Lecture 4b culture conditions and media

Scaling up the Scaling up the production processproduction process

Oxygen requirementsOxygen requirements- Supply of oxygen to satisfy cell Supply of oxygen to satisfy cell

metabolism is one of the major problems metabolism is one of the major problems associated with culture scale up.associated with culture scale up.

- O2 consumption rate: 0.06-0.6 O2 consumption rate: 0.06-0.6 mmole/hour for 10^6 cells/mlmmole/hour for 10^6 cells/ml

- For small volumes (< 1 litre) O2 For small volumes (< 1 litre) O2 diffusion form the headspace through diffusion form the headspace through the culture surfacethe culture surface

Lecture 4 Animal Cell BiotechnologyLecture 4 Animal Cell BiotechnologyCell Culture Conditions and MediaCell Culture Conditions and Media

Culture MediumCulture Medium animal cells require chemically complex liquid animal cells require chemically complex liquid

media suitable for growth of several media suitable for growth of several generationsgenerations

commercially made, supplied as 1x liquid ready commercially made, supplied as 1x liquid ready for use, 10x concentrated solution, or for use, 10x concentrated solution, or powdered formpowdered form

dilute concentrate with presterilized distilled dilute concentrate with presterilized distilled waterwater

dissolve powdered medium in water and filter dissolve powdered medium in water and filter sterilizesterilize


Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P56.


Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 22.


Components of a typical culture Components of a typical culture medium:medium:

1.1. CarbohydratesCarbohydrates2.2. Amino AcidsAmino Acids3.3. SaltsSalts4.4. Buffering systemBuffering system5.5. Vitamins and hormonesVitamins and hormones6.6. AntibioticsAntibiotics7.7. Phenol redPhenol red


1. Carbohydrates1. Carbohydrates glucose (25 mM) used most often as energy glucose (25 mM) used most often as energy

sourcesource may use alternative carbohydrate sources such may use alternative carbohydrate sources such

as fructose, galactose, or maltose (reduced as fructose, galactose, or maltose (reduced lactic acid production, more stable culture pH)lactic acid production, more stable culture pH)

precursor for biosynthesis ribose through the precursor for biosynthesis ribose through the pentose phosphate pathway (for nucleic acid pentose phosphate pathway (for nucleic acid synthesis)synthesis)

can also use glutamine (4 mM) for energycan also use glutamine (4 mM) for energy


Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 56.


2. Amino acids2. Amino acids 0.1-0.2 mM added as a source of precursors for 0.1-0.2 mM added as a source of precursors for

protein synthesisprotein synthesis glutamine (2-4 mM) used precursor for TCA glutamine (2-4 mM) used precursor for TCA

intermediates, purines, pyrimidines, amino intermediates, purines, pyrimidines, amino sugars, and asparaginesugars, and asparagine

ammonia is released as a by-product of ammonia is released as a by-product of glutamine breakdown, can be inhibitory to glutamine breakdown, can be inhibitory to growth (half-life of glutamine at 4growth (half-life of glutamine at 4ooC is 3 weeks)C is 3 weeks)

occurs occurs viavia cellular metabolism or thermal cellular metabolism or thermal breakdownbreakdown

asp glu asn ser gln his gly thr arg ala tyr met try val phe ile leu lys

Spe

cific

am

inio

aci

d co

nsum

ptio

n (u

Mol

/106

cells

/day

)

-1000

-800

-600

-400

-200

0

200

400

600

800

1000

Staionary cultureFluidised bed bioreactor -4000

-2000

0

2000

4000

Specific amino acid consumption in 2 cultures


Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P57.


to reduce accumulation of ammonia:to reduce accumulation of ammonia: → → use continuous feed of low [gln]use continuous feed of low [gln] → → use gln-containing dipeptides that use gln-containing dipeptides that

hydrolyze slowly in culturehydrolyze slowly in culture alanine accumulates as a result of alanine accumulates as a result of

ammonia uptake/removalammonia uptake/removal glutamate may be used as a substitute, glutamate may be used as a substitute,

results in lower ammonia producedresults in lower ammonia produced

Energy metabolismEnergy metabolismglucose-6PGlucose P-gluconate ribulose-5P

CO2

Ribose-5PRibose-5Pfructose-diP

P-enolpyruvate

pyruvate

LactateLactateacetyl CoA

citrate

2-oxoglutaratesuccinate

oxaloacetateTCAcycle

Glutamineglutamate

NH3NH3

CO2

CO2

AlanineAlaninetransamination

CO2

Radioactive glucose for Radioactive glucose for metabolic flux analysis metabolic flux analysis

