Cell Culture (Updated)

Bacteria (E. coli)

Binary fission.

Yeast (S. cerevisiae)

Budding.

Mold (A. niger)

Spore formation (conidia).

Plant & Animal Cells

Mitosis.

Mitotic Division in Plants

Involves interphase, preprophase, metaphase, anaphase, telophase, and cell plate formation.

Mitotic Division in Animal Cells

Involves prophase, metaphase, anaphase, telophase, and cytokinesis.

Cell Culture

Growing cells under controlled conditions outside their natural environment.

Suspension Culture

Cells grow freely in the culture medium without requiring attachment.

Examples of Suspension Culture

Bacteria, yeast, plant cells, hematopoietic cells.

Adherent Culture

Cells require cell-cell adhesion or attachment to a surface to grow.

Examples of Adherent Culture

Mammalian cells, plant cells.

Cell Passaging (Subculturing)

Process to transfer portion of cells from an existing culture to fresh medium.

Detachment

Using enzymes (trypsin, collagenase) or mechanical methods.

Neutralization/Washing

Remove enzymes using serum-containing medium.

Resuspension & Seeding

Cells are diluted and placed into fresh vessels.

Culture Medium

A nutrient-rich gel or solution used to support the growth, survival, and proliferation of cells.

Nutrients & Salts

Amino acids, vitamins, Na⁺, K⁺, Ca²⁺, etc.

Carbon and Nitrogen Sources (component)

Essential for cell growth and metabolism.

pH Buffers (component)

Bicarbonate, HEPES, phosphate buffers.

Growth Factors

Varies by cell type.

Types of Culture Media

Solid (agar-based), liquid (broth-based).

Defined Medium (type of culture media)

Exact composition is known.

Undefined Medium (type of culture media)

Complex components like yeast extract.

Specialized Medium (type of culture medium)

Designed for differentiation or regulatory compliance.

Biological Contaminants

Bacteria, fungi, viruses, mycoplasma.

Chemical Contaminants

Endotoxins, heavy metals.

Physical Contaminants

Dust, fibers, glass particles.

Antibiotics in Cell Culture

Used to prevent microbial contamination and maintain plasmid-bearing cultures.

Aseptic Techniques

Methods used to maintain a sterile environment in cell culture.

Sterile Workspace

HEPA-filtered laminar flow hoods.

Sterilization Methods

Autoclaving, ethanol disinfection.

Good Lab Practices

Standard protocols to prevent contamination.

Types of Monitoring Techniques

Microscopy, contamination checks.

Microbial Cell Culture

Growing bacteria, yeasts, or fungi in a controlled environment using solid or liquid nutrient-rich media.

Streak Plate

Isolate individual colonies.

Spread Plate

Evenly distribute bacteria for counting.

Plant Suspension Culture

Growth of free-floating plant cells used for metabolite secondary metabolite production.

Mammalian Cell Culture

The growth of cells in controlled conditions using a nutrient-rich medium.

Freezing Temperature for Cell Preservation

-80°C.

Long-Term Storage for Cell Preservation

-150°C to -196°C in liquid nitrogen.

Thawing Temperature for Cell Recovery

25-37°C.

Factors Affecting Cell Growth

Temperature, pH, osmolarity, oxygen levels.

Optimal Temperature for Mammalian Cells

37°C.

Optimal pH Range for Mammalian Cells

7.2-7.4.

Impact of pH on Protein Structure and Enzyme Activity

Disrupts hydrogen bonds and ionic interactions.

Impact of pH on DNA/RNA

Causes depurination and hydrolysis of phosphodiester bonds.

Impact of pH on Cell Survival and Proliferation

Increases ROS levels, leading to cell death.

Impact of pH on Membrane Integrity

Collapses proton gradients, affecting energy generation.

Effect of High Osmolarity

Causes cell shrinkage.

Effect of Low Osmolarity

Causes cell swelling.

Obligate Aerobes

Require oxygen for survival.

Obligate Anaerobes

Cannot survive in the presence of oxygen.

Facultative Anaerobes

Can grow with or without oxygen but prefer oxygenated environments.

Microaerophiles

Require oxygen but at lower concentrations than atmospheric levels.

Aerotolerant Anaerobes

Do not use oxygen but can tolerate its presence.

Batch Growth

Cell culture in a closed system where a fixed volume of medium is inoculated with cells, and no additional nutrients are added.

Phases of Batch Growth

Lag, log, stationary, death.

Net Growth Equation

Mu (net) = mu (gross) - k(death).

Growth Rate

The absolute increase in biomass in a system or cell over time (dX/dt).

Specific Growth Rate

The rate of biomass increase of a cell population per unit of biomass concentration (1/X * dX/dt).

Continuous Exponential Growth

Assumes cell division and biomass accumulation occur at every instant of time.

Monod Model

Describes microbial growth based on substrate concentration.

Monod Kinetics Assumptions

Single substrate, homogenous environment, no inhibition, steady state, cell uniformity, negligible cell death.

Zero-Growth Bioprocesses

Maintain cells in a non-growing state while still producing useful products.

Luedeking-Piret Equation

Describe product formation in zero-growth systems (dP/dt = BX, B = beta).

Biomass Yield (Yx/S)

Grams of cells per gram of substrate consumed.

Product Yield (Yp/S)

Grams of product per gram of substrate consumed.

Inoculum

A population of cells introduced into a medium to initiate a cell growth or fermentation process.

Direct Growth Monitoring Methods

Hemocytometer, automated cell counters, plate counting, flow cytometry.

Indirect Growth Monitoring Methods

Spectrophotometry (optical density), metabolic activity, dry weight.

Cell Immobilization

Trapping cells on a solid surface or within a matrix for reusability.

Adsorption

Cells adhere to a surface.

Ionic/Covalent Binding

Cells attached via chemical interactions.

Entrapment

Cells enclosed in a semi-permeable membrane.

Advantages of Cell Immobilization

Simplifies purification, reusability, protection from stress.

Disadvantages of Cell Immobilization

Mass transfer limitations, potential activity loss, cost.