XpertTM Animal Tissue Culture Teaching Kit
Product Code: CCK005
Contents
1.
2.
3.
4.
5.
6.
About the kit
Kit contents and storage instructions
Materials required but not provided in the kit
Aseptic techniques and good cell culture practices
Instructions for use
Protocols
6.1 Preparation of complete medium
6.2 Thawing of cryopreserved cells
6.3 Sub-culturing of the cells
6.4 Estimation of viability and enumeration of the cells
Two vials of CHO cells have been provided so that
one vial can be used a back up vial in case of failure
to revive or propagate the cells in the other vial.
CHO cells if cryopreserved at -180°C have an
indefinite shelf life. Shelf life of all other reagents is
minimum one year from the date of manufacture.
b.
Leibovitz’s Medium: : Leibovitz’s Medium is a
growth medium specifically designed to grow cells in
a CO2 free atmosphere. The standard sodium
bicarbonate/CO2 buffering system is replaced by a
combination of free base amino acids, phosphate
buffers and higher levels of galactose and sodium
pyruvate so that the medium does not require
supplementation with sodium bicarbonate and can be
used under conditions of free gaseous exchange with
atmosphere.
c.
Fetal Bovine Serum: Fetal Bovine Serum (FBS)
serves as a source of proteins, vitamins,
carbohydrates, lipids, hormones, growth factors,
minerals and trace elements. FBS contains growth
factors which promote cell proliferation and has an
antitrypsin activity which helps in cell attachment.
d.
Antibiotic Antimycotic Solution:
Antibiotic
Antimycotic solution helps to prevent microbial
contamination. This solution is a mixture of
Penicillin, Streptomycin and Amphotericin B in 0.9%
normal saline. It is effective against Gram positive
bacteria, Gram negative bacteria, Fungi and Yeast.
e.
Trypsin – EDTA Solution: Trypsin – EDTA
Solution is a cell dissociation solution containing
Trypsin and EDTA in Dulbecco’s Phosphate Buffered
1. About the kit
XpertTM Animal Tissue Culture Teaching Kit has been
developed for teaching basic animal tissue culture
techniques in educational organizations, where the
sophisticated facilities required for culturing of animal
cells may not be available.
CCK005, XpertTM Animal Tissue Culture Teaching Kit is
sufficient to perform 20 subcultures post thaw.
Long term cryopreservation of cells without loss in
viability requires storage in liquid nitrogen atmosphere at
-180oC. Hence to use the kit most effectively, it is
recommended to thaw the cells and start the procedure
immediately after receipt.
In our experience, CHO cells lost viability when stored in
the upper chamber (freezing compartment) of a frost-free
refrigerator.
a.
*Chinese Hamster Ovary (CHO) Cells: CHO cells
are fibroblast cells which are derived from the ovary
of the Chinese hamster. They grow as a monolayer
and can be cultured in a CO2 independent atmosphere.
*Cells supplied with this kit are strictly for educational activity in teaching institutions and should not be used for any other
commercial purpose.
Saline. Trypsin is a serine protease commonly used
for dissociation and disaggregation of adherent cells.
Ethylenediaminetetraacetic acid (EDTA), a chelating
agent is added to enhance enzymatic activity of
trypsin solution. EDTA acts by chelating calcium and
magnesium ions that enhance cell to cell as well as
cell to flask adhesion.
g.
Trypan Blue Solution: It is a vital stain. It selectively
stains the dead tissues or cells which after staining
appear blue in colour. Live cells or tissues with intact
cell membrane are selective for the compounds that
pass through the membrane as a result of which trypan
blue is excluded by viable cells and they remain
colourless.
2.
f.
Dulbecco’s Phosphate Buffered Saline (DPBS):
DPBS is a balanced mixture of synthetic salts that
maintains pH and osmotic pressure in the medium and
provides adequate concentration of essential organic
ions to the cells. It is used to wash the monolayer of
cells as it is free of calcium and magnesium. It does not
hinder the trypsin activity.
h.
