Cell Structure & Cells in Culture – Comprehensive Study Notes

The Cell Theory and Basic Cell Structure

• Objectives context: Understand cell structure, cell types, plasma membrane components and functions, cellular components, and autophagy.
• The Cell Theory (historical and modern):
  • 1839: Schleiden (botanist) and Schwann (zoologist) proposed core ideas:
  • All living things are composed of cells.
  • Each new cell arises from division of a preexisting cell.
  • A cell is the smallest functional unit with all life properties.
  • Modern cell theory adds: energy flow occurs within cells; hereditary information (DNA) passes cell-to-cell; all cells share basic chemical composition.

Basic Structure of the Cell and Cellular Organization

• Cellular morphology varies widely by cell type (examples):
  • Squamous, spheroid, stellate, fibrous shapes.
• Major components:
  • Plasma (cell) membrane: outer envelope.
  • Cytosol: fluid inside the cell.
  • Organelles: ultramicroscopic structures in cytosol that carry out cellular functions.
• Cell size considerations:
  • Limits to cell size relate to surface area-to-volume ratio; smaller cells exchange materials more efficiently.

Cell Size and Surface Area–Volume Considerations

• Surface area (SA) and volume (V) scale differently with diameter d:
  • SA \propto d^2, V \propto d^3
• For a cube with side length d, approximate formulas:
  • SA = 6d^2,\n V = d^3.
• Example (dimensionful):
  • For d = 10 μm:
  • SA = 6(10)^2 = 600\,\mu m^2, V = (10)^3 = 1000\,\mu m^3.
  • For d = 20 μm:
  • SA = 6(20)^2 = 2400\,\mu m^2, V = (20)^3 = 8000\,\mu m^3.
• Implications:
  • Larger cells contain more cytoplasm requiring more nutrients and waste removal, but have relatively less membrane surface area per unit volume for exchange.

Prokaryotic vs Eukaryotic Cells

• Eukaryotic cells:
  • Contain a nucleus; interior divided into functional compartments; typically have organelles and can undergo sexual reproduction.
• Prokaryotic cells:
  • Lack defined nucleus; nucleoid region instead.
  • Generally reproduce asexually.
• Cellular organization context: plasma membrane encases the cell; DNA resides in the cytoplasm or nucleus (in eukaryotes).

The Plasma Membrane: Structure and Function

• Double layer of phospholipids and proteins; encloses the cell; separates internal/external environments.
• Functions beyond barrier:
  • Provides attachment sites; mediates cell‑cell recognition; membrane proteins act as sensors of external signals.
• Membrane lipid composition (lipids make up ~50% of the membrane by mass):
  • Phospholipids form a bilayer; amphiphilic nature (
    hydrophilic heads, hydrophobic tails).
  • Cholesterol interspersed among phospholipid tails; modulates membrane cohesion and fluidity.
  • Glycolipids and cholesterol contribute to membrane dynamics and microdomains.

Lipids of the Membrane

• Phospholipids: most abundant membrane lipids; form bilayer; amphiphilic; dynamic.
• Cholesterol: interspersed among tails; stabilizes and tunes fluidity.
• Glycolipids: phospholipids with carbohydrate chains; located on extracellular surface; contribute to glycocalyx.
• Membrane fluidity and lateral mobility:
  • Lipids diffuse laterally within a monolayer at high rates (lateral diffusion ~ 10^7 moves/sec).
  • Transbilayer flip-flop (outer to inner leaflet) is rare and mediated by flippases, floppases, scramblases.
• Transbilayer transport proteins:
  • Flippase (P-type ATPase): moves phospholipids such as PE and PS from outer to cytosolic leaflet.
  • Floppase (ABC transporter): moves phospholipids from cytosolic to outer leaflet.
  • Scramblase: moves lipids in either direction toward equilibrium.

Lipid Rafts and Membrane Microdomains

• Lipid rafts are small (10–200 nm), dynamic, sterol- and sphingolipid-enriched domains in the plasma membrane.
• Characteristics:
  • Enriched in sphingolipids, cholesterol, and saturated fatty acids; reduced polyunsaturated fatty acids.
  • More ordered and tightly packed than surrounding bilayer, yet fluid.
  • Associated with cell signaling; act as platforms for signaling molecules.
• Functional significance:
  • Organize signaling, trafficking, and protein–protein interactions.
  • Linked to cancer hallmarks and various signaling pathways.
• Relevance to neuroscience and disease:
  • Lipid rafts influence neurotransmission, synaptic plasticity, and neurodegenerative disease processes; cholesterol metabolism impacts raft integrity and synapse maintenance.

