Animal Cell Culture Basics

Available Cell culture in MGT lab:

• KB cell lines – Oral Squamous Epithelial Cell Lines from National Center for Cell
Science

Entering the Cell Culture Room:

• Wash Your hands with the dettol soap solution

• Make sure that your garments do not touch the floor

• Open the first door and enter and close it properly. Wait for few seconds. Then open the
second door and close it properly.

• Then switch on the light and A/c (210c). Wipe your hands with 70% ethanol.

• Make sure that all the basic requirements such as Masks, parafilm, sterile glassware
media bottles, sterile 250ml beakers, etc are ready.

Requirements:

Glassware:

Glass Media Bottles, Blue Tip box, Yellow tip Box, 250ml Beaker (For waste disposal), Cell
culture flasks [t - flasks (tissue culture flasks)], Petri plates, falcon tubes and Cover slips.

Chemicals:

Pen-strp antibiotics, Gentamycin, Glycine, Glutamine, Sodium Bi Carbonate, Sodium Pyruvate,

Fetal Calf Serum, Trypsin, Phosphate Buffer Saline, Milli - Q water, 70% ethanol, 85% ethanol.
 STERILIZATION

Sterilization of Glassware:

• All the Glass wares should be washed with tap water. Then with distilled water 3 times.

• Keep the glass wares for drying in the hot air oven for O/N.

• Wrap the glass wares with the 4 times folded Aluminum foil. Cover them with news
paper tightly.

• Do the same kind of wrapping for the tip boxes also.

• For cover slips: Disperse the cover slips in Petri plate and wash it with 70% alcohol and
keep them in open under UV for 15 min and sterilize it after wrapping properly.

Sterilization of Chemicals:

Components needed for sterilization:

• Glycine – 200nM

 MW- 75.07g

 75.07g – 1000ml – 1M

 0.15 g – 100 ml – 20mM – take this as your stock I

 Take 1 ml from the stock I in 100 ml MQ.

 Take 10μl from the stock II in 100 ml Medium to get 200nM concentration

• Sodium Pyruvate - 0.1M is to be prepared in MQ and sterilize

 MW – 110.04 g

 110.04g – 1000ml – 1 M

 11 g – 100 ml – 1 M
  1.1 g – 100 ml – 0.1M

• Phosphate Buffer Saline (500ml):

100ml

NaCl 4.25 g 0.85g

Na2HPO4 3.18 g 0.636g

NaH2 PO4 1.10 g 0.22g

Weigh and dissolve the salts in 500ml Milli – Q. This is used to wash the cell lines.

• Distilled water

• MQ

Sterile plasticware available:

Cell culture t- flasks, Petri plates, falcon tubes

Sterile solutions available:

• Pen – strep, Gentamycin are already available in liquid form.

• Trypsin – EDTA:

From the stock of 10X (2ml), dilute to working concentration of 2X using sterile MQ
(10ml). This is used for detaching the cell lines from the surface of cell culture flask.

• Fetal calf serum.

• Sodium Bicarbonate.

• Gibbco’s MEM.

Components needed for filter sterilization:

Glutamine (200mM) has to be prepared in Sterile Milli – Q.

 • 0.6 g in 20 ml and filter Sterilize.

Note:

Gibbco’s MEM does not have the above chemical components which are needed for the KB cell
lines. So these components should be added to the 82ml of MEM medium for 100ml preparation
to reconstitute the medium. Prepare 100 ml stocks for each time.

Gibbco’s MEM (100ml):

Pen – strep 1ml

Gentamycin 200μl

Glycine (200nano Molar) 1ml

Glutamine (200milli molar) 1.5ml

Sodium Bicarbanate 2.92ml

Sodium Pyruvate (0.1M) 1ml

FBS 10ml

Reconstitution of the media:

• Take the MEM. Using the media bottle measurement, add 100ml of the media and take
18ml of the media and discard it.

• Add the above component in the same order.

Sterilizing condition for glassware and chemicals:

• Three whistles 45 minutes sim at 1200C.

Ethanol preparation:

• Prepare ethanol in Sterile Distilled Water.

*70% for surface cleaning the LAF and 85% for cleaning the CO2 incubator.
Surface Sterilization of LAF:

• Switch ON the Air flow wipe the surface of pipettman and tip boxes and floor of the LAF
with 70% ethanol.

• Switch ON the UV light, few seconds later switch off the air flow and keep the UV on for
15 min.

• Follow the same procedure when the UV light is off. Put the exhaust after switching off
the UV light and before starting the work in LAF.

INITIAL PASSAGE

KB Cell line

Human; Larynx

Growth Medium:

MEM (Eagle) with 2mM L- Glutamine and Earle’s BSS adjusted to contain 1.5 g/L Na
bicarbonate, 0.1 mM non essential amino acids, and 1.0 mM Na pyruvate ; FCS – 10%.

Growth conditions:

Temperature - 37ºC, CO2 – 5% atmosphere.

Initial passage:

o Remove medium, rinse with PBS, rinse with ~2.5 ml of Trypsin – EDTA solution and
allow cells to detach. Add same medium and aspirate and dispense into new flasks.

o Split ratio of 1:2 is recommended.

o Medium change: Twice a week.

