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Cell Culture Environment - PowerPoint PPT Presentation


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Cell Culture Environment. How to grow a plant ?. Water. Soil. Air (CO 2 and O 2 ). Nutrition. Water : Culture medium . Soil : substrate for cell growth . Air (CO 2 and O 2 ) : Air in incubation chamber. Nutrition : growth supplement. Culture Medium Functions of culture medium:

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slide2

How to grow a plant ?

Water

Soil

Air (CO2 and O2 )

Nutrition

slide3

Water : Culture medium

Soil : substrate for cell growth

Air (CO2 and O2 ) : Air in incubation chamber

Nutrition : growth supplement

slide4

Culture Medium

  • Functions of culture medium:
  • 1. Maintain the pH
      • 2. Maintain the osmolarity
      • 3. Provide nutrient
      • 4. Provide energy
slide5

Basic components of cell culture environment

  • The in vitro environment must meet the
  • fundamental physiological requirements of the cell
  • 1. Culture medium
  • 2 . Physiological factors
  • pH 7.27.5
  • osmolarity 280320 mOsmol/kg
  • CO2 25% in air
  • temperature 35oC-37oC
  • 3. Stationary versus dynamic media
  • supply enough
  • replenish at proper time
  • vol. 0.20.3ml/cm2
slide6

A complete culture media:

1. A basal medium specifies for all cellular

requirements

2. A set of components that specify other

cellular requirements and permit

growth of cells in the basal medium

slide8

Basal Medium

      • Types of basal medium
  • 1.Eagles medium and derivative
  • e.g. BME, EMEM, AMEM, GMEM, JMEM
  • 2. Media designed at Roswell Park
  • Memorial Institute ( RPMI)
  • e.g.RPMI 1629, RPMI 1630, RPMI1640
slide9

3. Basal medium designed for use after

serum supplement

e.g. Fischer’s Lieboutz, Trowell, Will

4.Basal medium designed for serum-free formulation

e.g. CMRL 1060

Ham’s F10 and derivatives

TC199 and derivatives

NCTC

Waymouth

slide10

5. For insects culture

  • e.g. Grace’s medium
  • Schneider’s medium
  • Mitsuhashi and maramerosch medium
  • IPL-41 medium
  • Chiu and black medium
  • D-22 medium
slide12

Basic components of the culture medium

1.Buffer system

ß - glycerophosphate

organic components

e.g. HEPES, Tes, Bes

2. Energy Source

glucose, maltose, sucrose, fructose, galactose,

mannose

3. Amino Acid

essential amino acid

slide13

4. Vitamins

precursor for the cofactors

e.g. biotin, choline, folic acid, nicotinic acid…

  • 5 . Hormones and growth factor
  • e.g. insulin, hydrocortisone, NGF, EGF
  • 6. Proteins and Polypeptide supplement with:
  • fetuin,-globulin, fibronectin, albumin, transferrin….

7. Fatty acid and Lipid

8. Accessory

e.g. Zn,Cu,Se….

slide14

10. Antibiotics

factors to be considered:

 absence of cytoxisity

broad anti-microbial spectrum

accepted cost

minimum tendency to induce

formation of resistant micro- organism

e.g. penicillin, streptomycin gentamycin.

slide15

Buffer System

Bicarbonate buffer

Recommended CO2 concentration and gas phase to use with common

basal media

basal medium NaHCO3 % of CO2

concentration gas phase

Eagle’s MEM(Hank's salt) 4

grace’s (Hank salt ) 4 atmospheric

IPL-41 (Hank salt ) 4 atmospheric

TC100 (Hank salt ) 4 atmospheric

Schneider’s (Hank salt ) 4 atmospheric

IMDM 36 5

TC199 26 5

DMEM/Ham’sF12 29 5

RPMI1640 24 5

Ham’sF12 14 5

DMEM 44 10

slide16

Choice of basal medium

      • from literature
  • e.g. BME: for HeLa, L-cells, BHK,
        • primary culture of human rodent and avian
        • fibroblast
        • e.g. RPMI: for human haemopoietic cells
        • e.g. Iscove’s modified Dulbecco’s medium( IMDM):
        • for haemopoietic origin, growth and
        • differentiation of human and murine primary
        • bone marrow culture
        • e.g. sf9,sf21: for Drosophilla and insect culture
slide17

Preparation of basal medium

  • Factors to be considered:
    •  Avoid contamination
    •  Use pure water
    •  Use analytical chemicals
    •  Glassware used must be cleaned
slide18

Equipment for preparation of media:

  • High purity chemicals and biologicals
  • Good analytical balance
  • Hot plate with magnetic stirrer

 Volumetric flasks of various volume

 pH meter

  • Osmometer
  • Autoclave and membrane filtrate
  • medium kept at 4oC
  • storage:
  • storage should not be over 3 months
slide19

Serum

Why use serum ?

