Cell Structure & Cells in Culture – Comprehensive Study Notes

Objectives – Session 1: Cell Structure

By the end of the section, you should be able to:
• Describe overall cell structure and major cell types (prokaryotic vs. eukaryotic).
• List plasma-membrane components and explain their functions.
• Recognize all principal organelles and state their roles.
• Define autophagy and outline its biological importance.

Cell Theory & Basic Principles

1839 (Schleiden – plants; Schwann – animals) → classical cell theory:
• All living things consist of cells.
• Each new cell arises from division of a pre-existing cell.
• The cell is the smallest unit that exhibits the full properties of life.
Modern additions:
• Energy flow occurs inside cells.
• Hereditary information (DNA) is passed cell-to-cell.
• All cells share a common chemical composition.

Cell Size, Shape & the S.A./Volume Constraint

Cells vary widely in form: squamous (epithelia), spheroid (leukocytes), stellate (neurons), fibrous (muscle), etc.
Surface area $A$ for a cube of side $d$ : $A = 6d^2$ ; volume $V = d^3$ .
As diameter doubles, $V$ rises $\propto d^3$ whereas $A$ rises $\propto d^2$ – volume grows faster ⇒ diffusion limits nutrition/waste exchange.
• Example (cube):
– Small cell $d = 10\,\mu m$ ⇒ $A = 600\,\mu m^2,\; V = 1000\,\mu m^3$ .
– Large cell $d = 20\,\mu m$ ⇒ $A = 2400\,\mu m^2,\; V = 8000\,\mu m^3$ .
– 8-fold cytoplasm increase but only 4-fold membrane growth.
Cells needing large exchange surfaces evolve microvilli, flattening, or elongated shapes.

Prokaryotic vs. Eukaryotic Cells

Prokaryotes:
• DNA in nucleoid; no true nucleus or membrane organelles; reproduce asexually.
Eukaryotes:
• True nucleus + endomembrane system; membrane-bound organelles; usually capable of sexual reproduction.

Plasma (Cell) Membrane – Overview

Universal phospholipid bilayer studded with proteins.
Functions: encloses cell & organelles; provides anchorage; houses receptors, enzymes, transporters; mediates cell–cell recognition.

Membrane Lipids

Phospholipids (≈ most abundant) – amphiphilic: polar head + 2 hydrophobic tails.
Cholesterol – inserts among tails, controls fluidity & mechanical stability.
Glycolipids – phospholipids with short carbohydrate chains; exclusively outer leaflet → glycocalyx formation.
Self-assembly: amphiphilicity drives spontaneous bilayer formation.

Fluidity & Leaflet Dynamics

Lateral diffusion: ~ $10^7$ exchanges s⁻¹.
Flip-flop rare; catalyzed by enzymes:
• Flippase (P-type ATPase) – PS, PE outer → cytosolic.
• Floppase (ABC) – phospholipids cytosolic → outer.
• Scramblase – bidirectional, Ca²⁺-activated, equilibrates.
Cis-double bonds create kinks → increased fluidity.

Lipid Rafts

10–200 nm microdomains enriched in cholesterol + sphingolipids; more ordered than surrounding bilayer.
Scaffold cell signalling (receptor clustering, ion-channel regulation, synaptic plasticity).
Implicated in cancer hallmarks, neurodegeneration (Alzheimer’s, Parkinson’s), immune signalling, autism (SLOS – DHCR7 mutation).
Heat-shock proteins stabilize rafts under stress.

Bilayer Asymmetry

Outer leaflet: phosphatidylcholine (PC), sphingomyelin (SM).
Inner leaflet: phosphatidylserine (PS), phosphatidylethanolamine (PE). Cholesterol ≈ symmetric.
Asymmetry critical for signal transduction & for marking apoptotic cells (externalized PS).

Fluid Mosaic Model Summary

Lipids = 90–99 % of molecules (by count): ≈75 % phospholipids, 20 % cholesterol, 5 % glycolipids.
Proteins = 1–10 % of molecules yet ≈50 % membrane mass.

Membrane Proteins: Classes & Functions

Integral (intrinsic): span bilayer; may form channels/pumps.
Peripheral (extrinsic): loosely attached to integral proteins or lipids.
Five key roles: 1 Transport, 2 Enzymes, 3 Receptors, 4 Cell-recognition, 5 Cell-adhesion (CAMs/Integrins).
• Channels: nongated, ligand-, voltage-, mechano-gated.
• Carrier pumps use ATP.
• Glycoprotein markers → self vs. foreign.

Cytoplasm, Cytoskeleton & Inclusions

Cytosol: aqueous matrix with ions, metabolites, proteins.
Cytoskeleton: microfilaments, intermediate filaments, microtubules – provide shape, motility, intracellular transport.
Inclusions: stored glycogen, lipid droplets, pigments, crystals.

Major Organelles (Eukaryotic)

Nucleus – double-membrane envelope with pores; stores chromatin; nucleolus synthesizes rRNA subunits.
Endoplasmic Reticulum:
• Rough ER – ribosome-studded; protein synthesis & folding.
• Smooth ER – lipid synthesis, carbohydrate & toxin breakdown, Ca²⁺ storage; forms transport vesicles.
Golgi apparatus – cis → trans stacks; modifies, sorts, packages proteins/lipids into vesicles; generates secretory vesicles.
Lysosomes – Golgi-derived, acidic, hydrolytic enzymes ("suicide bags"); digest macromolecules & organelles; involved in Ca²⁺ mobilization.
• Genetic defects ⇒ lysosomal storage diseases: Tay-Sachs (ganglioside build-up), Niemann-Pick (sphingomyelinase or NPC mutations): hepatosplenomegaly, neurodegeneration, no cure.
Peroxisomes – oxidative vesicles; degrade $H<em>2O</em>2$ , alcohol; β-oxidation of long-chain FAs.
Vacuoles (plant/large) – storage, turgor; in animal cells primarily small vesicles.
Ribosomes – rRNA + protein; free (cytosolic proteins) vs. bound (secretory, membrane proteins).
Mitochondria – double membrane, cristae, matrix; own circular DNA; prokaryote-like ribosomes using IRES; ATP production via aerobic respiration; maternal inheritance; mtDNA mutations → neuromuscular diseases.

