TISSUE CULTURE SYSTEM

HISTORY:
Tissue culture was first devised at the beginning of the century (20th century) by
Harrison (1907) and Carrel (1912), as a method for studying the behaviour of animal
cells free of systemic variations.
Harrison (1907) chose the frog as his source of tissue, presumably because it was a
cold-blooded animal and consequently incubation was not required.
TISSUE CULTURE:
Tissue culture system is that system in which tissue is kept in living condition by the
supplement of nutrition as/in the form of synthetic media in the laboratory.
Or
The term tissue culture designates those cell systems established with tissue fragments
or with masses of cells originating and migrating from the fragments.
TYPES OF TISSUE CULTURE
There are three main methods of initiating a culture
 Organ culture
 Primary explant culture and
 Cell culture
Organ culture: Organ culture implies that the architecture, characteristics of the
tissue in vivo, is retained at least in part, in the culture. Towards this end, the tissue is
cultured at the liquid-gas interface (gel, grid, etc.), which favors the retention of a
spherical or three dimensional shape.
Primary explant culture: In primary exaplant culture, a fragment of tissue is placed
at a glass-liquid interface. Where following attachment migration is promoted in the
plane of the solid substrate.
Cell culture: Cell culture implies that the tissue, or outgrowth from the primary
explant, is dispersed (mechanically or enzymatically) into a cell suspension, which
may then be cultured as an adherent manslayer on a solid substrate or as a suspension
in the culture medium.
PROPERTIES OF DIFFERENT TYPES OF CULTURES:

Category Organ Culture Explant Cell culture

Source Embryonic organs, Tissue fragments Disaggreated tissue,
adult tissue fragments primary culture
Effort High Moderate Low
Histology Informative Difficult Not applicable
Characterization Easy, histology Cytology and Biochemical,
marker molecular,
immunological and
cytological assays.
Propagation Not possible Possible Standard procedure
Homogeneity High intersample High intersample Low level of
variable variable variation
Quantitation Difficult Difficult Easy many
techniques are
available
Biochemical Possible Heterogeneous Lost, but may be
Differentiation reinduced.
ADVANTAGES:
(1) Direct access of the virus to the cell surface.
(2) Known cell populations derivable from single cells, may be produced in serial
culture.
(3) Cell lines from different species, organs and tissues are available or may be
cultivated.
(4) Metabolic studies and cytopathic changes may be studied conveniently.
(5) Cell cultures are free from antibody, hormonal and similar host factors.
(6) Standardized technical procedures may be established and employed.
(7) Many cell lines, continuously cultivated, may be frozen and stored.
(8) Control of the environment-
 Physicochemical- environment (pH, temperature osmotic pressure and
O2 and CO2 tension).
 Physiological environment (hormones and nutrient concentrations)
(9) Characterization and Homogeneity of sample
(10) Economy
DISADVANTAGES:
(1) Expatriation
(2) Quantity
(3) Origin of cells
(4) Instability
(5) Contamination
Notes: The following factors are accounted for the importance of tissue culture-

 The general recognition by virologist in 1950 that many viruses as they

multiply produce degenerative changes (cytopathic effect = CPE) in the
cultural cells which can easily be distinguished.

 The development of antibiotic and it use in tissue culture system for prevention
of secondary infection.
  The technique of using 2% trypsin for the preparation of suspension of
dispersed cell in establishing tissue culture system.
BASIC REQUIREMENTS FOR THE GROWTH OF CELL:
Temperature: The optimum temperature for most type of cells varies from 370-380C.
Hydrogen Ion concentration: The optimum pH varies from 6.6 ~ 7.8.
Gaseous Condition: O2 and CO2 are required for satisfactory growth of cells. The
required CO2 tension is produced by the cells metabolism provided the cells are grown
in tightly sealed or stoppered container.\
Constituents of Media: The precise role of inorganic salts incorporated in most of
cell culture media is not fully understood. Sodium salts are concerned with
maintaining osmotic pressure suitable for cells and both potassium (K) and
Magnesium (Mg) are activators of enzyme involved in the cells metabolism.
CELL CULTURE:
Culture prepared with dispersed cells are designated as cell cultures.
Types of cell culture:
Three types of monslayer cell cultures –
 Primary cell culture
 Diploid cell culture / cell line and
 Continuous / established / heteroptoid cell culture or cell lines
Primary cell culture:
Tissues/cells which are used one time for the preparation of cell culture after
collecting them from the animals.