Tissue Culture Flasks: Tissue Culture Flasks
provided in this kit have a vented cap, surface area of
25cm2 and a working volume of 5ml. These are
surface treated flasks which are specifically used to
culture adherent cells.
7.
8.
On receipt, remove the contents of the kit and place them in9.appropriate storage locations as per recommended
storage temperature.
10.
2. Kit contents and storage
Contents
Description
12.
13.
Cryopreserved Chinese Hamster Ovary
TCL095
Cells
14.
15.
Leibovitz's L-15 Medium
AL011A
16.
With L-Glutamine
Fetal Bovine Serum
17.
RM10432
18.
Antibiotic Antimycotic Solution 100X
19.
w/10,000U Penicillin, 10mg Streptomycin
A002
and 25µg Amphotericin in 0.9% normal
20.
saline
21.
Trypsin-EDTA Solution 1X
TCL007
0.25% Trypsin and 0.02% EDTA in22.
Dulbecco's Phosphate Buffered Saline
23.
Dulbecco's Phosphate Buffered Saline 1X
TL1006
Without calcium and magnesium 24.
Trypan Blue 0.5% solution in Dulbecco's
25.
TCL005
Phosphate Buffered Saline
26.
Tissue culture flask
TCG4
25cm2, Surface treated, vented cap 27.
28.
29.
∗
For long term storage
30.
•
Quantities supplied in excess to compensate operational losses.
31.
Code
Quantity
Store at
2 vials
Each vial contains
5X106 cells/ml
-20°C / ∗
170°C
90ml
2 - 8oC
10ml
-20oC
1ml
-20°C
•
-20°C
•
15-30°C
10ml
•
15-30°C
10Nos
15-30°C
15ml
25ml
3. Materials required but not provided in the kit
3.1 Equipments
•
•
•
•
•
•
Laminar air flow hood
Incubator at 37°C
Inverted microscope with 10X objective
Hemocytometer with cover slip
Centrifuge
Water bath at 37°C
3.2 Consumables
•
•
•
•
•
•
•
•
•
•
Micropipette
Tips (1000µl, 200µl) and tip boxes
Serological pipettes
15ml centrifuge tubes
1ml eppendorf tubes
Pipette aid (LA692)
Disposable gloves
Lab coat
Isopropanol spray
Tissue paper
4. Aseptic techniques and good cell culture
practices
a.
b.
Use Personal Protective Equipment (PPE), (laboratory
coat, gloves and eye protection) at all times while
working in a cell culture lab. Use head caps to cover
hair.
PPE for tissue culture facility should be kept separate
from PPE worn in general laboratory environment.
Before starting tissue culture work switch on the UV
light in the cabinet for 15-20 minutes.
d. Keep all the work surfaces free from clutter.
e.
All reagent and media bottles should be labeled
correctly with name and date of preparation and should
be kept at recommended storage temperatures.
f. Clean the working area of the laminar air flow hood
with 70% isopropanol.
g. Prior to starting work all reagent and media bottles,
pipettes, tip boxes should be sprayed with 70%
isopropanol.
h.
Arrange the work station in such a way that you have
an easy access to all the items and a wide clear space in
the centre of the bench.
i.
Keep all the reagents and media bottles to the left hand
side of work station and the consumables and discard
beaker to the right hand side of work station for
efficient working.
j. While working do not contaminate the gloves by
touching anything outside the cabinet (especially face
and hair). In case they become contaminated then,
respray with 70% isopropanol before proceeding.
k. In case of any spillage while working, mop up
immediately and swab the area with 70% isopropanol.
l. Avoid rapid movement within and immediately outside
the cabinet. Slow movement will allow the air within
the cabinet to circulate properly.
m. Avoid speaking, sneezing and coughing while working
in the cabinet to prevent the contamination.
n. Pipette tips, waste reagents and waste medium should
be discarded carefully into a separate discard beaker.
o. Once the work is finished, clear the working area and
clean with 70% isopropanol.
c.