Autophagy – Self-Eating Pathway

Lysosome-dependent degradation for survival, differentiation, development, homeostasis.
Inducers: starvation, hypoxia, stress.
Non-specific (bulk) vs. selective (protein aggregates, damaged mitochondria, pathogens).
Autophagosome formation: Atg9 vesicles → phagophore → expansion via Beclin1/PI3K → closure → fusion with lysosome (autolysosome).
Protective in neurodegeneration, infection control, cancer modulation. 2016 Nobel Prize to Yoshinori Ohsumi.

Objectives – Session 2: Cells in Culture

Explain rationale for culturing cells.
Distinguish primary, secondary, continuous cultures.
Understand embryonic/hematopoietic stem cells.
Outline hybridoma & monoclonal antibody production.

In Vitro Cell Culture Basics

In vivo = within organism; In vitro = on plastic/glass.
Conditions: $37^\circ C$ , 5 % CO₂, humid incubator; sterile laminar hood.
Uses: model cell biology, drug/toxicity testing, cancer research, virology (vaccine), genetic engineering, gene therapy.
Advantages: homogeneous environment, reproducibility, ethical alternative to animals.

Terminology & Cell-Line Types

Clone – genetically identical cell population from one progenitor.
Passage – one subculture; passage number tracks age.
Primary culture – directly from tissue; heterogeneous, limited lifespan, anchorage-dependent, contact inhibition.
Secondary culture – selected/cloned from primary; more homogeneous, still finite.
Continuous (immortal) lines – spontaneous or induced transformation (e.g., SV40 T antigen, EBV, HPV E6/E7, hTERT); infinite proliferation; lose anchorage & contact inhibition.
Common lines: 3T3 (mouse fibroblast), HeLa (human cervical), CHO (Chinese hamster ovary), 293 (human kidney, adenovirus-transformed)…

Culture Medium Components

Basal medium: glucose, essential salts, amino acids, phenol-red pH indicator.
• pH 7.2 = red; acidic → yellow; basic → purple.
Supplements: antibiotics (pen–strep), L-glutamine, buffers, growth-factor rich serum (fetal bovine, horse…).
Auxiliary reagents:
• PBS – washes serum before trypsinization.
• Trypsin-EDTA – detaches adherent cells (EDTA chelates Ca²⁺).
• Bleach – decontamination.

Immortalization Strategies

Viral oncogenes (SV40 T, EBV, Adenovirus E1A/E1B, HPV E6/E7) inactivate tumor suppressors $p53, Rb$ .
hTERT over-expression → telomere maintenance, bypass senescence.

Cellular Aging Mechanisms

Cellular clock (finite divisions), death genes, telomere attrition (TTAGGG repeats), free-radical DNA damage, mitochondrial decline.

Stem-Cell Potency Spectrum

Totipotent – zygote/early blastomeres; can form whole organism.
Pluripotent – inner cell mass (blastocyst days 5-14); can form > 200 cell types.
Multipotent – restricted lineages (e.g., hematopoietic, cord-blood, adult stem cells).

Reprogramming Pathways

Direct in vivo implantation of somatic biopsy.
In vitro re-differentiation of somatic-tissue stem cells → transplant.
Somatic nuclear transfer → ES cells → differentiation → transplant.

Hematopoietic lineage choice controlled by transcription factors (PU.1, GATA-1, etc.).

Cloning (Reproductive vs. Therapeutic)

Clone = genetically identical nuclear genome; mitochondrial DNA inherited from egg donor.

Hybridomas & Monoclonal Antibodies (mAb)

Fusion of B-lymphocyte (antibody-producing, mortal) + myeloma cell (immortal, non-Ig) ⇒ hybridoma (immortal & Ig-secreting).
Heterokaryon → nuclear fusion → single hybrid nucleus.
Hybridomas cultured with feeder cells or defined growth factors → unlimited mAb production for diagnostics & therapy.
Product types: monoclonal Ab (single epitope), polyclonal Ab, monospecific polyclonal Ab.

Ethical, Medical & Practical Considerations

Lipid-raft modulation → potential therapies for metabolic, neuro-, onco-, cardiovascular diseases.
Statins lower cholesterol; possible side-effects: impaired raft-dependent synaptic integrity, cognitive issues – weighs against proposals for mass statin administration (e.g., in water supply).
No current cure for Niemann-Pick; gene & enzyme-replacement approaches under study.
Autophagy-inducing small molecules (Baek et al., 2012) hold promise against cancer & degeneration.

Key Numerical & Formula Recap

Surface area-to-volume ratio (cube): $\frac{A}{V} = \frac{6d^2}{d^3} = \frac{6}{d}$ → inversely proportional to size.
Flipase ATP consumption: $ATP \rightarrow ADP + P_i$ to move $PS, PE$ across leaflet.

Take-Home Highlights

Cell size constrained by diffusion; membranes orchestrate organization, signalling, and energy.
Lipid rafts = functional nanodomains; asymmetry & fluidity underlie responses and disease.
Endomembrane system ensures directed trafficking; lysosomal defects produce severe inherited disorders.
Autophagy = protective recycling; 2016 Nobel recognized its mechanism.
Cell culture enables controlled experimentation, therapeutic protein production, immortal line development, stem-cell research, hybridoma mAb generation.