ANIMAL CELL CULTURE MANUAL

NATIONAL CENTRE FOR AQUATIC ANIMAL HEALTH

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY

FINE ARTS AVENUE, KOCHI-16

1. Washing glass wares for tissue culture work

 Remove the labels and wash the glass wares in tap water.
 Immerse the glass wares in boiling soap solution (non-toxic soap solution for tissue
culture) for 15 minutes.
 Next morning scrub with a clean brush. Wash in tap water at least 20 times and
leave in tap water overnight.
 Next day rinse in single distilled water and leave in single distilled water overnight.
 Take out the glassware next day, rinse with triple distilled water, dry it in hot air
oven, pack and sterilize.

2. Packing and sterilization

 Pipettes: Plug mouth of pipettes with cotton and arrange pipettes in pipette barrels after
burning off the cotton fibres projecting out. It will be easier if pipettes of different
volumes are arranged in different barrels. Slightly loosen the cap of the barrel and
sterilize in a hot air oven at 160C for two hours.

 Glass culture flasks: Cover the mouth of each bottle with aluminium foil and arrange in
a sterilization bucket. Open the vent of the bucket and sterilize in a hot air oven at
160C for three hours. Close the vent of the bucket after sterilization.

 Culture & media storage bottles, measuring cylinders and beakers, plastic
centrifuge tubes, Eppendorf tubes, screw cap vials & tubes : Arrange neatly all
these items in a plastic or glass beaker in the sterilization bucket with their caps
slightly loosened. Sterilize by autoclaving at 120C, 15 psi for 20min and dry in a hot air
oven at 100C for 2 hours. Remember to tighten the caps immediately after drying.

 Solutions: Slightly loosen the cap of the container, cover with paper and tie it with
twine. Sterilize by autoclaving at 15psi for 20 min or 10psi for 10 minutes as required.
Tighten the caps immediately after sterilization. Maintain at room temperature if the
storage is for a short duration or at 40C if it is for long term.

3. Preparation of Tissue culture Media

i) MEM

Dehydrated media- MEM with Earle’s salts, without L-glutamine, NaHCO3 and antibiotics
Dehydrated media - 9.54 g
Milliq water - 930 mL (autoclaved- 15 min. at 120 lbs)
NaHCO3 (3.5%) - 10 mL
L-Glutamine (30mg/mL) - 10mL
Antibiotic mixture (P/S) - 2 mL
Sodium pyruvate - 1 mL
Phenol Red - Few drops
Add FBS 10 % to prepare complete media (For 100 mL complete media - 90 ml in complete
media + 10 ml FBS) and check sterility of the media. Reagents preparations are following

1. L-Glutamine (30mg/mL)
Glutamine - 1.5g
Milliq water - 50mL
Sterilize by filtration through a 0.22µm membrane. Make 2mL aliquots and store at -200C.

2.Sodium bicarbonate(3.5%)

NaHCO3 - 3.5g
Milliq water - 100mL

Add a few drops of aqueous solution of Phenol red (1%) to the solution until it gives a purple
coloured solution. Pass CO2 through the solution taken in a 250mL conical flask for about 5
minutes. The color of the solution changes to orange red indicating the pH of 7.2 .Transfer it
into screw cap tubes leaving little space at the top. Autoclave at 10lbs for 10 minutes.

3. TPVG ( Trypsin Phosphate Versene Glucose)

PBS (1X) - 420mL

2% Trypsin - 25mL
0.2% Versene- 50mL
10% Glucose - 2.5mL
Antibiotic Mixture (p/s) - 1mL
pH: Adjust to 7.5 with 1N NaOH.

4. PBS (1X)

PBS (10X) - 50mL

Milliqwater - 450mL

Autoclave for 10 minutes at 10lbs.

5. PBS (10X)
NaCl - 80g
KCl - 2g
Na2HPO4- 11.5g
KH2PO4- 2.0g
Milliq water -1000ml

Autoclave for 15 minutes at 120 lbs.

6. Trypsin 2%
Trypsin - 2g
Autoclaved triple distilled water - 100mL
Sterilize by filtration through a 0.22µm membrane. Store at -200C

7. Versene (EDTA) 0.2%

Versene- 0.2g
Triple distilled water - 100mL
Autoclave for 15 minutes at 120lbs.

8. Glucose (10%)

Glucose - 10g
Triple distilled water - 100mL
Autoclave for 15 minutes at 120 lbs.