Membrane Asymmetry and the Fluid Mosaic Model

• Asymmetry: the two leaflets have distinct lipid compositions:
  • Outer leaflet enriched in choline-containing phospholipids (phosphatidylcholine, sphingomyelin).
  • Inner leaflet enriched in aminophospholipids (phosphatidylserine, phosphatidylethanolamine).
  • Cholesterol is thought to be distributed relatively evenly across leaflets.
• Maintenance by limited flip–flop, with active enzymes regulating distribution.
• Fluid mosaic model: a bilayer with mobile proteins embedded in a sea of lipids; membrane lipids comprise about 90–99% of membrane molecules (lipids ~75% phospholipids, ~20% cholesterol, ~5% glycolipids).

Membrane Proteins and Their Functions

• Proteins constitute 1–10% of total membrane molecules but ~50% of membrane weight due to size.
• Major classes:
  • Integral (intrinsic) proteins: span the membrane; can form channels.
  • Peripheral (extrinsic) proteins: attached to integral proteins or lipids on either surface.
• Canonical functions:
  • Transport (channels and carriers)
  • Enzymatic activity
  • Receptors for signaling ligands
  • Cell-cell recognition
  • Cell-adhesion functions
• Receptor proteins: bind ligands (e.g., hormones, neurotransmitters) to mediate intercellular communication; ligand specificity depends on receptor presence.
• Glycoprotein cell identity markers: part of the glycocalyx; identification tags for self vs foreign.
• Cell‑adhesion molecules (CAMs): Integrins; connect to other cells or extracellular matrix; support cell shape, adhesion, and movement.
• Enzymatic proteins: catalytic activity at membrane surfaces; example: brush-border enzymes in intestinal lining; degrade hormones/ neurotransmitters after their job

Cytoplasmic Organelles and the Endomembrane System

• Cytoplasm components:
  • Cytosol: intracellular fluid with solutes and proteins.
  • Cytoplasmic inclusions: glycogen, lipids, pigments.
  • Cytoskeleton: filaments and tubules for structural support and movement.
• Endomembrane system components:
  • Nuclear envelope
  • Endoplasmic reticulum (ER): rough and smooth
  • Golgi apparatus
  • Vesicles
• Function of the endomembrane system:
  • Compartmentalizes cellular reactions; restricts enzymatic reactions to specific compartments; facilitates transport between organelles.

Endoplasmic Reticulum (ER) and Golgi Apparatus

• ER: network of interconnected membranes continuous with the outer nuclear membrane.
  • Rough ER: studded with ribosomes; synthesizes and processes proteins; folds polypeptides.
  • Smooth ER: no ribosomes; lipid synthesis; carbohydrate and fat metabolism; detoxification; forms transport vesicles.
• Golgi apparatus: stack of flattened cisternae; functions include modification, packaging, and distribution of proteins and lipids for secretion or internal use; vesicles fuse with Golgi from ER; modified proteins packaged into secretory vesicles for targeted delivery; some secretory vesicles wait for signals (e.g., insulin release).

Lysosomes, Peroxisomes, and Vacuoles

• Lysosomes: acidic vesicles with hydrolytic enzymes; digest intracellular and extracellular materials; recycle worn-out organelles; “suicide bags.”; secretory lysosomes exist in immune cells and melanocytes; degrade bacteria, viruses, toxins.
• Lysosomal storage diseases:
  • Tay–Sachs disease: lysosomal enzyme defect; accumulation of gangliosides; affected children die by around age five.
  • Niemann–Pick disease: lipid and cholesterol metabolism defects; buildup of lipids in organs; three common forms (Types A, B, C) with severe liver disease, seizures, and neuromotor issues; currently no cure.
• Peroxisomes: enzymes that break down hydrogen peroxide, alcohol, and other toxins.
• Vacuoles: storage/ waste disposal compartments; larger than vesicles; can store water and other materials.

Ribosomes and Mitochondria

• Ribosomes: not membrane-bound; composed of protein and rRNA; sites of protein synthesis; two main types:
  • Free ribosomes: synthesize intracellular proteins.
  • Membrane-bound ribosomes: synthesize secreted proteins or those destined for membranes.
• Mitochondria: the powerhouse of the cell; site of respiration and ATP production.
  • Double-membrane structure with cristae; matrix inside inner membrane.
  • Contain own DNA and ribosomes; possess a prokaryotic-like protein synthesis machinery (mitochondrial genome).
  • Evidence of endosymbiotic origin; maternal mitochondrial DNA inheritance; mutations affect energy-demanding tissues (nerve and muscle).

