Lecture 6 Culture Vessels & Substrates

 Lecture 6 Culture Vessels & Substrates

• Culture Vessels
  • containers or devices used to grow and maintain microorganisms, cells, tissues, or plants in controlled laboratory conditions
  • provide a sterile environment and support the growth of biological samples
• Types
  • Petri dishes
  • Test tubes
  • Flasks
  • Multiwell plates
  • Roller bottles: Cylindrical bottles that rotate to increase surface area for cell growth.
  • Spinner flasks: Vessels with magnetic stirrers for suspension cultures.
  • Bioreactors: Specialized vessels for large-scale cell or microbial cultures, often with controlled temperature, pH, and oxygen levels.
  • Plant culture vessels: Specialized containers for plant tissue culture, often with ventilation systems.
• Function
  • Providing sterile environment
  • Supporting cell growth an proliferation
  • Facilitating gas exchange
  • Enabling nutrient supply and waste removal
  • Allowing easy monitoring and manipulation
• Culture substrate
  • medium or material used to support the growth of microorganisms, cells, or tissues in laboratory or industrial settings
  • provides essential nutrients, maintains appropriate pH levels, and creates suitable conditions for the growth and proliferation of the cultured organisms
  • choice of substrate depends on the specific cell type, research goals, and desired culture conditions
  • optimizing the substrate can significantly impact cell growth, differentiation, and function in culture
• Types
  • Plastic surfaces: Many adherent cells can attach directly to treated plastic surfaces of culture vessels.
  • Glass: Some cells prefer glass surfaces, which can be coated for better adhesion.
  • ECM proteins
    • Collagen: Promotes cell adhesion and growth for various cell types
    • Fibronectin: Enhances cell attachment and spreading
    • Laminin: Supports growth of epithelial and neuronal cells
  • Hydrogels
    • Matrigel: A complex mixture of ECM proteins
    • Synthetic hydrogels: Customizable for specific cell types
  • Microcarriers: Small beads for growing adherent cells in suspension culture
  • Scaffolds:
  • Feeder layers: Inactivated cells that support growth of other cell types
  • Agar or agarose: For microbial cultures or semisolid media
  • Methylcellulose: Used in hematopoietic stem cell cultures
  • Specialized coatings: Poly-L-lysine: Enhances cell adhesion & Gelatin: Promotes attachment of various cell types
• Function
  • Cell attachment and adhesion
  • Mimicking in vivo environments
  • Promoting cell differentiation
  • Controlling cell behaviors and morphology
  • Enhancing cell-cell interaction
• Substrate
  • normal cells→anchorage dependent
  • transformed hematopoietic cells→anchorage independent
• Common material
  • disposable plastics
    • PS
    • simple, reproducible, optical quality, flat growth surface=uniform distributed & monolayer
    • treat with corona discharge, gamma irradiation, chemically to produce hydrophilic, wettable surface
    • test for quality→growth rate & plating efficiency of cells in low serum/serum free media
    • PVC, PCO3,, PTFE, TPX, PMMA, Melinex
    • greater consistency & superior optical properties
  • glass
    • original
    • cheap, easily washed w/o lose growth-supporting properties, dry/moist heat
    • high optical quality is alkaline with high lead→reduce cell growth
    • neutralization with acid wash/coat
  • alternative
    • fibers: PLA, PGA and silk biodegradable→2D, 3D
    • derivatization: RGD peptide
    • metals: stainless steel
• Substrate coating
  • conditioning with spent medium, serum, purified fibronectin & collagen
  • polymers & nanoparticles: poly-D-lysine & diamond nanoparticle→proliferate & differentiation
  • denatured collagen: improve attachment
  • undenatured gelatin: improve exp of differentiated fx
  • matrices: Matrigel with laminin, fibronectin, proteoglycans
  • ECM: feeder layer
• Feeder layer
  • A population of connective tissue cells that are used to nourish cultured tissue cells
  • from mouse fibroblast
  • supply metabolites to the cells they support, do not grow or divide, and can be inactivated by gamma irradiation
• Choice of vessel
  • cell yield
    • proportional to surface area
    • small volume, multiple→multiwell plate, petri dish
    • large→flask with corrugated surfaces, multilayer flask
  • adherent/suspension
    • suspension w/o cell attachment
    • stirrer bottles for agitation
    • rotational speed 60rpm avoid damage from shear stress
  • venting
    • multiwell, petri dish loose-lid→humid atmosphere, control CO2 concentration
    • plug seal cap→vented by slackening caps one full turn to allow CO2 enter & escape
    • vented cap→permeable filter
  • sampling & analysis
    • samples process in same way→multiwell
    • sample process differ→separate vessels
    • phase contrast (meniscus size relative to well diameter) not easily perform on multiwell
  • uneven growth
    • vibration→cell density
  • cost→petri dish cheap but require humid, control CO2
  • sterilization of cheap soda glass labor intensive to plastic