This document provides information on various techniques for disaggregating animal tissue into single cells for cell culture, including mechanical, enzymatic, warm trypsinization, cold trypsinization, and using collagenase. It also discusses subculturing cells, cell counting using a hemocytometer, viability assays with trypan blue staining, maintaining aseptic technique to prevent contamination, and cryopreserving cells in liquid nitrogen with DMSO. The key steps and principles of each technique are explained.

1. By:
Dr. Fahad Khan
Assistant Professor
Department of Biotechnology
Noida Institute of Engineering & Technology, Greater
Noida
Basic of Animal Cell Culture
PART-III

3. *Process of disaggregation in which mechanical/physical forces
are applied for the dissociation of tissue into individual cells.
*This technique basically involves careful chopping or slicing of
tissue into pieces and collection of spill out cells.
*It can be achieved by:
*Pressing the tissue pieces through a series of sieves with a gradual
reduction in mesh size.
*Forcing the tissue fragment through a syringe and needle.
* Less expensive, quick and simple method.
* This process effects cell viability.

4. *Process of disaggregation of tissue with the help of enzymes.
*Enzymatic disaggregation is mostly used when high recovery
of cells is required from a tissue.
*Mostly used for embryonic tissues or tissues with fibrous
connective tissue and extracellular matrix.
*There are two important enzymes used in tissue
disaggregation- collagenase and trypsin.

5. *This method is widely used for disaggregation of cells.
*The chopped tissue is washed with dissection basal salt solution
(DBSS), and then transferred to a flask containing warm trypsin
(37°C).
*The contents are stirred, and at an interval of every thirty minutes,
the supernatant containing the dissociated cells can be collected.
*After removal of trypsin, the cells are dispersed in a suitable
medium and preserved (by keeping the vial on ice)
* Warm Trypsinization

6. *This technique is more appropriately referred to as trypsinization with
cold pre-exposure. The risk of damage to the cells by prolonged exposure
to trypsin at 37°C (in warm trypsinization) can be minimized in this
technique.
*After chopping and washing, the tissue pieces are kept in a vial (on ice)
and soaked with cold trypsin for about 6-24 hours.
*The trypsin is removed and discarded. However, the tissue pieces contain
residual trypsin.
*These tissue pieces in a medium are incubated at 37°C for 20-30 minutes.
The cells get dispersed by repeated pipettings.
*The dissociated cells can be counted, appropriately diluted and then used.
* Cold Trypsinization

7. *Collagen is the most abundant structural protein in higher animals.
*It is mainly present in the extracellular matrix of connective tissue and
muscle.
*The enzyme collagenase (usually a crude one contaminated with non-
specific proteases) can be effectively used for the disaggregation of
several tissues (normal or malignant) that may be sensitive to trypsin.
*The desired tissue suspended in basal salt solution, containing
antibiotics is chopped into pieces.
*These pieces are washed by settling, and then suspended in a complete
medium containing collagenase.
*After incubating for 1-5 days, the tissue pieces are dispersed by
pipetting.
*The clusters of cells are separated by settling. The epithelial cells and
fibroblastic cells can be separated.
* Disaggregation by Collagenase

8. • Subculturing or “passaging cells," is required to periodically
provide fresh nutrients and growing space for continuously
growing cell lines.
• The need to subculture a monolayer is determined by the following
criteria:
1. Density of Culture
2. Exhaustion of Medium
3. Time since last subculture
4. Requirement for other procedures

10. *Cell counts are necessary in order to establish or monitor growth rates as well as
to set up new cultures with known cell numbers.
*Hemocytometers are commonly used to estimate cell number and determine cell
viability.
*It is a fairly thick glass slide with two counting chambers, one on each side. Each
counting chamber has a mirrored surface with a 3 × 3 mm grid of 9 counting
squares.
*The chambers have raised sides that will hold a coverslip exactly 0.1 mm above
the chamber floor.
*Take average of cells in four quadrants and multiply it by 104 cells/ml
Hemocytometer

11. • Viability assays measure the number of viable cells in a
population.
• When combined with the total number of cells, the number
of viable cells provides an accurate indication of the health of
the cell culture.
• Trypan blue and erythrosin B stains are actively excluded by
viable cells but are taken up and retained by dead cells, which
lack an intact membrane.

12. Principle:
• The Trypan Blue dye exclusion test is used to determine the number of
viable cells present in a cell suspension.
• It is based on the principle that live cells possess intact cell membranes that
exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas
dead cells do not.
• When a cell suspension is simply mixed with the dye and then visually
examined to determine whether cells take up or exclude dye.
• A viable cell will have a clear cytoplasm whereas a nonviable cell will have
a blue cytoplasm.