Sterility check

Add 0.5 - 1.0 ml of filtered media to a tube containing sterile nutrient broth and
incubate for 48 hours. If the broth is clear after 48 hours the media is sterile

ii) Shrimp Cell culture Medium (SCCM)

For 1000 mL media, take 785 mL sea water (30 ppm) + 100 mL milliq water (total -885 mL) and
autoclave 20 min at 120 lbs. Add the following components in sterile condition

Amino acid mix A (100X) - 10 mL

Amino acid mix B (100X) - 10 mL
Sugar mix (100X) - 10mL
Cholestrol- 5 mL
Growth factor mix - 1 mL
Phenol red solution - 5 mL
Vitamin mix (MEM) - 10 mL
Bicarbonate - 150 mg
L-glutamine - 600 mg
Antibiotic mix(p/s) - 2 mL
Mix all the components in a 100 mL bottle and filter sterilize to the sea water previously
autoclaved. Adjust the pH 6.8 to 7.2 and osmolality 720±10 mOsm/gm.

Media components preparation

1.Amino mix A (100X) -50 ml

L-arginine - 225 mg
L-alanine - 350 mg
L-asparagine - 75mg
L-Cysteine - 5 mg
L-lysine - 300 mg
L-Histidine -75 mg
L-Methionine - 25 mg
L-proline -500 mg
L-serine -75mg
L-taurine -500mg
L-threonine -75 mg
L-valine -100 mg
L-glycine -100 mg

2. Amino acid mix 2 (100X) – 50 mL HCl

L-aspartic acid - 50 mg
L-cystine - 5 mg
L-leucine - 100 mg
L-phynyl alanine - 50 mg
L-tryptophan - 50 mg
L-tyrosine - 50 mg
L-isoleucine - 50 mg
L-glutamic acid -50 mg

3. Sugar mix (100X) – 100 mL

Glucose -1000 mg
Ribose - 10 mg
Tichalose - 10 mg
Sodium pyruvate -500 mg

4.Growth factor mix -20 ml

IGF 1 (100 ng/mL) - 40 µl

IGF II (150 ng/mL) - 30 µl

Mix in a 20 mL SCCM and keep it as stock.

 5.Cholostrol(100 X )

0.4 mg in 20 mL SCCM

iii) L-15 medium

4. Primary cell cultures from different donor tissues

Primary culture can be defined as the cells derived directly from the donor tissue or organ. There
are three stages to be considered:

1) Dissection and removal of donor tissue asceptically

2) Isolation of the tissue and short term maintenance
3) Seeding culture flask and cell propagation

i) Primary lymphoid cell culture from shrimp

 Animals take in crushed ice-box

 Surface sterilize the animal using sodium hypochlorite (800 ppm: 8 mL sodium
hypochorite solution make in to 80 mL using distilled water)
 Wash with sterile sea water 2 to 3 times to remove the residues of chlorine.
 Then dip in a ice-cold ethanol for a while and immediately transfer in to sterile sea
water.
 Dissect the animal using sterile dissection sets and keep the lymphoid organ in a
holding medium (SCCM without FBS and with anitbiotics).
 Mince the tissue using sterile blade and transfer the 1 mm 3 tissue pieces into cell
culture flask.
 Add FBS to surrounding of the tissue and incubate for 3 h.
 Discard the excess FBS and add medium (SCCM) to tissue surrounding.
 After 3-6 h add 3-5 ml medium (SCCM) to the culture flask and observe after 12 h.

ii) Primary culture from fish using fin as donor tissue

 Cut the fin from animal without sacrificing.

 Tween 80 was used as detergent to wash the external tissues such as fin and gill.
 It was prepared in concentration (5:5) by mixing Tween 80 and PBS containing
0.5% of glucose.
 Transfer the tissue in to holding medium (L-15without FBS containing Penicillin
(500 IUmL-1) Streptomycin (500 µg mL-1) and Nystatin (1000 U mL -1)
Amphotericin B (2.5 µg mL-1 ).
 Wash the tissue 2-3 times in holding medium.
  Tissues were minced into small pieces (approximately 1 mm 3 in size) using sterile
surgical blade.
 Ex-plants were seeded in 25 cm2 cell culture flasks (Greiner bio one, Austria) by
spreading them uniformly over the flask.
 FBS was added to facilitate attachment of the tissue to the flask.
 After incubation of the flasks for 3hrs added 3 ml L-15 and kept for incubation at
250Cas closed system.
 On attaining 80% confluence (4-5 days) cells were passaged using 0.25% trypsin-
EDTA solution.

