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  Section: General Biotechnology / Animal Biotechnology
 
 
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Animal Cell, Tissue and Organ Culture

 
     
  Content
Requirements for animal cell, tissue and organ culture
  Substrates for cell culture
  Substrate treatment
  Culture media
    Natural media
    Synthetic media
  Sterilization of glassware, equipments and culture media
  Isolation of animal material (tissue)
    Disaggregation of tissue
    Establishment of cell culture
Cultivation of animal cell en masse in bioreactor
Immobilized cell culture
Insect cell culture
Somatic cell culture
Organ culture
  Organ culture on plasma clots
  Organ culture on agar
  Organ culture in liquid medium
  Whole embryo culture
Valuable products from cell cultures
  Monoclonal antibodies
  Production of commercial products from insect culture

Culture Media
Culture of animal cells and tissue is rather more difficult than that of microorganisms and plants because the later synthesize certain chemical constituents from inorganic substances. However, the culture media provide the optimum growth factors (e.g. pH, osmotic pressure, etc) and chemical constituents (unlike microbes). There are two types of media used for culture of animal cell and tissue, the natural media and the synthesized media.

Natural media
Natural media are the natural sources of nutrient sufficient for growth and proliferation of animal cells and tissue. These are of three types : (i) coagulans or plasma clots (it is used since long time but now available in market in the form of liquid plasma kept in silicon ampoules or lyophilized plasma. Plasma may also be prepared in laboratory taking out blood from male fowl and adding heparin to prevent blood coagulation), (ii) biological fluid (it is obtained in the form of serum from human adult blood, placental, cord blood, horse blood, calf blood or in the form of biological fluids such as coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour (from eyes), etc. The most commonly used fluids are human placental, cord serum and foetal calf serum. Before use its toxicity should be checked, and (iii) tissue extract (extract from some tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow, etc. are also used for culture of animal cells, where embryo extract is of most common use. Tissue extract should be used before a week or stored at 27°C.


Synthetic media
Synthetic media are prepared artificially by adding several nutrients (orgtmie and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors, etc. However, different types of synthetic media may be prepared for a variety of cells and tissues to be cultured. It can be prepared for different functions. Basically, synthetic media are of two types, serum-containing media (i.e. the media containing serum) and serum-free media (i.e. media devoid of serum). Example of some of the media are: minimal essential medium (MEM) (Eagle, 1955), 199 (Morgan et al. 1950), CMRL 1066 (Parker et al, 1957), RPMI 1640 (Moore et al, 1967) and F12 (Ham, 1965). Some of these media are given in Table 6.1.


Table 6.1. Chemical composition of different media (with serum) used for animal cell and tissue culture (quantities are in mg/l).

 

Chemical constituents

Eagle's MEM

Dulbecco's modification

Ham's F12

 

1.

Amino Acids

 

 

 

 

L-asparagine

126

84

211

 

L-cystine

24

48

-

 

L-glutamine

292

584

146

 

Glycine

-

30

7.5

 

L-histidine HC1.H2O

42

42

21

 

L-isoleucine

52

42

21

 

L-leucine

52

105

13.1

 

L-lysine.HCl

73.1

146

36.5

 

L-methionine

15

30

4.48

 

L-phenylalanine

33

66

4.96

 

L-proline

-

-

34.5

 

L-serine

-

42

10.5

 

L-threonine

48

95

11.9

 

L-tryptophan

10

16

2.04

 

L-tyrosine

36

72

5.4

 

L-valine

47

94

11.7

2.

Vitamins

 

 

 

 

Biotin

-

-

0.0073

 

D-Ca-pantothenate

1

4

0.48

 

Choline chloride

1

4

14

 

Folic acid

1

4

1.3

 

Inositol

2

7.2

18

 

Nicotinamide

1

4

0.04

 

Pyridoxal.HCl

1

4

0.062

 

Riboflavin

0.1

0.4

0.038

 

Thiamine.HCl

-

-

1.36

 

Vitamin B12

-

-

1.36

 

Pyridoxin.HCl

-

-

0.062

3.

Inorganic Salts

 

 

 

 

CaCl2 (anhydrous)

200

200

-

 

CaCl2.2H2O

-

-

44

 

Fe(NO3)3.9H2O

-

0.1

-

 

KC1

400

400

221

 

MgCl2.6H20

-

-

122

 

MgSO4.7H2O

200

200

-

 

NaCl

6800

6400

7599

 

NaHCO3

2200

3700

1176

 

Na2H2PO4.H2O

140

125

-

 

Na2HPO4.7H2O

-

-

268

 

CuSO4.5H2O

-

-

0.00249

 

FeSO4.7H2O

-

-

0.834

 

ZnSO4.7H2O

-

-

0.863

4.

Other Chemicals

 

 

 

 

D-glucose

1000

4500

1802

 

Lipoic acid

-

-

0.21

 

Phenol red

10

15

12

 

Sodium pyruvate

-

110

110

 

Hypoxanthine

-

-

4.1

 

Linoleic acid

-

0.084

-

 

Putrescine.2HCl

-

-

0.161

 

Thymidine

-

-

0.73

5.

CO2 (gas phase)

5%

10%

5%

Source : Freshney (1987).

When the synthetic media are devoid of serum in culture medium, it is called serum-free media. Example of some serum-free media for certain cells and cell line are given in Table 6.2. By doing so the medium could be made selective for a particular type of cells because each type of cells requires different chemical constituents and physical factors. Serum-free media should not be used commonly until cheap and better serum-free media are available. Serum itself has several disadvantages as given below: (i) it deteriorates within a year and differs with batches, (ii) a number of batches are required if more than one cell types are used which make difficult for maintaining and co-culturing of cells difficult, (iii) supply of serum is less than its demand, therefore, medium becomes several times costly, and (iv) undesirable growth stimulation and inhibition may occur. Fiechter (1996) has enlisted the advantages and disadvantages of using the serum in culture media (Table 6.3).

Table 6.2. Serum-free medium for certain cell and cell lines.

 

Serum

Serum-free medium

Cell or cell lines

1.

CS

MCDB 202

Chick embryofibroblasts

 

 

CMRL 1066

Continuous cell line

 

 

MCDB 110, 202

Fibroblasts, human diploid fibroblasts

 

 

MCDB 402

Fibroblasts, mouse embryofibroblasts, 3T3 cell

2.

FB

MCDB 130

Endothelium

 

 

F12

Skeletal muscles

 

 

HoS

Mouse leukemia, mouse erythroleukemia, skeletal muscles

Source : based on Freshney (1987).


Table 6.3. Advantages and disadvantages of serum in culture media.
1. Advantages
Serum contains a complete set of essential growth factors, hormones, attachment and
spreading factors, binding and transport proteins.
It binds and neutralizes toxins.It contains protease inhibitors.It increases buffering capacity.It provides trace elements and other nutrients.

2. Disadvantages
It is not chemically defined and, therefore, it is of variable composition lot to lot.It may be a source of contamination by viruses, mycoplasma, prions, etc.Its components may bind , inactivate, antagonise or mimic the action of added medium
ingredients.
It increases difficulties and cost of down stream processing.It is most expensive ingredient of the culture media   
Source : based on Fiechter (1996).
 
     
 
 
     



     
 
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