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).