Cell Experiment Protocol Ch 2. Cell Experiment Overview 2nd
Higher category: 【Biology】 Cell Experiment Protocol Index
1. Overview
2. Types of Animal Cell Culture
1. Overview
⑴ Purpose
① Large-scale quantitative culture of cells
② Mass production of cell-derived substances
○ From animal cells: vaccines, antibodies, enzymes, hormones
○ From plant cells: pharmaceuticals, food additives, flavors, pesticides, plant hormones, enzymes
⑵ Animal Cells
① Size of animal cells: diameter 10–30 µm
② Proliferation of animal cells: doubling time of ~10–50 hours
③ Waste products of animal cells: ammonium, lactic acid
④ Mostly adherent cells.
⑶ Terminology
① Cell line: the cells with which culture was first initiated
② Passage number: the number of times subculture has been performed
○ Stem cells: a passage number of 4–8 is appropriate
○ Cancer cells: no limit on passage number
③ Generation number: the number of times the cells have divided
④ Split ratio: into how many portions the culture is divided during subculture
⑤ Relationship between generation number and passage number
Ntotal = 2(Generation Number) × N0 = (Split Ratio)(Passage Number) × N0
∴ Generation Number = Passage Number × log(Split Ratio) / log(2)
cf. log(Split Ratio) / log(2) ≈ (Split Ratio) / 2 for (Split Ratio) = 2, 3, 4, 5
∴ Generation Number = Passage Number × (Split Ratio) / 2
⑷ Factors
① Factor 1. Support material
○ As most cells are adherent: the surface-area-to-volume ratio of the support should be high.
② Factor 2. Mass transfer
③ Factor 2-1. Control of external environmental conditions: temperature, pH, DO, nutrients, redox potential, CO2 concentration
○ Temperature: 37 ± 0.2 °C; narrow acceptable range, particularly sensitive to high temperatures.
○ pH: tends to rise early in culture; as cells proliferate, lactic acid accumulates and pH decreases.
○ Dissolved oxygen (DO)
○ DO decreases as metabolism proceeds.
○ High DO can be cytotoxic
○ When DO is low: sparge with oxygen gas
○ When DO is high: sparge with nitrogen gas
○ Nutrients: supply continuously via medium exchange
○ Mode 1. Batch culture
○ Mode 2. Continuous culture
○ Aeration: use aeration methods that maximize oxygen transfer
④ Factor 2-2. Minimize accumulation of toxic metabolites such as lactic acid and ammonium.
⑤ Factor 3. Mechanical stimulation
○ Minimize agitation speed to reduce mechanical damage to cells.
2. Types of Animal Cell Culture
⑴ Types of liquid culture: surface culture, deep (submerged) culture, dialysis culture
⑵ Liquid culture, suspension cells
① Method 1. Stirrer flask
○ Minimize stirring speed to reduce shear stress on cells.
② Method 2. Biostat
○ Supplies fresh medium and removes products to maintain constant conditions.
③ Method 3. Mixing & aeration
④ Method 4. Rotating chamber
○ Component 1. Cell chamber: where cells accumulate
○ Component 2. Semipermeable membrane
○ Component 3. Sampling port: outlet for cell products
⑤ Method 5. Hollow fiber
⑥ Method 6. Perfused suspension culture
○ Definition: a system in which cells are confined to narrow compartments along the periphery of hollow fibers; fresh medium is perfused (flow-through) and spent medium is discharged along the tubes.
○ Advantages: markedly improves cell proliferation; induces multilayering.
⑦ Method 7. NASA bioreactor
○ Definition: a rotating culture that creates a simulated microgravity environment for cell growth
○ Advantages: minimal physical damage due to microgravity conditions
○ When rotation stops, cells settle, allowing medium exchange.
⑶ Liquid culture, adherent cells
① Method 1. Nunc Cell Factory
○ Stacked rectangular Petri-dish-like units
② Method 2. Flask
○ Drawback: only one side of the total surface area is utilized.
③ Method 3. Roller bottle
○ Advantage: can utilize nearly the entire surface area.
④ Method 4. Microcarrier
○ Definition: culture method in which cells are grown attached to microbeads.
○ Microbead size: 90–300 µm
○ Packed-bed bioreactor: microbeads are immobilized.
○ Fluidized-bed bioreactor: microbeads are in motion.
⑤ Method 5. Ceramic matrix
⑥ Method 6. Multi-tray
○ Feature: typically composed of 10 chambers.
⑦ Method 7. Plastic film
○ Definition: cells are cultured within a film.
○ Film materials: PET–Teflon (fluoroethylene propylene)
⑧ Method 8. Heli-cell
○ Definition: cells are cultured on a helical ribbon.
○ Uses polystyrene formed into twisted ribbons as packing material.
⑨ Method 9. Stack plate
○ Drawback: higher medium-volume-to-surface-area ratio.
⑷ Solid-state culture
① Definition: cells are cultured directly on the surface of a medium containing appropriate moisture.
② Examples: mushroom cultivation, compost fermentation, production of fermented soybean products
3. Cell Immobilization
⑴ Definition: fixing adherent cells in place
⑵ Advantages
① Promotes cell proliferation.
② Increased stability enables long-term culture.
③ Enables suspension culture of adherent cells: increases surface area per unit volume; mimics 3-D tissue.
④ Protects from shear stress.
⑤ Surface treatment can obviate the need for continuous addition of growth factors.
⑶ Type 1. Use of particulate carriers: cells are attached to carrier surfaces for culture.
① Advantages: increased surface area; more uniform growth environment
⑷ Type 2. Microencapsulation: cells are enclosed within capsule membranes.
① The capsule has a defined molecular weight cutoff.
② Advantages: prevents immune-cell infiltration; protects from shear stress.
③ Drawbacks: limits diffusion of dissolved oxygen and nutrients.
4. History of Cell Culture
⑴ Mid-19th century: Pasteur proposed fermentation by microorganisms.
⑵ 1870s: Koch proposed heat sterilization methods
⑶ 1933: Kluyver proposed suspension culture using flasks.
⑷ 1940: Submerged culture methods were used for mass production of penicillin during World War II.
Input: 2019.10.07 13:13