Cell Culture & Fluorescence Imaging Study Guide

Overview of Cell Culture and Imaging

• Module Context: Cell Biology 5BBB0209, King's College London (School of Cardiovascular and Metabolic Medicine and Sciences, Department of Biochemistry).

• Lead Lecturer: Prof Elisabeth Ehler.

• Core Pedagogical Objectives:

  • Gain comprehensive technical insight into cell culture methodology.

  • Master the fundamental physical and chemical principles of fluorescence imaging.

  • Analyze exemplary applications of fluorescent proteins in cell biology research.

Fundamentals of Animal Cell Culture

• Primary Rationale for Culturing Cells:

  • Defined System: Provides an in vitro environment where variables (nutrients, pH, temperature, gas partial pressures) can be controlled precisely, and its known what’s in the culture dish.

  • Ease of Experimental Manipulation: Enables direct pharmacological interventions, adding drugs, and live-cell high-resolution microscopy.

  • High Reproducibility: Continuous cell lines supply a virtually inexhaustible source of cells → better reproducibility of experiments

• Disadvantages and Limitations:

  • Artifact Generation: High risk of morphological and physiological artifacts, particularly when growing cells as standard two-dimensional (2D) artificial monolayer cultures.

  • Lack of Systemic Context: Absence of tissue architecture, systemic hormonal regulation, metabolic cross-talk, mechanical loading, and multi-lineage immune interactions present in whole organisms.

Technical Requirements: Media, Environment, and Substrates

• Cellular Nutrition ("Food"):

  • Basal Medium: Inorganic salt solutions; pH sensor; glutamine added as cells can’t produce it themselves (e.g., Dulbecco's Modified Eagle Medium / DMEM) providing osmolality, essential ions, glucose, and essential amino acids including L-glutamine.

  • Growth Factors and Serum: usually coming from the addition of animal serum —typically Fetal Calf Serum (FCS), Horse Serum, or Chick Embryo Extract—provides vital growth factors, hormones, attachment factors, and trace nutrients, keeps cells more differentiated.

  • Supplements: Antibiotics (e.g., Penicillin/Streptomycin) and hormonal additives such as Insulin. induced pluripotent stem cells have to be cultured without antibiotics otherwise they wont differentiate well

• pH Regulation and Sensor System:

  • Mammalian cell media typically rely on a bicarbonate buffer system functioning alongside controlled atmospheric $\text{CO}_2$ (usually $5.0\text{\% } \text{CO}_2$).

  • Phenol Red Indicator: Acts as a visual pH sensor in the medium:

    • $\text{pH } 6.5$: Bright Yellow (indicates acidic accumulation from metabolic overgrowth, starving cultures, or bacterial/yeast contamination). cause for worry cells may be damaged due to acidity or yeast contamination

    • $\text{pH } 7.0$: Orange-Yellow (slightly acidic shift).

    • $\text{pH } 7.4$: Orange-Red (optimal physiological pH for most mammalian cell lines). cells are happy

    • $\text{pH } 7.6$: Deep Red/Purple (alkaline shift due to $\text{CO}_2$ depletion, low cell density, or gas exchange failure). catastrophy, co2 supply of incubator depleted/bacteria contamination

• Culture Vessels ("Accommodation"):

  • Petri dishes (shallow plastic vessels, used for non-sealed short-term incubations).

  • Tissue culture flasks (engineered with plug-seal caps or gas-permeable vented filter caps).

  • Multiwell plates (6-well to 96-well formats for high-throughput quantification).

  • Specialized glass-bottom dishes and chambered coverslips for optical clarity in fluorescence microscopy.

• Substrates and Matrix Coatings: better attachment of cells

  • Hydrophilic tissue-culture-treated polystyrene plastic.

  • Extracellular Matrix (ECM) biological proteins: Fibronectin (FN), Laminin (LAM), Collagen IV (COL IV).

  • Charged synthetic/natural amino acid polymers: Poly-L-lysine (PLL), Poly-ornithine (PLO).

  • Advanced matrices: Hydrogels, fibrin networks, and biocompatible 3D scaffolds.

  • Substrate-Specific Cell Responses: LNCaP prostate cancer cells display marked structural variations depending on matrix coating: depending on which carpet its grown on the morphology differs. when growing new cells, test different substrates to find which ones best

Historical Progression of Cell Culture Technology

• 1931: Primitive cell culture executed on open laboratory benches without sterile containment, leading to widespread bacterial contamination and short culture survival.