 Primary cells are the first generation cell preparations that grow from the
tissues of origin.

 Predominantly epithelial cells

 Normal diploid chromosomes of the same number as the parent tissue.

 eg. Primary rhesus monkey kidney (pRMK), primary human embryonic

kidney (pHEK)
Diploid cell culture:
Diploid cell lines as their name suggests are those which continuously maintain their
diploid chromosome number throughout serial passages, these cells usually die out
after 50th passage. eg. Human Lung fibroblast (MRC-5 or WI-38).
Continuous Cell Culture: CCC demonstrates heteroploid chromosome numbers
during repeated subculturing.

 Originate from malignancies.

 Replicate vigorously.

 Usually not difficult to culture.

 Being capable of indefinite passaging.

eg. Human cervical carcinoma (HeLa)
DETERMINATION OF GROWTH OF VIRUS
 Qualitative Method and
 Quantitative method
 Qualitative methods for determination of a virus in the cell culture are as follows-
1. Sampling of the virus in the liquid and/or cellular phage of the culture for
infectivity tests in various host systems.
2. Cytopathologic investigations.
3. Studies of cellular metabolism.
4. Most of action of viruses with antibody and other antiviral substances
5. Co-existent viral infections
6. Haemagglutination lest
7. Serological lest and
8. Microscopy
 Quantitative Methods:
1. Cytopathic effects observed from microscopic examination.
2. Lack of metabolic activity as evidenced by calorimetric or pH change of the
medium.
3. The production of plaques with the agar overlay technique.
CELLS USED FOR CULTIVATION OF CERTAIN VIRUSES
Cells Viruses
i) Hela Polio, equine abortion, NDV, measles, herpes
simplex.
ii) Human embryonic intestine Some arboviruses
iii) Human amnion Polio, adenovirus, measles
iv) human embryonic skin Varicella, Measles
v) Mouse fibroblast Herpes simplex
vi) rabbit kidney Herpes simplex and admo
vii) Canine kidney Canine distemper, canine hepatitis
viii) Bovine fetus kidney FMD, Infectious-bovine rhinotrachetis, bovine
enterovirus.
ix) Whole chicken embryo Herpes simplex, NDV, influenza
x) Chicken embryo CAM NDV, Influenza, Infections bronchitis of chicken
xi) Chicken embryo lung NDV, Influenza
xii) chicken embryo kidney Infections bronchitis of chicken.

CONTAMINATION OF CELL CULTURE

All cell cultures, whether commercially purchased or originated and maintained in
house, may be subject to contamination by bacteria, fungi or mycoplasma.
Sources for Contamination:
The sources for contamination are many and include-

 the usual throat flora of laboratory personnel

 clinical specimens

 contaminated cell culture reagents and additives such as bovine sera.