5. Instructions for use
Prepare complete medium as per protocol no. 6.1
Thaw the frozen cells as per protocol no. 6.2
Seed the cells in T-25 flask as
described in protocol no. 6.2, step (f)
After 2hrs
Check for cell attachment
Cells attached
Cells not attached
Incubate the cells for another two hours
Give medium change as described in protocol no. 6.2, step (j)
Incubate at 37oC
Flask will be 80 – 90% confluent
Sub-culture the cells as per protocol no 6.3
Estimate the viability & enumerate the cells as per protocol no 6.4
Seed the cells in T-25 flask for further maintenance as per protocol no 6.3.2
Flask will be 80 – 90% confluent
6. Protocols
Read the entire procedure carefully before starting
the experiment.
6.1 Preparation of complete medium
Thaw Antibiotic Antimycotic Solution (A002) and
Fetal Bovine Serum (RM1112) by keeping the
bottles at room temperature for 30 minutes or in a
37°C water bath.
Note: Temperatures higher than 37°C can result in
deterioration of reagents.
b) Once Antibiotic Antimycotic Solution and FBS are
thawed completely, remove the bottle of
Leibovitz’s Medium (AL011A) from the
refrigerator.
c) Disinfect all bottles with 70% isopropanol on the
outside with a quick spray or wipe with 70%
isopropanol before placing them in laminar air flow
hood.
d) Add 10ml of RM1112 and 1ml of A002 to 90ml of
Leibovitz’s medium (AL011A) using all
precautions. Swirl the bottle to ensure uniform
mixing.
e) The complete medium is now ready for use.
Note: Complete medium should be stored at 2-8oC.
It should be used within one month from the day of
preparation.
a.
b.
c.
d)
e)
a)
f)
g)
h)
i)
j)
6.2 Thawing (Revival) of cryopreserved cells
Requirement:
• Frozen vial of cryopreserved cells
• Complete growth medium
• Tissue culture flask (T-25)
• Pipettes
• Laminar air flow hood
• Water bath at 37oC, Incubator at 37oC
• 70 % isopropanol
Procedure:
a) Do not remove the vial of cryopreserved cells
from -20oC until the culture flask is ready as
below.
b) Warm the complete medium by keeping the bottles
at room temperature for 30 minutes or in a 37°C
water bath.
Note: Temperatures higher than 37°C can result
in deterioration of the medium.
c) Label one T-25 flask and keep it ready in laminar
air flow hood. Label should contain the following
information.
k)
l)
m)
Name of the cell line
Passage number
Date
Aseptically add 5ml of pre-warmed complete
medium to the flask.
Place the frozen vial of cells in a water bath at
37oC. Hold the frozen vial of cells in the water
bath with lower half immersed in water. Keep
shaking the vial until the frozen clump inside
it thaws completely.
Note: Avoid getting the water up to the cap of
the ampoule to decrease the chance of
contamination.
Swab the vial thoroughly with 70%
isopropanol and open it in a laminar hood.
Immediately transfer the contents of the
ampoule to T-25 flask containing complete
growth medium.
Rock the flask gently to ensure proper mixing
of cell suspension and the medium and
incubate at 37ºC.
Note: Flask should be always placed
horizontally in the incubator. Do not incubate
the flask in upright position.
Two hours after incubation, check for the
attachment of cells by observing the flask
under an inverted microscope.
Place the flask upright in the laminar air flow
hood and carefully aspirate entire medium
from the flask using a pipette. This is done to
remove the cryoprotectant DMSO. DMSO at
frozen temperature protects the cells by
preventing formation of ice crystals.
However, at room temperature, it is toxic to
the cells.
Add 5ml of complete medium to the flask and
further incubate it at 37ºC till it becomes 80 –
90% confluent.
Change the medium after every 48 hours.
After every 24 hours check for the attachment
and confluence of the cells by observing the
flask under an inverted microscope.
Precautions:
•
Do not use incubator or the palm of your hand
to thaw the cell cultures since the rate of
thawing achieved is too slow resulting in loss
of viability. Use water bath as prescribed in
the procedure.