Autophagy: Self-Eating and Cellular Recycling

• Autophagy is a lysosomal degradation pathway essential for survival, differentiation, homeostasis, and defense against pathogens.
• Triggers: starvation, hypoxia, stress; nutrients recycled from bulk cytoplasm to sustain cells.
• Selective autophagy targets damaged organelles, protein aggregates, intracellular pathogens.
• Autophagosome biogenesis (overview):
  • Atg9-positive vesicles seed the process and fuse into a phagophore (double-membrane sheet).
  • Beclin1 and PI3K stabilize and extend membrane; the cup engulfs mitochondria, peroxisomes, ribosomes, inclusions, and cytosol.
  • The phagophore closes to form the autophagosome, which then fuses with lysosome to form autolysosome where hydrolases digest contents.
• Molecular players mentioned:
  • LC3, p62, Beclin1, PI3K, Atg proteins.
• Medical relevance:
  • Clearance of toxic protein aggregates linked to neurodegenerative diseases (e.g., Parkinson’s).
  • Autophagy as a defense mechanism against pathogens; significant interest in therapeutic modulation.
• Nobel Prize (2016): Yoshinori Ohsumi awarded for discoveries of autophagy mechanisms.

Cells in Culture: Overview and Rationale

• Definition: cells from living organisms grown on plastic or glass in controlled conditions (in vitro) vs in vivo in organisms.
• Conditions for growth:
  • Incubators maintain body temperature and CO2; sterile laminar flow hoods to maintain asepsis.
  • Special media with nutrients to support growth and division.
• Needs and purposes of cell culture:
  • Model systems for cell biology, disease interactions, drug effects, aging and nutrition studies.
  • Toxicity testing and pharmacology.
  • Cancer research: compare normal vs cancerous cell responses; study signaling.
  • Virology: virus cultivation for vaccines and studying replication.
  • Genetic engineering: protein production, vaccine manufacturing.
  • Gene therapy applications.
• Benefits:
  • Uniform, controlled environment reduces organismal variability; reproducibility across experiments; ethical advantages over animal use.

Cultures, Subcultures, and Cell-Line Nomenclature

• Key terms:
  • Clone: population derived from a single cell; genetically identical.
  • Sub-culture: transferring cells from one vessel to another.
  • Primary culture: cells taken directly from tissue.
  • Secondary culture: derived after initial culture.
  • Established/Stable/Continuous cell line: immortalized, often tumor-derived or transformed with viral elements; widely used (e.g., CHO cells).
  • Passage number: count of successive subcultures from primary culture.
• Primary vs Secondary vs Immortalized lines:
  • Primary: finite lifespan; maintain differentiated phenotype; anchorage-dependent; contact inhibition.
  • Secondary: derived from primary; more homogeneous; still finite lifespan.
  • Immortalized: indefinite propagation; loss of anchorage dependence and contact inhibition; often homogeneous;
    can be transformed by viral genes or oncogenes.
• Commonly used cell lines: e.g., CHO, HeLa, MDCK, 3T3, COS, HEK293, etc. (Table 8-1 examples).

What Cells Need to Grow In Culture

• Requirements:
  • An adhesion surface and a suitable liquid medium in culture vessels.
  • Appropriate environment: CO2, temperature around 37°C, humidity; oxygen tension typically ambient.
  • Sterility: aseptic technique; antibiotics/antimycotics as needed.
• Medium components:
  • Basal media: provide pH/osmolarity balance, energy source (glucose), salts, minerals; pH indicator (phenol red).
  • Supplements: antibiotics (penicillin/streptomycin) to prevent bacterial contamination; non-stable amino acids like glutamine; buffers.
  • Serum: provides growth factors and nutrients; derived from cow/horse/sheep.
  • Additional nutrients: amino acids, vitamins, lipids, growth factors.
• Common washing and detachment steps:
  • Phosphate Buffered Saline (PBS): wash to remove serum that inhibits trypsin; warmed to avoid shock.
  • Trypsin-EDTA: detaches adherent cells; EDTA chelates Ca2+ to enhance trypsin activity; serum in medium can reduce trypsin efficiency.
  • Bleach for disposal: used to sterilize surfaces and dead cells before disposal.