• 1961: Implementation of open-flame aseptic fields, glass culture flasks, and dangerous mouth-pipetting practices.

• 2013–Present: Advanced sterile processing utilizing Class II Laminar Flow Biosafety Cabinets (e.g., SafeFAST hoods), motorized electronic pipettors, full personal protective equipment (PPE), automated $\text{CO}_2$ incubators ($37.0^\circ\text{C}$, $5.0\% \text{ CO}_2$), regulated dual-gas manifolds, and cryogenic liquid nitrogen storage Dewars ($-196.0^\circ\text{C}$).

Human Cell Lines and the History of HeLa Cells

• Henrietta Lacks (1920–1951):

  • An African American woman treated for aggressive cervical adenocarcinoma at Johns Hopkins Hospital in Baltimore.

  • Tissue biopsy harvested in 1951 without her knowledge, authorization, or informed consent.

• Establishment of the HeLa Line:

  • Isolated by technician Mary Kubicek in the laboratory of Dr. George Gey and Dr. Margaret Gey.

  • Gey's original growth medium comprised chicken plasma, calf fetus puree, special balanced salts, and human umbilical cord blood.

  • HeLa became the first continuously proliferating, immortalized human cell line, featuring a rapid population doubling time of approximately $24\,\text{hours}$.

• Ethical Implications:

  • Triggered decades of bioethical debate concerning donor autonomy, biological ownership, patient privacy, and uncompensated medical commercialization, as chronicled in Rebecca Skloot's book The Immortal Life of Henrietta Lacks.

Primary Cultures, Continuous Cell Lines, and Cell Dynamics

• Central Dogma of Cell Culture Dynamics:

  • Low Cell Density + Serum Mitogens $\rightarrow$ Active Cell Proliferation.

  • High Cell Density + Differentiation Factors + Cell-Cell/Matrix Interactions $\rightarrow$ Proliferative Arrest and Lineage Differentiation.

• Primary Culture vs Immortalized Cell Lines:

  • Primary Cultures: Cells isolated directly from native animal tissues (e.g., primary cardiomyocytes). Retain tissue-specific function and ultrastructure but possess a finite lifespan in culture (typically maintained for approximately $1\,\text{week}$).

  • Continuous Cell Lines: Immortalized cells capable of infinite subculture; can be cryopreserved indefinitely; may undergo genetic drift, karyotypic alteration, and loss of specialized differentiation markers. for a cell to grow forever it has to be immortalized, but may have lost differentiation markers

  • Multistep Transformation: Progressive transition from pre-immortal primary tissue cells to immortal lines, culminating in transformed cells characterized by loss of contact inhibition and focus formation.

• Standard Cell Growth Curve Phases:

  1. Lag Phase: Period following subculture where cells attach, spread, re-establish homeostasis, and synthesize factors required to enter the cell cycle (minimal population increase).

  2. Exponential (Log) Phase: Period of steady logarithmic population doubling. Optimal phase for experimental assays and subculturing.

  3. Plateau (Stationary) Phase: Confluency is reached ($100\%$\text{ coverage}); growth rate matches cell death rate due to contact inhibition and nutrient exhaustion.

• Subculturing (Passaging) Protocol:

  1. Aspirate spent growth medium from the culture vessel.

  2. Wash monolayer with Calcium/Magnesium-free Dulbecco's Phosphate-Buffered Saline ($\text{DPBS-A}$), with or without EDTA, to clear serum trypsin inhibitors.

  3. Add Trypsin-EDTA enzyme solution; incubate at $37.0^\circ\text{C}$ for approximately $10\,\text{minutes}$ until cell-substrate focal adhesions cleave and cells round up.

  4. Neutralize trypsin by adding complete serum-containing media; gently pipette to disperse cells into a single-cell suspension.

  5. Count viable cells and reseed into fresh vessels at target density.

• Cellular Senescence:

  • Replicative limit observed in primary cells (Hayflick limit); characterized by growth arrest, cell enlargement, cytoplasmic flattening, and vacuolization.

• Representative Mammalian Cell Lines:

  • HeLa: Human cervical epithelial adenocarcinoma.

  • MDCK: Madin-Darby canine kidney epithelial cells (polarized transport model).

  • COS-7: African green monkey kidney fibroblast-like cells (transfection host).