13. Preparing the hemocytometer
1. If using a glass hemocytometer and coverslip, clean with alcohol before use. Moisten the
coverslip with water and affix to the hemocytometer. The presence of Newton’s refraction rings
under the coverslip indicates proper adhesion.
2. If using a disposable hemocytometer (eg INCYTO DHC-N01), simply remove from the packet
before use.
Preparing cell suspension
1. Gently swirl the flask to ensure the cells are evenly distributed.
2. Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a 5 mL
sterile pipette and place in an Eppendorf tube.
3. Take 100 μL of cells into a new Eppendorf tube and add 400 μL 0.4 % Trypan Blue (final
concentration 0.08%). Mix gently.
PROCEDURE
Counting
1. Using a pipette, take 100 μL of trypan blue-treated cell suspension and apply to the
hemocytometer. If using a glass hemocytometer, very gently fill both chambers underneath
the coverslip, allowing the cell suspension to be drawn out by capillary action. If using a
disposable hemocytometer, pipette the cell suspension into the well of the counting chamber,
allowing capillary action to draw it inside.
2. Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.

14. 3. Using a hand tally counter, count the live, unstained cells (live cells do not take up trypan blue) in
one set of 16 squares (Figure 1). When counting, employ a system whereby cells are only counted
when they are within a square or on the right-hand or bottom boundary line. Following the same
guidelines, dead cells stained with trypan blue can be also be counted for a viability estimate if
required.
4. Move the hemocytometer to the next set of 16 corner squares and carry on counting until all four
sets of 16 corner squares are counted.
Viability
To calculate number of viable cells per mL: – Take the average cell count from each of the sets of
16 corner squares. – Multiply by 10,000 (104). – Multiply by five to correct for the 1:5 dilution
from the trypan blue addition. The final value is the number of viable cells/mL in the original cell
suspension.
Example If the cell counts for each of the 16 squares were 50, 40, 45, 52,
the average cell count would be
– (50 + 40 + 45 + 52) ÷ 4 = 46.75
– 46.75 x 10,000 (104) = 467,500
– 467,500 x 5 = 2,337,500 live cells per mL in original cell suspension
Figure 1. Hemocytometer gridlines
diagram indicating one of the sets of 16
squares that should be used for counting.
CONTD………

15.  Maintaining asepsis is one of the most difficult challenges to work with living
cells.
 Contamination is the presence of a minor and unwanted constituent
(contaminant) in material, physical body, natural environment, at a workplace.
 In biological sciences accidental introduction of foreign material
(contamination) can seriously distort the results of experiments where small
samples are used.
 In cases where the contaminant is a living microorganism, it can often multiply
and take over the experiment, especially cultures, and render them useless.
 Potential routes to contamination including failure in the sterilization
procedures for solutions, glassware and pipettes, turbulence and particulates
(dust and spores) in the air in the room, poorly maintained incubators and
refrigerators, faulty laminar-flow hoods, the importation of contaminated cell
lines or biopsies, and lapses in sterile technique.
 Common Contaminants are Yeast, Fungi, Viruses, Bacteria & mycoplasma.

16. • In general indicators of contamination are turbid culture media,
change in growth rates, abnormally high pH, poor attachment,
multi-nucleated cells, graining cellular appearance, vacuolization,
inclusion bodies and cell lysis.
• Yeast, bacteria & fungi usually shows visible effect on the culture
(changes in medium turbidity or pH).
• Mycoplasma detected by direct DNA staining with intercalating
fluorescent substances e.g. Hoechst 33258
• Mycoplasma also detected by enzyme immunoassay by specific antisera
or monoclonal abs or by PCR amplification of mycoplasmal RNA.
• The best and the oldest way to eliminate contamination is to discard the
infected cell lines directly.

17. • The best method for cryopreserving cultured cells is storing them in liquid
nitrogen in complete medium in the presence of a cryoprotective agent such as
dimethylsulfoxide (DMSO).
• Cryoprotective agents reduce the freezing point of the medium and also allow a
slower cooling rate, greatly reducing the risk of ice crystal formation, which
can damage cells and cause cell death.
• DMSO is known to facilitate the entry of organic molecules into tissues.
Handle reagents containing DMSO using equipment and practices appropriate
for the hazards posed by such materials. Dispose of the reagents in compliance
with local regulations.