CH2OH

C

C

C

O

C

HC.OH

OH

OH

OHH

H

H H

1-14C

6-14C

3-3H

Lecture 4.5 Animal Cell BiotechnologyLecture 4.5 Animal Cell BiotechnologyCell Culture Conditions and MediaCell Culture Conditions and Media

3. Salts3. Salts included to make solution isotonic and to included to make solution isotonic and to

maintain balance with the intracellular contentmaintain balance with the intracellular content OsmolarityOsmolarity – – measure of the total number of measure of the total number of

particles dissolved in solutionparticles dissolved in solution a control parameter, can affect property of a a control parameter, can affect property of a

cultureculture

solute of molesionsor particles of molesx molarityOsmolarity


1 Osmolar ( 1 Osm/l) has 6.023x101 Osmolar ( 1 Osm/l) has 6.023x102323 particles in 1 liter of waterparticles in 1 liter of water

ionization increases the number of ionization increases the number of osmotically active particlesosmotically active particles

based on total number of particles based on total number of particles dissolved in solutiondissolved in solution

nature of particles is irrelevantnature of particles is irrelevant Ex. Ex. 1 mM glucose → 1 mOsm/l1 mM glucose → 1 mOsm/l 1 mM NaCl → 2 mOsm/l1 mM NaCl → 2 mOsm/l


osmolality (mOsm/kg of water) = osmolarity (in osmolality (mOsm/kg of water) = osmolarity (in dilute solution)dilute solution)

osmolarity of standard culture medium osmolarity of standard culture medium (isotonic) (isotonic) ~ 250-350 mOsm/l (300 mOsm → ~ 250-350 mOsm/l (300 mOsm → optimal)optimal)

→ → hypertonic – osmolarity is too high (cells hypertonic – osmolarity is too high (cells shrink)shrink)

→ → hypotonic – osmolarity is too low (cells burst)hypotonic – osmolarity is too low (cells burst) osmolarity of medium should be within osmolarity of medium should be within 10% of 10% of

optimal valueoptimal value


higher osmolarity, the lower the freezing higher osmolarity, the lower the freezing pointpoint

water has a freezing point of 0water has a freezing point of 0ooC, a saline C, a saline solution with an osmolality of 1 Osm/kg has a solution with an osmolality of 1 Osm/kg has a freezing point of -1.858freezing point of -1.858ooCC

can use osmometer to determing freezing can use osmometer to determing freezing point of a solution relative to waterpoint of a solution relative to water


4. Buffering system4. Buffering system Bicarbonate is typically included to act as a Bicarbonate is typically included to act as a

buffer system in conjuction with a CObuffer system in conjuction with a CO22 atmosphere (5-10%)(enriched air), maintains atmosphere (5-10%)(enriched air), maintains pH range of 6.9-7.4pH range of 6.9-7.4

Advantage: cheapAdvantage: cheap Disadvantage: cultures may become alkaline Disadvantage: cultures may become alkaline

very quickly upon removal from incubatorvery quickly upon removal from incubator alternate buffers may be used alternate buffers may be used → → HEPES (pKa = 7.3 at 37HEPES (pKa = 7.3 at 37C)C) → → MES (pKa = 6.5 at 37MES (pKa = 6.5 at 37C)C) → → CHES (pKa = 9.5 at 37CHES (pKa = 9.5 at 37C)C)

10-20 mM HEPES is optimal, cultures can be 10-20 mM HEPES is optimal, cultures can be grown without an enriched COgrown without an enriched CO22 atmosphere, atmosphere, expensiveexpensive


5. Vitamins and hormones5. Vitamins and hormones present in micromolar amounts, used as present in micromolar amounts, used as

metabolic cofactorsmetabolic cofactors varies from one medium formulation to varies from one medium formulation to

anotheranother different cell lines have different different cell lines have different

requirementsrequirements


6. Antibiotics6. Antibiotics often included in media for short-term often included in media for short-term

cultures in order to reduce the risk of cultures in order to reduce the risk of bacterial or fungal contaminationbacterial or fungal contamination

often used in combinations as an antibiotic often used in combinations as an antibiotic cocktailcocktail

→ → Penicillin G (100 u/mL) inhibits Gram-Penicillin G (100 u/mL) inhibits Gram-positive bacteria by inhibiting cell wall positive bacteria by inhibiting cell wall synthesissynthesis

