Cell Biology & Tissue Culture: Biology of Cultured Cells and HeLa

Learning Objectives for Biology of Cultured Cells and Tissue Culture

• Distinguish major types of mammalian cells in culture and explain their laboratory applications.
• Analyse healthy and unhealthy cell morphology to assess cell condition and suitability for subculture.
• Evaluate confluence levels and justify decisions related to passaging and experimental timing.
• Interpret morphological changes in response to culture conditions and potential contamination.
• Document cell culture observations using accurate terminology, annotated images, and compliant laboratory records.
• Reflect on the ethical considerations associated with human-derived cell lines such as HeLa.

Fundamental Concepts of Cell Morphology

• Definition of Morphology: In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features.
• Importance in Tissue Culture:     - It is essential for regular examination to ensure successful experiments.     - Routine daily observation using an inverted microscope is standard practice.     - Visual assessment allows a technician to determine if cells are unhealthy, require splitting, or need feeding.     - Accurate visual assessment can prevent contamination outbreaks and preserve the integrity of the cell line.

Phase Contrast Microscopy

• Definition: A type of light microscopy that enhances contrasts of transparent and colourless objects by influencing the optical path of light.
• This is a critical tool for looking at unstained cultured cells.

Growth Modes in Cell Culture

Cell cultures take one of two forms based on их growth mode, which typically reflects the tissue from which they were derived:

• Adherent Culture:     - Cells grow as a monolayer attached to the tissue culture flask.     - Typical of cells derived from solid tissues (e.g., lungs, kidney).
• Suspension Culture:     - Cells grow as single cells or small free-floating clumps.     - Typical of cell lines derived from blood (e.g., leukemia, lymphoma).

Confluence and Cell Density Coverage

• Confluence: A measure of the number of cells attached to a substrate; it refers to the coverage of the dish or flask by the cells.
• Confluent (Confluent Monolayer): Occurs when the substrate of the culture vessel is completely covered with cultured cells, and there is no room for cells to grow further as a monolayer.
• Confluence Levels:     - Low: $10\%$, $20\%$, or $30\%$.     - Moderate: $50\%$ or $70\%$.     - High: $80\%$ or $90\%$.     - Full: $100\%$.
• Over-confluent state:     - Characterized by unclear edges.     - Large areas of cells begin growing on top of each other.     - The monolayer structure is lost.

Classification of Cell Morphology Types

Cells in culture are categorized into four primary morphology types:

1. Fibroblasts

• Characteristics:     - Principal active cell of connective tissue.     - Irregularly shaped and adherent.     - Known for producing large amounts of extracellular matrix (ECM) proteins such as collagen, glycosaminoglycans, and proteoglycans.
• Appearance:     - May appear bipolar (two extensions), multipolar (many extensions), and elongated.     - Frequently form swirls in heavy (dense) cultures.
• Applications:     - Active immune regulators that amplify inflammation by recruiting immune cells.     - Used to model disease mechanisms, study cell-cell interactions, and test therapeutics for chronic inflammation.

2. Epithelial Cells

• Characteristics:     - Line internal surfaces of the body (e.g., gastrointestinal tract, respiratory tract, nephron tubules).     - Specialized functions include protection (skin), absorption (nutrients in the gut), and secretion (hormones, mucus).
• Appearance:     - Attached to a substrate.     - Appear flattened and many-sided (polygonal).     - Growth Pattern: They form sheets or patches rather than spreading individually.
• Examples: HeLa, MDCK, Caco-$2$.

3. Endothelial Cells

• Characteristics:     - Form the tunica intima (interior surface of blood vessels).     - Functions as a selective filter for gases, fluids, and immune cells.     - Possess a smooth anticoagulant surface.
• Appearance:     - Grow as a single layer.     - Display a typical ‘cobblestone’ monolayer pattern at stationary density.
• Example: HUV-EC-C (HUVEC), isolated from the human umbilical vein, used in cardiovascular research.

4. Neuronal Cells

• Characteristics:     - Responsible for sending and receiving neurotransmitters.     - Categorized into sensory neurons, motor neurons, or interneurons.
• Culture Challenges:     - Particularly difficult to culture because mature neurons do not undergo cell division.
• Applications: Fundamental to understanding the functioning of the nervous system.

5. Lymphoblast Cells

• Characteristics:     - Hematopoietic origin (blood cells).     - Do not attach to a substrate (remain in suspension).     - Spherical shape.
• Example: Raji line (established in $1963$ from an $11$-year-old male with Burkitt's lymphoma).

Historical Timeline of Mammalian Tissue Culture

• $1907$: Nervous cells isolated from the spinal cord.
• $1912$: Cell cultures made viable for longer periods through asepsy and nutrients.
• $1951$: HeLa cell lineage established.
• $1956$: Cell agglomerates formed on rat-tail collagen.
• $1975$: $3D$ cell culture using human progenitor cells.
• $1980$ - $1990$: Development of "organoids" (neuroblastoma and lung).
• $1981$: Isolation of mouse ESCs (Embryonic Stem Cells).
• $1993$ - $1997$: $3D$ culture used for transplantation (cornea and skin).
• $1998$: Isolation of human ESCs.
• $2006$: Establishment of iPSCs (induced Pluripotent Stem Cells).
• $2009$: Description of mini-guts (small intestine organoids).
• $2012$: Establishment of iNSCs (induced Neural Stem Cells).

Summary Table of Common Cell Lines

Passage Number

• The passage number indicates the total number of times a cell line has been sub-cultured, passaged, or split since its original isolation.

The HeLa Cell Line and Henrietta Lacks

• The Origin Story:     - Henrietta Lacks was an African-American mother of five who visited Johns Hopkins Hospital in $1951$ for vaginal bleeding.     - Dr. Howard Jones discovered a large malignant tumor on her cervix.     - During her treatment (radium therapy), a biopsy sample was sent to Dr. George Gey.
• Scientific Breakthrough:     - Dr. Gey found that while other samples died, Lacks' cells doubled every $20$ to $24$ hours.     - They became the first immortalized human cell line.
• Biological Characteristics:     - Telomerase: HeLa cells have an active version of telomerase during division, preventing the shortening of telomeres.     - Hayflick Limit: By maintaining telomeres, HeLa cells circumvent the Hayflick Limit (the finite number of divisions a normal cell can undergo before senescence).     - Genome: Horizontal gene transfer from HPV$18$ created the HeLa genome, which differs from Henrietta Lacks' original genome.     - Chromosome Count: They are hypertriploid ($3n+$), containing approximately $76$ to $80$ chromosomes instead of the normal human diploid count of $46$.
• Global Impact:     - Used to test human tissue outside the body for the first time.     - Sent to space to study the impact of zero gravity.     - Used to study effects of radiation from nuclear explosions.     - Utilized in the beauty industry for cosmetic side-effect testing.     - Essential for studying genetic mutations, developing cancer therapeutics, and treating infectious diseases.

Pathological Morphology Changes

• Viral Infection (e.g., HSV-$1$):     - Normal corneal epithelial cells display a "cobblestone" appearance.     - Upon infection, a Cytopathic Effect (CPE) is observed.     - Symptoms include increased space between infected cells, appearing as early as $8$ hours post-infection ($8\,h\,p.i.$).