•
Thawed cells should be immediately
transferred to growth medium.
6.3 Sub-culturing off the cells
The process of suub-culturing is
i divided intto two
stagees:
6.3.11 Dissociationn of cells from
m culture vesseel.
6.3.22 Splitting (diiluting) the diissociated cells into
apprropriate ratio and
a seeding inn fresh medium
m.
6.3.1 Disssociation of cells
c
from cullture vessel
Requirement:
• Dulbecco’s Phosphate Buffered Saline
(TL1006)
• Trypsin- EDT
TA solution (T
TCL007)
• Complete groowth medium
• Pipettes
• Laminar air flow
fl hood, Inccubator at 37oC
• 70 % isopropanol
Procedure:
a) Exam
mine the cuulture flask under
u
an innverted
micrroscope careffully for conffluency as well
w
as
signss of contamiination or cuulture deterioration.
Splitt the flask if itt is 80-90% coonfluent.
Notee: It is impoortant to exam
mine your cuultures
dailyy and always prior
p
to sub-cculture. Alwayys split
the cells
c
before they reach 100%
% confluency..
b) Place the flask upright and remove the spent
medium from the T-25 flask byy aspiration.
c) Add 1ml Dulbeccco’s Phosphaate Buffered Saline
(TL11006) to the flask. (This step is requiired to
remoove the tracess of serum and
a divalent cations
c
like calcium and magnesium which
w
hinder action
of tryypsin.)
d) Rinsse the cell sheeet by rockingg the flask for 1 to 2
minuutes so that the whole monolayer
m
is briefly
b
washhed with PBS.
e) Disccard the wash solution by asspiration.
f) Add 500µl of Trrypsin-EDTA solution (TC
CL007)
to thhe flask.
Notee: The volumee should be sufficient
s
enouugh to
comppletely cover the
t monolayerr of the cells.
g) Rockk the flask to ensure thhat the dissocciation
soluttion covers the cell sheet.
h) In adddition to roccking gently, flasks of celll lines
that are characteriistically difficcult to removee from
subsstratum may be tapped to exxpedite removval.
i) If neecessary, incuubate the flaskk at 37°C for 1 to 2
minuutes. Monitorr the processs by observinng the
When
flaskk under inverted
i
m
microscope.
dissoociation is complete, cells
c
will be
b in
suspension and apppear roundedd.
Note: The exact tim
me needed to
o dissociate cells
c
will vary
v
accordiing to the cell line. The
dissociiation processs should be monitored
m
cloosely
to avoid cell damagee.
j) Once thhe cell dissocciation is com
mplete, add 5m
ml of
compleete medium too the flask to inhibit
i
the tryyptic
activityy which may ddamage the ceells.
k) Dispersse the cells innto a single ceell suspensionn by
slow, repeated pipettting.
l) Estimaate the viabillity and enum
merate the ceells.
(Refer Protocol no. 66.4)
Fig a. CHO cell
monoolayer before
tryypsinization
Fig b.
b CHO cells
after trypsinization
t
utions:
Precau
•
•
Doo not exposee the cells to
o Trypsin-ED
DTA
sollution for lonnger time. Pro
olonged expossure
cann damage the cells.
It is
i very importtant to neutrallize the trypsinn by
addition of serum
m (or complette medium in this
f
casse) prior to seeding the ceells into the flask
forr proper attachhment of cellss to the flask.
i
6.3.2 Splitting the dissociateed cells into
appropriatte ratio and seeding in fressh medium
Requiremeent:
• Cell suuspension withh a known con
ncentration
• Complete growth meedium
• Pipettes
• Laminaar air flow hoood, Incubator at 37oC
• 70 % issopropanol
Procedure::
a) Determ
mine the conceentration of th
he trypsinized cell
suspension as per prootocol no. 6.4
4.2
b) Using the
t following formula, calcculate the amoount
of cell suspension thhat should be added to the new
n
T-25 fllask to get a reequired cell deensity.
