All living organisms descended from a single common ancestral cell. The complexity and branching of the resulting evolutionary tree are still being understood.

• Foundational Quote: The course textbook, Molecular Cell Biology, opens with the quote: "Nothing in biology makes sense except in the light of evolution."

• Pillar of Discipline: A foundational pillar of cell biology is "seeing is believing," emphasizing the role of morphology and light microscopy in shaping our understanding of internal cellular compartments.

• Historical Timeline of Cell Theory:

  • 1839: Matthijs Schliden (focused on plants) and Theodor Schwann (focused on animal cells) published Microscopical Researches into the Accordance in the Structure and Growth of Animals and Plants.

  • Original Tenets of Cell Theory:

    1. Cells are the elementary parts of all tissues.

    2. A universal principle of development of organisms is that they are formed of cells.

    3. Synthesis of these ideas: All living organisms are composed of cells, and the cell is the most basic unit of life.

  • 1855 to 1858: Rudolf Virchow established the third tenant: omnis cellula e cellula (every cell from a preexisting cell).

  • Rejection of Spontaneous Generation: Virchow's tenant formally established that cells make other cells and biological matter is not generated spontaneously.

Implications of Cell Theory and Cellular Pathology

• Total Activity Principle: Theodor Schwann articulated that if cells are the basic unit of life, the activity of an entire organism depends on the total activity of its independent constituent cells.

• Input/Output Models: This led to models where cell behavior is determined by inputs from the extracellular environment, which are then coded into cellular outputs.

• Homeostasis: The response mechanism of the cell to the outside environment, which must be controlled constantly, introduced the concept of homeostasis.

• Cellular Pathology: Rudolf Virchow defined pathology through the lens of cell biology in his book Cellular Pathology as Based upon Physiological and Pathological Histology.

  • Virchow proposed the cell as the basic element of all pathological processes.

  • His early drawings of tumors demonstrated that changes in structure and morphology are the basis of disease.

  • This remained the dominant concept for understanding disease until the modern molecular pathology revolution.

Modern Organelles and Comparative Model Systems

• Definition of Organelles: According to the National Cancer Institute dictionary, an organelle is a "small structure in a cell that is surrounded by a membrane and has a specific function."

• Subfunctionalization: The compartmentalization of cellular processes into specific membrane-bound units allows for specialized functions.

• Model Organisms: Integrative cell biology relies on various model systems, selected for their fit at different levels of biological causality.

  • Krogh's Principle: August Krogh introduced the idea that for a large number of problems, there will be a specific animal (or small group of animals) of choice on which it can be most conveniently studied.

  • Drosophila and Zebrafish: Often used to study how evolution impacts physiology at the tissue and organism levels.

  • Molecular and Structural Biology: These fields often require models with more sequence information available than traditional organismal models.

Fundamental Principles of Cell Culture

• HeLa Cells: The first human-derived cell line was taken from a malignant cervical tumor in 1952. While used for foundational observations, these cells were obtained without the consent of the patient, Henrietta Lacks. This underscores modern ethical issues regarding the ownership of biological materials.

• Core Definitions:

  • Primary Culture: The stage where cells are initially isolated from the tissue of interest and allowed to proliferate.

  • Cell Line: Established after cells are transferred from the primary culture to a new media. Initially has a limited lifespan.

  • Cell Strain: A subpopulation of a cell line where there is positive selection for a particular component or feature, leading to genotypic or phenotypic uniformity (e.g., better attachment or faster growth).

• Kidney Cell Lineage Example:

  • Primary Kidney Cell Culture: Freshly derived, finite lifespan.

  • 293 Cell Line: Immortalized version of kidney tissue isolation, grows indefinitely.

  • HEK 293 2 sus Cell Strain: Selected from the 293 line specifically for its ability to grow in suspension without attaching to surfaces.

• Primary Cells vs. Immortalized Cell Lines:

  • Primary Cells: Benefit from high biological relevance (mimics in-organism physiology); drawback is they are time-consuming, finite, and have high variability between donors.

  • Immortalized Lines: Benefit from infinite lifespan, ease of use, low expense, and minimum variability; drawback is potential biological "drift" over time.

• Cell Transformation: In primary cultures, some cells may undergo spontaneous oncogenic mutations, becoming tumorigenic. This process, called transformation, allows cells to grow indefinitely.

• Contact Inhibition: Noncancerous cells stop proliferating when they touch each other, maintaining parallel orientation. Transformed cells lose contact inhibition, growing randomly or on top of one another.