5. Cell Lines

Once a primary culture is sub cultured it is called a cell line. Cell lines with limited culture life
spans are known as finite cell lines. They are called diploid cell lines as they have diploid sets of
chromosomes. A cell line having the capacity for infinite survival is called a continuous cell line
previously known as ‘established’ and often referred to as immortal.

i) Routine maintenance

Once a culture is initiated it will need a periodic medium change or feeding followed eventually
by subculturing on attainment of full growth of cells. In nonproliferating cultures, the medium
will still need to be changed periodically, as the cells will still metabolize and some constituents
of the medium may become exhausted or degrade spontaneously. Four factors indicate the need
for the replacement of the culture medium.

1. A drop in pH: Most cells stop growing as the pH falls from pH 7.0 to pH 6.5 and start to
lose viability between pH 6.5 and pH 6.0. Therefore, the medium goes from red through
orange to yellow, it should be changed immediately.
2. Cell concentration: Cultures at a high cell concentration exhaust the medium faster than
those at a lower concentration.
3. Cell type: Normalcells( e.g. Diploid fibroblasts) usually stop dividing at a high cell
density due to cell crowding, growth factor depletion, and other reasons. The cells block
in the G1 phase of the cell cycle and deteriorate very little, even if left for two or three
weeks or longer.
4. Morphological deterioration: This factor can be monitored by regular examination and
familiarity with the cell line. If deterioration is allowed to progress too far, it will be
irreversible.

ii) Changing the medium or feeding

Examine the culture by eye and through an inverted microscope. If indicated, e.g. by a fall in pH,
remove the old medium and add fresh medium.
Materials

Pipettes
Pre-warmed Growth Medium
 1. Prepare the hood, bring the reagents and materials necessary for procedure and place
those required immediately in the hood, after swabbing it with 70% alcohol.
2. Examine the cultures carefully for signs of contamination or deterioration.
3. Take the culture inside the hood after swabbing with 70% alcohol. Carefully swab the
neck of the bottle with 70% alcohol.
4. Remove and discard the medium.
5. Add the same volume of fresh medium, prewarmed to 370C.
6. Examine the culture under microscope and return to the hood.

iii) Subculture

The growth of cells in culture usually follows a standard pattern. A lag phase following seeding
followed by a period of exponential growth, called the log phase. When the cell density
((cells/cm2) reaches a level such that all of the available space is occupied) exceed the capacity of
the medium, growth ceases or is greatly reduced. In such situations either the medium must be
replaced more frequently or the culture must be divided (sub cultured). For an adherent cell line
sub culturing involves removal of the medium and dissociation of the cells in the monolayer with
trypsin.

The attachment of cells to each other and to the culture flaskis mediated by cell surface
glycoproteins, and Ca2+. Other proteins, and proteoglycans derived from the cells and from the
serum, become associated with cell surface and the surface of the substrate and facilitate cell
adhesion. Subculture usually requires chelation of Ca2+ and degradation of extracellular materials
and potential cell adhesion molecules.

Sub culturing of cell culture using TPVG

1. Prepare the hood, and bring the reagents and materials to the hood to begin the procedure.
2. Examine the culture carefully for signs of contamination or deterioration.
3. Take the culture inside the hood and discard the medium.
4. Add PBS prewash (0.2ml/cm2) to the side of the flask opposite the cells so as to avoid
dislodging of cells, rinse the cells, and discard the rinse. This step is designed to remove
the serum and Ca2+ that would inhibit the action of trypsin.
5. Add TPVG (0.1ml/cm2) to the side of the flask opposite the cells. Turn the flask over and
lay it down. Ensure that the monolayer is completely covered. Leave the flask for 15-30s,
and then withdraw all but a few drops of the trypsin, making sure that the monolayer has
not been detached.
6. Incubate, with the flask lying flat, until the cells round up. Do not leave the flask longer
than necessary. Tap heavily on the flask to facilitate release/ detachment of cells from the
flask.
7. Add medium (0.1-0.2mL/ cm2), and disperse the cells by repeated pipetting over the
surface bearing monolayer. Finally, pipette the cell suspension up and down a few times,
with the tip of the pipette resting at the bottom corner of the bottle, taking care not to
create foam. The degree of pipetting required will vary from one cell line to another;
some cell lines disperse easily, while others require vigorous pipetting in order to
 disperse them. Pipette the suspension up and down sufficiently to disperse the cells into a
single cell suspension.
8. A single- cell suspension is desirable at subculture to ensure an accurate cell count and
uniform growth on reseeding.
9. Count the cells using a hemocytometer.
10. Dilute the suspension to the appropriate seeding concentration by adding the appropriate
volume of cells to a premeasured volume of medium in a culture flask.

iv)