Bacterial/Fungal Contaminants:
Usually produce gross changes in the appearance of the culture. Culture media
become turbid, the pH of the media becomes acid, cells appear granular and may
detach from the culture vessel.
Detection: Bacterial and fungal contamination are not always obvious. Inoculate
samples from suspicious cultures on to blood and/or Sabourauds agar and into
thioghycollate or similar broth medium to test for growth of contaminating bacteria or
fungi are usually discarded because contamination is difficult to eliminate.
Mycoplasmal Contaminants:
Mycoplasma contamination may go undetected through many generations of cells.
Detection: Mycoplasma isolation requires the use of broth or plate media that are
enriched with yeast extract and hones serum. An incubation period of 48 hours to 7
days may be required for growth of the organisms on agar and subculturing of broth
media to agar plates at 10 ~ 14 days is recommended for detection of small quantities
of the organisms.
CELL CULTURE MEDIA AND STOCK SOLUTION:
Many different cell-culture media can be used and all have the following basic
composition:
1) A balanced salt solution
2) A protein supplements such as serum or amniotic fluid, or protein
derivatives such as lactalbumin hydrolysate or tryptoe-phosphate broth,
or amino acid.
3) An antibiotic mixture to control accidental microbial contamination.
 Balanced Salt Solutions (BSS): + PBS
Hanks BSS (HBSS) and Earles Bss are frequently used as bases for growth media.
Their function is to maintain a physiologic pH (7.2-7.6) and osmotic pressure and to
provide water, glucose and inorganic ions needed for a normal cell metabolism.
BSS and PBS are also frequently used to wash inocula and dead cells from cultures to
remove serum-containing media before trypsinization and to dilute trypsin solutions.
BSS consists of essential inorganic chemicals dissolved in glass-distilled or deionized
water to form isotonic solutions.
Table: Composition of some BSS and PBS

Dulbecco’s
Solution Component HBSS EBSS PBS
BSS
A Water 800 800 800 1000
NaCl 8.0 6.8 8.0 8.0
KCl 0.4 0.4 0.2 0.2
MgSO4.7H2O 0.2 0.2 - -
KH2PO4 0.06 - 0.2 0.26
Na2HPO4.2H2O 0.06 - 0.14 0.57
NaH2PO4.H2O - 0.14 - -
Glucose 1.0 1.0 - -
Phenolred, Na 0.017 0.017 0.017 0.017

B Water 100 100 100 -

CaCl2 0.14 0.2 0.1 -

C Water 100 100 100 1.8

MgCl2.6H2O - - 1.0 -
NaHCO3 0.35 2.2 -

 Body Fluid: Sera, plasma and other body fluids were the earliest nutrient media
employed for cell culture. They are still widely employed but usually in
combination with other natural substances or as supplements to chemically
defined media.

 Sera: Horse, cattle, and lambs calves about 6 months of age is satisfactory.
Function: are not clearly understood. However certain serum proteins
promote attachment and spreading of cells on glass surfaces. Fetal bovine
serum is particularly rich in protein.

 Plasma: Chicken plasma is preferable specially from cockerels (< 1 year of age).
Use: i) For physical support
ii) Nutrient supplement for a variety of cells.

 Bovine Amniotic Fluid: incorporated in media for supporting cell growth.

 Embryo extract: Saline extract of chick or bovine embryo is used to provide

growth factor.
 Bacteriological Media: Sources of amino acids e.g. I. Lactalbumin hydrolysate,
II. Yeast extract, III. Trypotase phosphate and IV. Peptones.
 Cell Dispersing Agents: Proteolytic enzyme Trypsin 0.5% solution, PBS without
Ca and Mg ions. The solution is sterilized by filtration.
Others are: Collagenase
Profeinase
 Antibiotic solution: Antibiotic-antimycotic® solutions are commercially available
(e.g. from GIBCO-BRL). These solutions contain