Generating Stable Cell Lines and Immortalization

• Immortalization methods:
  • Transduction with viral genes: SV40 T antigen, EBV, Adenovirus E1A/E1B, HPV E6/E7.
  • SV40 T antigen is a common, reliable transformational agent.
  • Viral genes inactivate tumor suppressors (p53, Rb) to bypass senescence.
  • Telomerase (TERT) transfection maintains telomere length, enabling extended replication; immortalized lines often retain key phenotypes.
• Concept of senescence: loss of the ability to divide; immortalization bypasses this.
• Table example: commonly used cell lines include CHO, HeLa, HEK293, MDCK, and others; many are tumor-derived.

Stem Cells, Differentiation, and Cloning Concepts

• Aging and cellular aging factors:
  • Cellular clock (finite divisions), death genes, DNA damage at telomeres, free radicals, and mitochondrial damage contribute to aging.
  • Telomeres and telomerase: TTAGGG repeats protect chromosome ends; telomerase maintains telomere length in some cells.
• Stem cells by differentiation potential:
  • Totipotent: can form an entire organism; early embryo cells (1–3 days).
  • Pluripotent: can form any cell type (over 200); cells from the blastocyst stage (5–14 days).
  • Multipotent: restricted to a limited set of lineages; fetal tissue, cord blood, adult stem cells.
• Fate of embryonic stem cells:
  • Blastocyst cells are embryonic stem cells with potential to form various lineages.
• Hematopoietic lineage: transcription factors regulate myeloid, lymphoid, erythroid differentiation.
• Reproductive and therapeutic cloning concepts:
  • Clone: genetically identical; considerations about mitochondrial DNA (mDNA) from egg cytoplasm.
  • Nuclear transfer strategies: in vitro reprogramming and in vivo transplantation routes (somatic cell nuclear transfer, i.e., cloning).
• Hybridoma technology for monoclonal antibodies:
  • Hybridoma production involves fusing a specific antibody-producing B cell with an immortal myeloma cell to yield a cell line that produces a single type of antibody.
  • Monoclonal vs polyclonal antibodies: monoclonal is specific to one epitope; polyclonal is a mixture.
  • Feeder cells provide growth factors to support hybridoma growth.

Practical Notes on Hybridoma Preparation and Antibodies

• Production steps include fusion of B cells and myeloma cells, selection, cloning, and expansion of hybridomas.
• Monoclonal antibodies are produced by hybridomas and are used in diagnostics and therapeutics; polyclonal antibodies are derived from multiple B cell clones.

Summary of Key Takeaways

• Cells are organized into a hierarchy from molecules to organelles to membranes to whole organisms; the plasma membrane is a dynamic, selectively permeable barrier with lipids and proteins.
• The endomembrane system compartmentalizes cellular processes; organelles contribute to protein synthesis, processing, and degradation.
• Autophagy is a central cellular recycling process with roles in development, immunity, and disease; Nobel Prize in 2016 recognized its mechanisms.
• Cultured cells are essential tools in biology and medicine, enabling controlled studies of cell behavior, drug effects, and disease processes; immortalized cell lines and stem cell technologies expand experimental possibilities.
• Stem cells offer potential for regenerative medicine; cloning and hybridoma technologies underpin advances in therapeutics and diagnostics.
• Lesion-based considerations (Tay–Sachs, Niemann–Pick) underscore the clinical relevance of lysosomal function and lipid metabolism in human disease.

Important Numerical and Formula References (recap)

• Surface area vs volume relationships:
  • SA \propto d^2, \quad V \propto d^3
• For a cube with side length d:
  • SA = 6d^2,\\
    V = d^3
• Lipid raft size: 10\,\text{nm} \leq L \leq 200\,\text{nm}
• Lipid diffusion: \text{lateral diffusion} \approx 10^7\; \text{moves/sec}
• Nuclear DNA content conventions: Humans have 46\text{ chromosomes} \; (23\text{ pairs})
• Telomere sequence: ext{TTAGGG}
• Embryonic stem cell potency: totipotent, pluripotent, multipotent definitions and examples as described
• Major differentiation potentials and lineage examples noted in stem cell sections
• 2016 Nobel Prize: Yoshinori Ohsumi for autophagy mechanisms

References to Clinical and Experimental Context (as taught)

• Lipid rafts and signaling in cancer and neurobiology contexts.
• Cholesterol metabolism impacts raft stability and synaptic function; statin effects discussed in broader lectures.
• Pathologies connected to lysosome function highlight the importance of cellular recycling in health and disease.
• Hybridoma technology underpins monoclonal antibody production for diagnostics and therapeutics.