Maintenance of Cell lines

Initial Passage

KB cell lines were obtained from NCCS, Pune in sterile conditions at an approximate confluency
of 80% in a cell culture T-25 flask filled with medium.

1. The medium from the T-25 flask was aseptically transferred to a falcon tube and kept
aside for later use.
2. The monolayer of cells was washed with 1 ml of PBS twice.
3. 1 ml of Trypsin-EDTA was added to the flask and swirled for 3- 5 minutes until the
monolayer is detached.
4. 2.5 ml of MEM reconstituted with 10% FCS was added to the flask to deactivate trypsin.
The cells were pipetted vigorously in order to disaggregate cell clumps.
5. 1 ml of the cell suspension was put into 2 new flasks.
6. To this 2 ml of old medium (MEM kept aside earlier) and 1 ml of new medium were
added to the flasks to make up the total volume to 4 ml per flask.
7. The flasks are then closed and stored at 37°C in the incubator at 5% CO2

The old medium was added for the first 2 passages or sub culturing with a gradual decline in
content. This was done in order to acclimatize the cells from the old medium to the new medium.

Passaging – Sub culturing:

• Whenever you are using LAF follow the above procedure strictly.

• Whenever you are taking your hands you have to wash with alcohol and keep your hand
inside the LAF.

• Whenever you are taking a medium containing bottle and other utensils all should be
surface sterilized and kept in to LAF.
Requirements:

• Reconstituted MEM medium.

• PBS

• Trypsin

• 250 ml beaker

• Parafilm

• t – flasks, Blue tips

• 100% ethanol to make 70% and 85% ethanol.

Procedure:

• Take the culture medium, PBS, Trypsin from the fridge and keep them outside to bring
them to RT.

• Wipe the LAF with 70% alcohol and switch on UV for 15 min.

• After 15 min, take the culture medium, PBS & Trypsin , surface clean them & keep them
inside the LAF

• Bring the culture from the CO2 incubator

• Do not speak while opening

• Observe the cell confluency under the Phase contrast microscope.

• Wipe the bottom surface with 85% alcohol and keep it in the LAF after surface
sterilization.

• First remove the old medium from the culture flask.

• Wash cell layer with 1 ml PBS for at least 3 times by pouring on the surface of the cell
• Then remove the PBS.

• Add 1 ml of Trypsin by keeping the culture flask vertically

• Then gently shake (~ 30 seconds) it by keeping the flask horizontally for Trypsin to touch
the cells

• Leave it for 2- 3 min

• In the mean time keep ready the flask with media and two sterile culture flasks

• *In the trypsinized flasks add double the amount of media to inactivate the Trypsin i.e, ~
2.5ml of medium.

• *Make sure the tip does not touch the culture flasks and media flasks

• *Then mix the medium with the Trypsin by gentle pippetting in order to disperse the cell
microfilm equally throughout the medium.

• Take the new sterile culture flasks

• *Split the old cell culture in equal amount to the new culture flask (from one old culture
during passaging we can subculture in two culture flasks)

• *Add 3 ml of fresh medium in to each of the flasks (culture flasks having the capacity of
4 ml of culture medium)

• *All these steps should be done by keeping the culture flasks in vertical position.

• Then gently keep them horizontally and spread evenly till the border and do not allow the
medium to reach perforation (a white whole which helps in air passaging for the cells).

• Again wipe the bottom surface wipe and keep them in CO2 incubator.

• Next day observe the adherence of cells under the microscope. If needed take the old
culture medium and add fresh medium next day itself. The adherence of the cells can be
seen within 12 h.

• 1 ml of inoculum will give cell confluency in 3 days. Check it frequently after 3 days.
Culturing in Coverslips:

Take sterile coverslips and add 50μl - 100 μl of inoculum + make up the total volume to 500 μl
by adding fresh medium.

Culturing in Petriplates:

Take sterile Petriplates and add 100μl - 500 μl of inoculum + make up the total volume to 2ml by
adding to the fresh medium.

Fumigation of the Lab:

• Do the fumigation before you establish the cell culture

• Do not do it in the presence of any cell culture

• Take 5 petriplates and load it with appropriate amount of cotton

• Douse it fully with *formic acid (100%)

• Keep in the corner of the culture room

• For a single room 2 petriplates are needed.

• The other one is to keep in side the hood.

• Then leave it O/N

• Open the room for 3 hrs in order to eliminate the fumes from the room

*Be cautious! The formic acid is corrosive and an irritant to your eyes and skins

For cleaning and fumigating of CO2 incubator:

• Use 100% alcohol to wipe the racks.

• For fumigation use 100% alcohol in the same way as mentioned for culture room
fumigation.
• Prepare 0.37 g of CuSO4 in 100 ml of sterile MQ and fill the tray and keep it in the last
rack of the incubator.

• Check once in a week the pressure gauge of CO2 cylinder whether the inlet and outlet
showing 5 and 0.5 pressure respectively . If it drops a little from the prescribed one, order
for the new cylinder.