  • BHK-21: Baby hamster kidney fibroblasts.

  • HEK-293: Human embryonic kidney cells (adenovirus transformed).

  • A2780: Human ovarian carcinoma cells.

  • HL-60: Human promyelocytic leukemia cells (grown non-adherently in suspension culture).

Cryopreservation and Cell Culture Contamination

• Cryopreservation Standard Operating Procedure:

  1. Harvest cell monolayer via trypsinization and resuspend at high density ($2 \times 10^6 - 2 \times 10^7\,\text{cells/mL}$).

  2. Add freezing medium containing a penetrating cryoprotectant (e.g., $10\%$\text{ Dimethyl Sulfoxide / DMSO} or Glycerol) to prevent lethal intracellular ice crystal formation.

  3. Dispense aliquots into pre-labeled cryovials.

  4. Cool slowly at a controlled rate of approximately $-1.0^\circ\text{C/min}$ using a rate-controlled freezer, an isopropyl alcohol freezing container (Mr. Frosty), or an insulated pipe-lagging foam sleeve at $-80.0^\circ\text{C}$ for $> 4\,\text{hours}$.

  5. Transfer cryovials into liquid nitrogen Dewars ($-196.0^\circ\text{C}$) for permanent storage in liquid or vapor phase.

• Cell Culture Contaminants ("The Undesirables"):

  • Mould/Fungi: Multi-cellular filamentous hyphae network visible under low-power phase-contrast microscopy.

  • Yeast: Round or ovoid budding single-celled structures causing media turbidity.

  • Bacteria: Rapidly proliferating motile cocci or bacilli; causes rapid medium acidification (turning phenol red yellow) and turbidity.

  • Mycoplasma: Minute, wall-less bacteria ($0.1 - 0.3\,\mu\text{m}$) invisible under standard light microscopy. Alters host cell expression, metabolism, and behavior without causing turbidity; detected via DAPI staining or PCR.

Advanced Three-Dimensional (3D) Tissue Models

• Organotypic Co-Culture:

  • 3D dermal model combining epidermal keratinocytes and dermal fibroblasts in a collagen gel matrix maintained at an air-liquid interface.

  • Recapitulates stratified tissue architecture, validated via immunofluorescence against stratum corneum markers (Involucrin) and basement membrane zone markers (Collagen VII).

• Engineered Heart Tissue (EHT):

  • Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) cast within 3D hydrogel or fibrin matrices.

  • Generates functional tissue constructs capable of spontaneous, synchronized, contractile beating.

Principles of Fluorescence and Chemical Fluorochromes

• Physical Principles of Fluorescence:

  1. Photon Absorption: Fluorophore electrons absorb light energy at a specific excitation wavelength ($\lambda_{\text{ex}}$), jumping to a higher energy state.

  2. Excited Lifetime: Fluorophore remains in the excited state for a brief period (fluorescence lifetime, typically $1 - 10\,\text{ns}$).

  3. Photon Emission: Electron returns to ground state, releasing energy as an emitted photon of lower energy and longer wavelength ($\lambda_{\text{em}}$).

  • Stokes Shift: The difference in nanometers between the peak absorption and peak emission wavelengths ($\Delta \lambda = \lambda_{\text{em}} - \lambda_{\text{ex}}$).

  • Quantum Yield: Indicator of fluorophore efficiency, defined as ratio of emitted photons to absorbed photons ($\Phi = \frac{\text{Photons Emitted}}{\text{Photons Absorbed}}$).

• Chemical Fluorophores:

  • Fluorescein: Small aromatic organic dye producing green fluorescence.

  • Fluorescein Isothiocyanate (FITC): Reactive derivative featuring an isothiocyanate group ($\text{N=C=S}$) that covalently reacts with primary amines ($-\text{NH}_2$) on lysine residues of antibodies and target proteins.

*   *Alexa Fluor Dyes*: Highly photostable synthetic dyes covering the visible spectrum (e.g., Alexa 488, Alexa 546, Alexa 633).
*   *Phalloidin*: Bicyclic peptide toxin from the death cap mushroom (*Amanita phalloides*). Binds selectively and tightly ($K_d \approx 10^{-8}\,\text{M}$) to filamentous actin (F-actin) without binding globular actin (G-actin) monomers; conjugated to Alexa dyes for cytoskeletal staining.