Advantages of using serum

  • Serum represent a cocktail of most of the factors
  • Required for cell proliferation and maintenance.
  • 3. An universal growth supplement which is effective
  • with most cells.
  • 4. Serum buffers the cell culture system against a variety
  • of perturbation
slide20

Types of serum

1. FBS ( Fetal Bovine serum) from abattoir

2. Horse serum, calf serum from donor )

should pass virus test)

slide21

Constituents of serum

  • 1.Growth factors ; 5—30 kDa polypeptide
  • 2.Albumin
      • a. a carrier protein
      • b. carry thyroxin and metal
      • c. pH buffering

3. Transferrin-ion transport protein

  • 4. Anti-protease: prevent cell from proteolytic damage
  • a1-trypsin
  • b2-macroglobulin
slide22

Nutritional and protective factors which may be supplied by serum

    • 1.Specific growth factors--
    • EGF,PDGF,IGF,FGF, IL-1,IL-6
    • 2.Trace elements--
    • Iron, Zinc, Selenium
    • ( Co,Cu,I,Mn,Mo,Cr,Ni,V,As,Si,F,Sn)
    • 3. Lipids-- cholesterol, linoleic acid, steroids
    • 4. Polyamines--putrescine, ornithin, spermidine
slide23

5. Attachment factors-- fibronectin, fetuin, Laminin

    • 6. Mechanical protection– albumin
    • 7. Buffering capacity– albumin
  • 8. Neutralization of toxic factors– albumin
    • 9. Transport of metals
    • transferrin/Fe+3,
  • ceruloplasmin/Cu+2
  • 10.Protease inhibitors– antitrypsin,1macroglobulin
slide24

Potential problems with the use of serum

    • 1. Lack of reproducibility
      •  Serum batches varies considerably
      • The presence of specific antibodies may
      • also affect the results
      • Serum may vary during the process of collection
      •  Sterity
slide25

2. Risk of contamination

  •  To prevent viral infection:
  • -propiolactone
      • -irradiation
      • heat inactivation( 56oC, 1hr)
      • 3. Availability and cost
      • 4. Influence of downstream process
  •  difficulties in purifying proteins
        • serum protein 4—8 mg/ml,
        • recombinant protein---- 0.1ug/ml
  •  difficulties in purifying monoclone antibody
slide26

Sourcing and selection of serum

 sourcing:

dealing with reputable supplier selection:

test the capability for growth on cheaper serum type

test different batches of serum for growth performance

test of cell growth from very low seeding

test of cloning efficiency

slide27

Serum storage and use

kept frozen, -20oC

thaw rapidly, mix gently

kept at 4oC once thawed

slide28

Replacement of serum in medium

 Replace by Controlled Process Serum Replacement

( CPSR)

CPSR: derived from bovine plasma,

lower endotoxin

lower protein

 Replace by supplement or fortified serum

fortified serum: supplement with mitogen, growth factor,

hormones, proteins, protein stabilizer, trace elements

slide29

Serum Free medium

A properly designed serum free medium

 is reproducible

is not reliant on economics of the world cattle market

 simplifies down stream purification

has no unknown factors e.g. viruses, growth inhibitors

slide30

The most common requirement of serum free medium

e.g. polypeptide hormone, insulin, iron transport protein,

transferrin

  • Supplement of serum free medium
  • e.g. steroid growth factors, trace elements reducing
  • agents, diamines, vitamins albumin complexes with
  • unsaturated fatty acid
slide31

Design of serum free media

      • Reduce serum gradually
      • Adding other components
      • Growth assay study in the presence of added
      • supplements for basal medium
  • Add components singly or in combination to a basal
  • medium in a step wise manner
slide32

Mechanical stabilizers and adhesion factors

 For suspension culture:

 To prevent shear damage: damage by air bubble, stirrer,

shaker…

improved by increase viscosity of the medium by adding:

carboxy methyl cellulose ( viscosity modifier) and

prolyvinylpyrrolidone( PVP), pluronic-F68

principles: formation of an interfacial

structure of adsorbed molecule on cell

surface)

slide33

Considerations :

 Energy source of basal medium

Carrier of lipids

Proper environment for metabolism

Growth function of cells ( good buffering capacity)

Good maintenance of electrolyte balance

 Fe+2---Zn+2----Cu+2trace elements: Na+,K+,

Ca+2,Mg+2,Cl-,HPO4-2…..

slide34

 For anchorage dependent cells

  • substrate treat with adhesive glycoprotein,

fibronectin, laminin, chondrotin, serum spreading

factors

slide35

Difficulties that may encountered with serum free medium

  • a. Design of a dedicated medium for each cell type
  • b. Culture condition become more critical
  • c. Serum free medium has a reduced capacity to inactivate
  • or absorb toxic materials
slide36

Selection of components

e.g. transport protein,stabilizing proteins growth

regulators, growth factors, attachment proteins,

crude extracts, essential nutrient

Practical hints on solubilizing specific components

e.g. riboflavin, folic acid, tyrosine, cystein

dissolve in NaOH

e.g. fatty acid, lipids, fat soluble vitamins

in alcohol solutions

e.g. pluoronic F-68

soluble in cold water

e.g. hypoxanthine

heat to dissolve