Formu
ula:
C1xV11 = C2xV2
Therefo
fore, V1 = C2xxV2
C
C1
Wheere,
C1 = Cell concenttration obtaineed per ml
C2 = Required celll concentratioon per ml
V1 = Volume of cell
c suspensionn to be added
V2 = Total volum
me of seeding
Exam
mple,
C1 = 0.5 x106 cellls per ml
C2 = 0.1 x106 cellls per ml
V2 = 5ml
Thenn, V1 = 0.1 x1106 cells per ml
m x 5ml
0.5 x1106 cells per ml
m
= 1ml
Therrefore, 1ml of
o the cell susspension shouuld be
addeed to the T-255 flask to obtaain the requireed cell
denssity of 0.1 x1006 cells per ml.
c) Add the requiredd amount of cell suspensioon (as
calcuulated in step no. 2) to the T-25
T
flask.
d) Add the completee medium to thhe T-25-Flaskk while
mainntaining the tootal volume of
o the seedingg. (i.e.
as per the example add 4ml off complete medium
m
to maintain
m
the tottal volume of 5ml)
e) Incuubate the flaskk at 37°C annd observe affter 24
hourrs.
f)
Noote: Do not leaave the slide for
f more than 1-2
miinutes, as viabble cells may die and beginn to
takke up the stainn.
Plaace the slide oon the microsscope and usinng a
100X objective, ccount the totaal number of cells
c
(sttained as weell as unstaained cells) and
nuumber of deadd cells (staineed cells) from
m all
thee four WBC cchambers.
Stained (Blue colored)
dead
d
cell
Unstained (collorless)
viable cell
6.4.1 Estim
mation of viab
bility of cells
Calcuulate the perccentage of viiable (unstainned)
cells as
a per the folloowing formula.
Form
mula:
Percentage of viabble
cells
= No. of
o viable cells X 100
Totall no. of cells
6.4 Estim
mation of viab
bility and enu
umeration off
cells
Requirement:
• Trypsinized cell
c suspensionn
• Trypan Blue solution (TCL
L005)
• Hemocytomeeter with coverrslip
• Inverted Micrroscope with 10X
1 objectivee
• Pipettes
• Tips
Proccedure:
a) Mix the dissoociated cell suuspension in the T25 flask thorooughly by pipeetting.
b) Take 50µl of this suuspension intto an
eppendorf tubbe.
CL005)
c) Add 50µl of Trypan Blue solution (TC
in 1: 1 proportion to it andd mix it propeerly by
pipetting.
d) Prepare the hemocytometerr by gently cleeaning
the slide surfa
face with 70%
% isopropanol taking
care not to scrratch the semii silvered surfface.
Place the covver slip over the hemocytoometer
counting cham
mber.
e) Using a pipettte place a droop of cell suspension
at the edgee of the chhamber. Thee cell
suspension when
w
expelled from the tip will
w be
drawn under the
t cover slip by capillary action.
a
Note: To get accurrate percentag
ge viability, each
e
viability assay shouuld be perform
med in triplicaates,
follow
wed by takingg an average of all the thhree
readinngs.
6.4.2 Enum
meration of th
he cells
Calcuulate the conceentration of ceells per ml as per
the following formuula.
Countin
ng chamber
Form
mula:
C = A x D x 104
Where,
C = Concentratiion of cells peer ml
A = Average nuumber of cells
D = Dilution facctor i.e. 2 (1:1 dilution)
1004 = Conversioon factor for counting
chamber
Example:
Precautions:
If total count in four chambers is 100,
Then, A= 100/4 = 25 and D = 2
•
C = 25x2x104
= 50 x 104
= 0.5 x106 cells per ml
•
Trypan blue is toxic and a potential
carcinogen. Avoid direct contact with skin
and eyes.
The following points should be considered
while loading the cell suspension onto a
hemocytometer chamber.
1. Avoid getting air bubbles while loading
the cell suspension.
2. Do not overfill the chamber as this will
cause the sample to run into the other
chamber.
3. Incompletely filled chamber will result in
uneven distribution of cells.
Revision No. 0/2011-07
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