Fluorescent Proteins and Genetic Tagging

• Green Fluorescent Protein (GFP):

  • $27\,\text{kDa}$ native fluorescent protein isolated from the jellyfish Aequorea victoria.

  • Excited by UV/blue light ($\lambda_{\text{ex}} \approx 395\,\text{nm} / 475\,\text{nm}$) and emits green light ($\lambda_{\text{em}} \approx 509\,\text{nm}$).

• Nobel Prize in Chemistry (2008):

  • Osamu Shimomura: First isolated GFP from Aequorea victoria and identified its light emission property under UV radiation.

  • Martin Chalfie: Demonstrated GFP as a non-invasive expression marker in living organisms (coloring 6 neurons in Caenorhabditis elegans).

  • Roger Tsien: Resolved chromophore chemistry and engineered point mutations yielding expanded spectral variants (CFP, YFP, mCherry, eGFP).

• Chromophore Structure:

  • Enclosed inside an 11-stranded $\beta$-barrel shield.

  • Formed autocatalytically by post-translational cyclization and oxidation of tripeptide sequence $\text{Ser65}-\text{Tyr66}-\text{Gly67}$.

• Recombinant Fusion Proteins:

  • GFP sequence is cloned in-frame with cDNA encoding a target protein within an expression vector driven by a promoter (e.g., CMV).

  • Transfected host cells express a single chimeric fusion protein (e.g., cMy2-GFP localization at M-bands, $\alpha$-actinin at Z-disks).

• Multicolor Labeling Techniques:

  • Rosa26FP Transgenic Mice: Combinatorial expression of CFP, YFP, and mCherry for tissue lineage tracking.

  • Tetbow Method: High-density neuronal tracing using fluorescent proteins mTurquoise2, EYFP, and tdTomato.

Advanced Live-Cell Imaging: FRET and FRAP

• Fluorescence Resonance Energy Transfer (FRET):

  • Mechanism: Non-radiative transfer of excitation energy from a donor fluorophore to an acceptor fluorophore via dipole-dipole coupling.

  • Strict Operational Requirements:

    1. Significant spectral overlap between donor emission spectrum and acceptor absorption spectrum.

    2. Ultra-close physical distance between donor and acceptor fluorophores ($< 10\,\text{nm}$).

  • cAMP Biosensor Application:

    • Protein Kinase A (PKA) tagged with Cyan Fluorescent Protein (CFP, donor) on regulatory (R) subunits and Yellow Fluorescent Protein (YFP, acceptor) on catalytic (C) subunits.

    • Low cAMP (Basal State): Holoenzyme complex remains intact ($< 10\,\text{nm}$ proximity); donor excitation at $405\,\text{nm}$ yields FRET emission at $545\,\text{nm}$ (yellow).

    • High cAMP (Forskolin Stimulation): $\text{cAMP}$ binds regulatory subunits, causing dissociation of catalytic subunits ($> 10\,\text{nm}$ distance); FRET is abolished, causing increased donor emission at $470\,\text{nm}$ (blue).

• Fluorescence Recovery After Photobleaching (FRAP):

  • Mechanism: Measures intra-cellular mobility, lateral diffusion rates, and binding dynamics of fluorescently tagged molecules.

  • Experimental Protocol:

    1. Record baseline fluorescence intensity of a defined target region.

    2. Expose the Region of Interest (ROI) to a high-power laser beam to photobleach fluorophores permanently.

    3. Monitor the rate and extent of fluorescence signal recovery over time as unbleached molecules diffuse back into the ROI.

  • Structural Application: Applied to evaluate structural protein mobility at cardiac Z-discs (comparing exchange rates of YFP-myotilin, YFP-$\alpha$-actinin, and YFP-telethonin).

Summary and Key Literature

• Key Applications of Fluorescent Proteins:

  1. Subcellular protein localization and organelle tracking.

  2. Cell fate mapping during embryonic development.

  3. Real-time analysis of protein-protein interactions (FRET).

  4. Quantification of protein mobility and exchange kinetics (FRAP).

• Recommended Core Reading:

  • Snapp (2009): Fluorescent Proteins: A Cell Biologist’s User Guide. Trends Cell Biol 19, 649-655.

  • Chao et al. (2019): Imaging cAMP nanodomains in the heart. Biochem Soc Trans 47, 1383-1392.

  • Sakaguchi et al. (2018): Bright multicolor labelling of neuronal circuits with fluorescent proteins and chemical tags. eLife 7, e40350.