CCBPE Lec 3: Cell Culture Maintenance & Bicarbonate

Culture environment of CO2 Incubator

1) Temperature: maintained within limits of natural habitat of animals (i.e. 37oC)

2) Oxygen: 1-10%

3) CO2: get dissolved in the mdium and remains in equillibrium with Bicarbonate Ions HCO₃^- → Lower pH

Why should antibiotics not be used for routine culture of cell?

• Toxicity effect

• Mask the presence of low levels of microbial contamination

Why is serum added to basal medium?

• contains factors for cell proliferation & maintenance

• buffer against perturbations and toxic effects (like pH/presence of heavy metal ions/endotoxins)

Disadvantages of Serum-containing medium

• Lack of standardisaion (variation in serum effects)

• Risk of viral/prion/mycoplasma contamination

• Availability and cost (expensive)

• Make purification of proteins (in serum) more difficult in downstream processing

• Serum Ab → Potential cytotoxicity

Challenges of Serum-free medium

• Chemical contaminants in serum-free medium may be more toxic to cells

• More specific to certain cell types

• Adaptation to serum-freee medium takes time

Serum-Free Adaptation

• Have cells in logarithmic phase of growth

• Consistently monitor cells

• Methods:

  1) Sequential adaptation

  → Slowly increasing serum conc.

  2) Starve and save adaptation

When to use Dulbecco’s modified Eagle’s medium (DMEM)?

Serum supplementation for high cell growth

When to use Ham’s F12 or F10 and the MCDB media series?

Growing specific cell types at low density with minimal amount of serum

Methods to adapt adherent cells to a suspension culture

1) Incubate cells in serum-free medium (using Sequential adaptation)

2) Coat surface with anti-sticking agent (e.g. Teflon)

Partial pressure and Henry’s law constant

H = 29.4 atm/M

Henderson-Hasselback Equation (determine pH)

Note: The concentrations of the base/acid must be in moles/L

Importance of Bicarbonate buffer

• Maintaining pH is Human body and Cell culture

• Counters CO2 to create an equillibrium

What are some factors to look out for when Observing a Cell Culture?

• Drop in pH → Colour change

• Cell concentration (Higher conc → Medium used up faster)

• Morphological deterioaration

What method can be used for Protein determination?

• Bradford Assay

• Specific enzyme activity

What methods can be used for DNA determination?

Staining of cells with fluorescent that bind to DNa

• DAPI

• Hoescht 33258

What factors can be used indirect monitoring of Metabolic assessment?

• Glucose concentrations

• Lactic Acid production

• Oxygen Consumption

Name at least 3 Cell Viability Assays

(name at least one of the 3)

• Dye exclusion (e.g. Trypan Blue)

• MTT Assay

• Lactate Dehydrogrenase (LDH) Determination

• Colony forming assay → Calculate plating efficiency

• Rate of protein and nucleic acid synthesis

• Energy level: intracellulcar energy charge

Limitation of Dye Exclusion assay

Cannot distinguish between healthy viable cells and unhealthy viable cells

Describe the concept of the MTT Assay in indirect monitoring of cell viability

• MTT added to culture medium and taken in by the cells

• MTT (yellow) is reduced by NAD(P)H dehydrogenase in mitochondria to purple formazan derivative

• Upon, solubilisation, purple formazan quantified colorimetrically by UV-Vis spectrophotometer

• Strength of purple colour directly proportional to the metabolically active cells

Describe the concept of the LDH Dehydroogenase Determination Assay in indirect monitoring of cell viability

• When a cell is damaged, LDH is produced

• More lactate → pyruvate via NAD⁺ reduction to NADH

• Diaphorase uses NADH to convert INT to Red Formazan

• More cell damaged (cytotoxicty)→ More LDH released → More Red Formazan formation

Describe the concept of the Colony forming Assay in indirect monitoring of cell viability

• Known no. of cells at LOW density seeded and grown in wells

• Each cell will produce a cell colony

• Count no. of cell colonies

• PE = No. of colonies/cells plated * 100%

Describe the concept of the Rate of protein and nucleic Assay in indirect monitoring of cell viability

• Use radio-labelled amino acids/nucleotides

• Add to log phase cultures

• Rate of protein synthesis measured by scintillation counter

• More protein content → More cell growth

What radio-labelled amino acids/nucleotides usually used in rate of protein nucleic acid determination?

• 3H-leucine

• 35S-menthionine

Intracellular energy charge caluclation

Energy=ATP+\frac{0.5\left(ADP\right)}{ATP+ADP+AMP}

• Lower values → Lower viability

• Normal cells have 0.7 - 0.9