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Cell Experiment Protocol 1. Basics of Cell Culture [1]

Higher category: 【Biology】 Cell Experiment Protocols

1. Classification of incubation

⑴ Batch culture

① Grows cells by limiting the amount of culture medium.

○ Growth-limiting substrate : Substrates with limited amounts that affects cell growth

② 1^st. Lag phase

○ In order for microbes to recognize and adapt to new molecules, the number of cells does not increase and the level of internal change is taken.

○ Shortening strategy : Adapting to incubation environment before cell culture, Metabolic improvement substance(e.g., Mg), Cell factor concentration optimization

③ 2^nd. Exponential phase

○ Balanced growth : A pattern of growth in which all cells multiply at a certain rate

④ 3^rd. Deceleration phase

○ Unbalanced growth starts.

⑤ 4^th. Stationary phase

○ Useful secondary metabolites such as antibiotics begin to be produced, i.e. non-replicating metabolism begins.

○ As the stage where oxygen and nutrients are insufficient, sufficient supply of oxygen and nutrients can delay the stationary phase.

⑥ 5^th. Death phase or decline phase

○ Errors are severe because dead cells are counted during absorbance analysis (using a spectrophotometer).

⑵ Fed-batch culture

① Cultivation method that increases productivity of products by adding organic nutrition ingredients only during incubation but not extracting the culture fluid.

○ The volume of culture fluid continuously increases.

○ The continuously supplied nutrient is called ‘feed’.

② Advantages : Overcomes substrates inhibition problem.

○ If E. coli grows at maximum speed using glucose, it will produce organic acids, a by-product, which will inhibit growth. In the fed-batch culture, it is possible to reduce the production of by-products and cultivate highly concentrated E. coli by maintaining the concentration of substrate and maintaining the growth of E. coli properly.

⑶ Chemostat

① An open-system culture method that maintains environmental conditions while continuously providing nutrients and removing wastes.

② CSTR(continuous stirred-tank reactor model) is generally used for incubator.

③ Parameter

○ Cell density(X) : g cells / L

○ Dilution rate : F(feed) / V(incubator volume)

○ Productivity of biomass : D × X

○ Space time : The duration of the reactant’s stay in the reactor, V(incubator volume) / F(volume flow)

○ Space velocity : Inverse number of space time, Relative velocity concept

⑷ Basic equation

① Parameter

○ Biomass yield : Indicates how much more cells can multiply per substrate of unit mass.

○ Unit : g cells / g substrates

○ Generally marked as Y_x/s 

○ Product yield : Indicates how much product per substrate of unit mass can be produced.

○ Unit : g products / g substrates

○ Generally marked as Y_p/s 

○ Production speed : The rate of formation of acellular products, and the rate of formation of products per cell of unit mass

○ Unit : g products / g cells · h

○ Generally marked as q_p

② Biomass’s rate of change equation

③ Substrates’ rate of change equation

④ Products’ rate of change equation

⑤ Monod equation : Well applied when cell concentration is low.

○ Similar to Michaelis-Menten formula.

○ Residual substrate concentration(S) is independent of the incoming substrate concentration(S0).

⑥ Contois equation

○ Developed from Monod equation.

○ Residual substrate concentration(S) is independent of the incoming substrate concentration(S0).

⑸ Cell recycle : If there is cell recycle in a chemostat, dilution rate can be greater than the specific growth rate.

① General phenomenon : μ = D

② μ_m < D : Generally cells continue to escape.

③ Proof

⑹ Equation abount amount of expression recombinant proteins from recombinant microorganisms in fed-batch culture.

P : concentration of cloned-gene protein (mg proteins / mg cells)

t : time(h)

k_e : maximum rate of protein synthesis (mg proteins / me cells per hour)

p : intracellular plasmid concentration (mg plasmids / mg cells)

K_t : transcription rate saturation constant (mg / mg cells)

k_-p : cloned-gene protein denaturation rate constant (1st-order reaction) (h^-1)

μ : specific growth rate (h^-1)

2. Biofuel process

⑴ Conducted in the order of pre-treatment, saccharification, and fermentation.

① Example of pre-treatment : Cellulase and hemicellulase production.

② Example of saccharification : Hydrolysis that converts cellulose and hemicellulose into monomer sugars.

③ Example of fermentation : Fermentation of hexose and pentose.

⑵ SHF(separate hydrolysis and fermentation)

① Traditional process consisting of 1st stage(pre-treatment, saccharification) and 2nd stage(fermentation).

② Advantage : Since each stage is executed under optimal conditions, it is executed without the cleaning pre-treated biomass, eliminating poison and supplying nutrients. 

⑶ SSF(simultaneous saccharification and fermentation)

① Process in which the preparation phase occurs first and the fermentation takes place simultaneously with saccharification.

② Advantage 1. Biomass hydrolysis rate is high due to removal of final product inhibition.

③ Advantage 2. Cleaning pre-treated materials improves biomass conversion rate due to removal of inhibitory mixtures.

④ Advantage 3. Use of heat-resistant yeast is permitted.

⑷ CBP(consolidated bio-processing)

① The pre-treatment, saccharification and fermentation are treated as a single process.

② Advantage : Provides low cost, high efficiency potential for biomass decomposition enzyme production.

3. Conservation of strains

⑴ Method for long-term preservation of strains : Cryopreservation

① Note. -70 ℃ deep freezer, liquid nitrogen (-196 ℃)

⑵ Method for short-term preservation of strains : Incubation in agar plate, agar slant, etc. → Refrigerate them(0-5 ℃)

① Agar molecules function as contact-dependent growth inhibition.

Input : 2019.03.14 08:59