Penicillin-G sodium  10,000 IU/ml

In sterile saline Streptomycin sulfate  10,000 µg/ml

Amphotericin-B (Fungizone®)  25 µg/ml

Dose: 1 ml. of this solution to 100 ml. of medium
 Buffering system: Incorporation of a buffering system in the medium is
mandatory to aid in maintaining proper pH. Media with HBSS contain a low
concentration of sodium bicarbonate and function effectively in a closed system
(the culture flasks are incubated with caps tightly closed in an ambient air
incubation). Media with EBSS contain a higher concentration of sodium
bicarbonate and function well in an open system (incubated with caps loosely in
place in an incubator with a 5% CO2 environment).
 pH Indicator: All media, both outgrowth and maintenance, contain a pH
indicator, usually phenol red.
 A salmon-pink colour indicates a pH of 6.9-7.2, optimal for cell culture
maintenance.
 Colour changes to bright pink, indicating increased pH, when the buffering
system is disturbed (as when caps are not tightly closed) or when cell are
not metabolizing well.
 Colour change to bright yellow indicates excess acid production and
usually is seen in cultures infected by bacteria or fungi or in cutters that
proliferated excessively and are need of refeeding or subculturing.
 BASIC PROCEDURE OF CELL CULTURE
(1) Equipments and its preparation
(2) Solutions and constituents of media
(3) Culture media
(4) Techniques for culturing cells
(5) Control of contamination
(6) Infection of cell cultures
 Subculturing
 Freezing (Preservation)
 Reactivation (Thawing)
Culture Media:
a) Media containing biological substances
b) Synthetic media e.g. M-199 and Earle’s Minimum essential media (EMEM).
I. Outgrowth media/growth promoting media: Employed to aid the attachment of
cells to glass and to initiate growth. Basic media enriched with 10% serum serve
as outgrowth media for freshly subcultured or newly established cell cultures
when rapid cell proliferation is desired. The amino acid L-glutamate is also added
in a concentration of 3% outgrowth media to stimulate cell growth.
II. Maintenance Media: In this case, the serum content is reduced to 2% and used
for cultures that have reached confluency and are to be maintained in a steady
state.
Techniques for culturing cells: Cells are used in two main forms-

 as sheet about one cell thick  Monolayer

 as suspension of either tissue fragments or dispersed cells.
Tissue and organs from which cell cultures are to be derived should ideally be taken
from fetuses or from gnotobiotic animals to minimize the risk of contamination with
latent viruses.
 PRACTICAL
CHICKEN EMBRYO FIBROBLAST CELL CULTURE

Materials Required:
(1) Embryonating chicken eggs 8-10 days
(2) HBSS
(3) Medium-199
(4) Petridishes
(5) Scalpels
(6) Scissors
(7) Pipettes
(8) Forceps
(9) Conical flask
(10) Magnetic stirrer
(11) Containers for cells

(12) Incubator (separate)  absence of CO2 incubator, Hepes buffer is used instead
of bicarbonate buffer
(13) 0.25% trypsin in the BSS
(14) Sterilizer (Autoclave/oven)
(15) Refrigerator (separate)
(16) Freezer (-200C)
(17) Inverted microscope
(18) Soaking and water bath
(19) Pipette, cylinder and washer

Disinfection with sodium hypochlorite-24 hrs.

Potassium dichromate/sulphuric acid-2 hrs.

Repeated washing in tap water
 
Rinsing in distilled H2O – thrice (Single distilled)

Drying (oven)

Sterilization in pipette container
(20) Deionized and distillation apparatus (Deionized double distilled water)
(21) Bench top centrifuge (Refrigerated)
(22) Liquid nitrogen freezer for continuous cell line
(23) Laminar air flow/hood/clean cabinet
(24) Haemocytometer
(25) Vacuum pump
(26) pH meter
(27) Electronic balance
(28) Automatic micropipette – 20-200 µl

(29) Calf serum  specially fetal calf serum

(30) Tryptose phosphate broth
(31) Autifungal agents (Fungizone, Mycostain)

(32) Antibiotic  Penicillin + Streptomycin or Gentamycin

PREPARATION OF 10% FCS CONTAINING MEDIA:

* 4.47 gm Mix MEM (500 ml) + L-glutamate (5 ml)

MEM/500 ml + antibiotic (1+ 1 cryovial)

Adding 50 ml serum and well mixing
(When 10 ml serum is used, it is
maintenance media (2%)

Adjustment of pH (7.0-7.2) by adding
Sodi-bi-carb up to light red colour
PREPARATION OF CHICKEN EMBRYO FIBROBLAST CELL:
8-10 days embryonated egg

Soaking with Tr. of iodine

Shell rupture

Membrane removed

Embryo on a petridish containing PBS

Separate of head, extremites and intestine from the body

Washing with PBS

Body is cut into small pieces

Taking in a conical flask

Adding to Trypsin 0.3 ml (Warm trypsinzation 0.25%)

Stirring on hot plate stirrer with magnetic bar for 10-15 minutes

Sieving by delipidized gauze in funnel

Neutralization of trypsin by MEM

Centrifugation 3-5 minutes to remove MEM and trypsin (1000-1200 rpm)

Discard the supernatant

Washing the cell with PBS for 3 times to remove
RBC and trypsin  clear cells are found

METHOD OF CELL CULTURE:

Taking of 0.3 ml cell in each cell culture tube

Adding of MEM in culture tube around 3 ml

Observation of the cell under microscope

Placing in the incubator

After 24 hour observe in the inverted microscope
Viability test: Viability test of cells can be made at this stage by trypan blue stain
0.25% Trypan blue stock.