Environmental and Nutritional Requirements of Cultured Cells

• Incubation Conditions: Most human cell cultures require an artificial environment maintained at $37\,^{\circ}C$ in a carbon dioxide incubator.

• pH Buffering: Cells give off $CO_2$ and require it for growth. Bicarbonate is added to the media to buffer the $CO_2$.

  • Concentration: Roughly $5\%\,CO_2$.

  • Target pH: Physiological range of $7.2 - 7.4$.

• Types of Cultures:

  • Adherent Cultures: Anchorage-dependent cells that must attach to a substrate.

  • Suspension Cultures: Cells that float in the media.

• Essential Media Components: A delivery device for salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones, trace elements, and potentially antibiotics.

• Phenol Red: A pH indicator used to monitor the acidity of the media:

  • Deep Red/Orange: $7.0 - 7.4$ pH (Healthy).

  • Yellow: Acidic conditions.

  • Pale Pink: Basic conditions.

• Serum: A supplement used to deliver essential growth factors and hormones to the culture.

• HEPES: A chemical buffer used to maintain stable pH outside of a $CO_2$ incubator by accepting or donating hydrogen ions to minimize fluctuations.

Morphological Classifications and Visual Monitoring

• Five Major Morphological Groups:

  1. Epithelial-like: Columnar or cuboidal in shape.

  2. Endothelial: Elongated cells.

  3. Fibroblasts: Spindle-shaped and spread out, making them ideal for imaging.

  4. Lymphoblast-like: Spherical, ranging from small to large (typically grow in suspension).

  5. Neuronal: Extended pyramidal or spindle-shaped.

• Indicators of Unhealthy Cells:

  • Blebs/Ripples: Found at the cellular edge.

  • Granulations: Often indicates necrotic cells or programmed cell death.

  • Multinucleated: Common in stressed or transformed cells.

• Biological Contamination:

  • Visible/Macro: Bacteria, yeast, and fungal filaments (molds).

  • Unseen: Viruses and Mycoplasma.

  • Mycoplasma Detection: Mycoplasma is a common contaminant from human skin. It is detected using non-visual methods like PCR amplification of species-specific amplicons.

The Cellular Growth Cycle and Subculturing Protocols

• Growth Curve Phases:

  1. Lag Phase: Occurs immediately after seeding; growth is slow as cells recover from the stress of subculturing.

  2. Exponential (Log) Phase: Period of rapid proliferation until the surface is occupied or nutrients are depleted.

  3. Stationary Phase: Proliferation slows and eventually stops.

• Confluency: The percentage of the field of view covered by cells.

• Subculturing Timing: Subculturing (passaging) must occur near the end of the exponential phase—typically at $70\text{--}80\%$ confluency—to avoid a "culture crash" or irreproducible changes.

• The Detachment Process:

  • Dissociation Reagent: Typically a combination of Trypsin (a protease) and EDTA (a divalent cation chelator).

  • Mechanism: Trypsin breaks proteins anchoring cells to the surface; EDTA binds $Ca^{2+}$ and $Mg^{2+}$ ions used as cofactors by cell adhesion molecules.

Practical Case Study: Demonstration of Subculturing Procedures

• Step-by-Step Subculture Protocol (conducted by TA Libby):

  1. Aseptic Technique: Sterilization of gloves and surfaces with ethanol; use of a biosafety cabinet with unidirectional HEPA-filtered airflow.

  2. Inspection: Checking cell confluency and health via light microscopy.

  3. Reagent Preparation: Warming media and trypsin to $37\,^{\circ}C$ in a water bath.

  4. Aspiration and Washing: Removing old media and washing with buffered saline to remove residual serum (which inhibits trypsin).

  5. Enzymatic Treatment: Adding trypsin and incubating at $37\,^{\circ}C$ for approximately $5\text{--}15$ minutes until cells round up and detach.

  6. Inhibition: Adding "complete media" (containing serum) to the dish to neutralize the trypsin activity.

  7. Trituration: Pipetting the solution up and down to ensure all cells are in suspension and disaggregated from the surface.

  8. Harvesting: Transferring the suspension to a tube and using a centrifuge to spin down a cell pellet.

  9. Resuspension: Aspirating the supernatant (trypsin/media mix) and adding fresh complete media to the pellet.

  10. Seeding: Placing the resuspended cells into fresh culture vessels (flasks, dishes, or 96-well plates).

• Long-term Storage: Cells can be cryopreserved in liquid nitrogen at approximately $-140\,^{\circ}C$.