→ → Streptomycin (50 mg/L) inhibits Gram-Streptomycin (50 mg/L) inhibits Gram-positive and Gram-negative bacteria by positive and Gram-negative bacteria by inhibiting protein synthesisinhibiting protein synthesis

→ → Amphotericin B (25 mg/l) as an antifungal Amphotericin B (25 mg/l) as an antifungal agent by inhibiting ergosterol synthesisagent by inhibiting ergosterol synthesis


not used for routine subculture or stock not used for routine subculture or stock cultureculture

low levels of bacteria or fungus may be low levels of bacteria or fungus may be maskedmasked

may allow for selective retention of may allow for selective retention of antibiotic resistant contaminantsantibiotic resistant contaminants


7. Phenol red7. Phenol red pH indicator of the mediumpH indicator of the medium sensitive to slight pH changes around sensitive to slight pH changes around

the growth optimum for cellsthe growth optimum for cells at lower pH, the phenol red turns orange at lower pH, the phenol red turns orange

(pH 7.0) or yellow (pH 6.5)(pH 7.0) or yellow (pH 6.5) overnight change from red to yellow overnight change from red to yellow

indicates bacterial contaminationindicates bacterial contamination

Lecture 4 Animal Cell BiotechnologyLecture 4 Animal Cell BiotechnologyCell Culture Conditions and Media - Cell Culture Conditions and Media -

SerumSerumSerumSerum added as a supplement (added as a supplement (10% v/v10% v/v)) supernatant of supernatant of clotted bloodclotted blood, contains , contains

undefined materials essential for cell undefined materials essential for cell growth, esp. for anchorage dependent growth, esp. for anchorage dependent cellscells→ → pH 6.85 – 7.05pH 6.85 – 7.05→ → osmolarity – 250-295 mOsmol/Losmolarity – 250-295 mOsmol/L→ → protein content – 60-80 mg/mLprotein content – 60-80 mg/mL→ → albumin content - 30-50 mg/mLalbumin content - 30-50 mg/mL

Lecture 4 Animal Cell BiotechnologyLecture 4 Animal Cell BiotechnologyCell Culture Conditions and Media Cell Culture Conditions and Media

- Serum- SerumSerum provides:Serum provides: Growth factorsGrowth factors LipidsLipids Attachment factorsAttachment factors Metal transporting proteins (i.e. transferrin)Metal transporting proteins (i.e. transferrin) Protease inhibitors (neutralizes trypsin)Protease inhibitors (neutralizes trypsin) High protein content protects against toxins, High protein content protects against toxins,

shear damage by stirring in fermentersshear damage by stirring in fermenters

Lecture 4 Animal Cell BiotechnologyLecture 4 Animal Cell BiotechnologyCell Culture Conditions and Media - Cell Culture Conditions and Media -

SerumSerum cow or horse serum most commonly usedcow or horse serum most commonly used fetal calf serum (FCS) most effective due to fetal calf serum (FCS) most effective due to

high content of high content of embryonic growth factorsembryonic growth factors

→ → newborn calf serum (under 10 days)newborn calf serum (under 10 days)

→ → donor calf serum (under 8 months)donor calf serum (under 8 months) serum is tested for performance and serum is tested for performance and

cytotoxicity effects on plating efficiency cytotoxicity effects on plating efficiency and cell growthand cell growth

filtered and tested for bacterial, fungal, filtered and tested for bacterial, fungal, and viral contaminationand viral contamination

Lecture 4.5 Animal Cell BiotechnologyLecture 4.5 Animal Cell BiotechnologyCell Culture Conditions and Media - Cell Culture Conditions and Media -

SerumSerumDisadvantages of serum:Disadvantages of serum:1.1. ExpensiveExpensive2.2. Chemically undefinedChemically undefined3.3. High protein content High protein content 4.4. Source of contaminationSource of contamination5.5. Ethical considerations?Ethical considerations?

Lecture 4.5 Animal Cell BiotechnologyLecture 4.5 Animal Cell BiotechnologyCell Culture Conditions and Media - Cell Culture Conditions and Media -

SerumSerum want to use serum-free mediawant to use serum-free media supplement media with insulin, supplement media with insulin,

transferrin, ethanolamine, and selenitetransferrin, ethanolamine, and selenite specific for different cell typesspecific for different cell types

Component Component characteristics of media characteristics of media

formulationsformulationsSerum based media

Serum-free media

Protein free media

Animal-component free media

Chemically-defined media

Media formulated goal:-Chemically defined-Protein-free-Animal component-free

Lecture 4b culture conditions and media

Technology

Transcript of Lecture 4b culture conditions and media