0.9 ml Trypan blue stock

+ WBC counting chamber Note: For 1 ml cell
suspension, 1% 1 drop
0.1 ml cell
trypan blue is enough

* Dead cell appear blue

* Usually 2 × 106 cell/ml

Hot trypsinization  200-370C G Trypsin Gi activities ‡ewk ZvB 10-15 minutes wKsev
Max. 30 minutes n‡jB P‡j| (Short term trypsinization)

Cold trypsinization  40-00C G Kiv nq| G‡¶‡Î overnight ivL‡Z nq| (Long term)

PREPARATION OF INOCULUM:

 Swab samples (150 µl) and Antibiotic (75 µl) in test tube

Seated by parafilm and remain 20-30 min. at room temperature

Keep at 40C until use

 Take 10 × PBS (100 µl) and 10 × PBS (300 µl) in a series of test tube

Inoculate feces of pigeons to make 10-20% suspension (eye estimation)

Seated the test tube with parafilm and centrifuge at 2500 rpm for 5 minutes

Collection of supernatant (200 µl) in another test tube

Add antibiotic (100 µl) and PBS (50 µl) seated with parafilm

INFECTION OF CELL BY VIRUS:

 10% MEM (growth promoting) and 2% (maintenance) MEM is made.

 Cell culture flask (test tube) is taken from incubator


Slight burning of cap by flame and remove fluid from the cell culture flask (2.5 ml)

Add 0.2 ml inoculums and remain
40-60 minutes in room temperature
 
Remove all fluid from cell culture flask by syringe

Slight burning of the cap by flame

Add 2% MEM of 2-2.5 ml in each flask

Again slight burning of the cap by flame

SUB CULTURE:
Removal of media

Washing with PBS

0.25% trypsin (break intercellular junction) + 0.02% EDTA
(detach the cell from wall of bottle)

0
Incubation at 37 C/room temperature for 5-10 minutes

Shaking bottle for detachment

Reduction of trypsin activity by using 10% fetal calf serum

Centrifugation

Wash with PBS

Tabbing of cell suspension

Counting

2 passage 8 × 106 cell/ml
nd

PRESERVATION:
1. Check the culture for the followings:
a) healthy
b) freedom from contamination
c) Specific characteristics
2. Grow the culture up to the late log phase.
3. Dilute the preservatives in growth medium to make freezing medium
 a) Add dimethyl sulphoxide (DMSO) 5-10% or
b) Add glycerol (10-15%)
4. Resuspend the cells freezing medium at approximate 1 × 106 - 1 × 107 cells/ml
5. Dispense the cell suspensions into cryovials/ampoules.
6. Place the cryovial/ampoules on canes for the canister storage.
7. Lay the ampoules on cotton wool in a polystyrene foam box with a wall thickness
of 15 mm.
8. The box is placed at -200C for overnight, then at -700C for overnight and finally
transfer them to a liquid nitrogen (N2) freezer.

10% DMSO ---- 10 ml

5-10% FCS ---- 10 ml

2 ml + 2 ml cell suspension

Over Over
O0C -200C -800C
Night Night
Over Night

-1960C
THAWING:
(1) Preparation of medium.
(2) Retrieve the ampoule from the freezer, and place it in 10 cm water bath at
370C in a bucket.
(3) When the ampoule is thawed, then soak the ampoule thoroughly with 70%
alcohol and open it.
(4) Transfer the contents of the ampoule to a culture flask.
(5) Add medium slowly to the cell suspension.
(6) The cells in the ampoule may be stained with naphthalene black or trypan
blue to determine their viability.