PetakaG3
Cell Culture Common
Quick Protocols

www.petaka.com

life in it!

TMPetakaG3

TMBasics
Maximum volume of media permitted
in Petaka isMedia injection and withdrawal
should always be done holding
Petaka in a VERTICAL POSITION

Maximum
volume20ml(more will alter Petaka’s performance!)

PetakaG3

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Ready to hostPetakas (RTHP)
PREPARING “READY TO
HOST” PETAKAS
Hold the Petaka in a vertical position!
1. Inject 0.5 ml of Antibiotic solution
2. Pump 16 ml of Medium

Total

Keep 3.5 ml
free to add
cells and serum
later16.5ml

3. Keep these Petakas refrigerated at4ºC in a vertical position in stands
or in a plastic bag (zip bag)
Dehydration and pH should becontrolled if RTHP are kept In
refrigerator for months!

PetakaG3

SEEDING CELLS IN “READYTO HOST” PETAKAS
Holding Petaka a in vertical position
1. Disinfect port with 90% Ethanol &a flame
2. Warm the Petakas in an incubator,
at 37ºC for 20 -30 min in aVERTICAL position
3. Inject 1.5ml of serum (*)
4. Inject up to 2ml of cell suspension
5. Whilst holding the Petaka in avertical position, squeeze it until
the level of media reaches the air
exit & then release until the air input
stops. Then the inside/outsidepressure will be balanced
6. Shake gently for 10-15 seconds
7. Place in a stand & allow to incubate
in a HORIZONTAL position

TMCulturing cells in media
supplemented media
SEEDING THE CELLS
Holding Petaka in a vertical position!
1. Inject 1 ml of Serum for 5%
concentration
2. Inject 0.5 ml of Antibiotic solution
3. Inject X ml of Cell suspension
4. Inject (18.5 –X) ml of Medium
Total20ml

5. Whilst holding the Petaka in avertical position, squeeze it until
the level of media reaches the air
exit & then release until the air inputstops. Then the inside/outsidepressure will be balanced.
6. Incubate Petaka according tothe Cell type (see INCUBATIONPOSITIONS)

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TMHarvestinganchored cells
ADHERENT CELLS WITHTRYPSIN-EDTA
1. Disinfect the port with 90% Ethanol& a flame
2. Completely extract all the media
from the Petaka
3.Inject 4 ml of 0.25% Tyrpsine-EDTAsolution
4. Shake thoroughly
5. Incubate at 37ºC for 3 min in ahorizontal position (both sides)
6. Check Cell detachment under themicroscope
7. WHEN CELLS ARE DETACHED!
Tap the Petaka 2-3 times
8. Pellet the cells using a Petaka
centrifuge (see centrifugation)

PetakaG3

9. To pellet the cells in the bottom
right hand corner of Petaka,position it with the port being theupper most corner of the device
10. Introduce Petaka into thecentrifuge rotor pocket
11. Set desired speed & time & begin
centrifuge
12. After centrifuge, the cell pellet will
be visible in the bottom right handcorner
13. To extract the pellet, use a micro
pipette with a 1mm tip
14. Transfer the cells to a conical tube

TMHarvesting freesuspended cells
NON ADHERENT CELLS
1. Disinfect port with 90% Ethanol & aflame
2. Tap the Petaka 2-3 times
3. Pellet the cells using a Petaka
centrifuge (see centrifugation)
5. To pellet the cells in the bottom righthand corner of Petaka, position itwith the port being the upper mostcorner of the device

PetakaG3

6. Introduce Petaka into the centrifuge
rotor pocket
7. Set desired speed & time & begincentrifuge
8. After centrifuge, the cell pellet will
be visible in the bottom right handcorner
9. To extract the pellet, use a micro
pipette with a 1mm tip
10. Transfer the cells to a conical tube

TMKeeping cells in “invitro dormancy”(only adherent cells)

PUTING THE CELLS IN“IN VITRO DORMANCY”

DO NOT CHANGE
THE MEDIUM!

1. After 75% confluent cell growth,
remove the Petakas from the incubator
2. Keep the Petakas at 20-25ºC in a
vertical position, away from direct
sunlight, or Infra-Red sources.
3. Keep in a Polyspan Shield
4. Periodically Check Cell shape underthe microscope

In-dormancy survival time depends on:a) Cell typeb) Culture Mediac) Type of Buffer (in the media)d) Stability of the environmental variables

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TMRecovering cells
from “dormancy”
RECOVERING CELLSFROM “DORMANCY”



1. Disinfect port with 90% Ethanol & aflame
2. Slowly withdraw the medium
3. Inject the same volume of NEWmedium with the same additives
4. Whilst holding the Petaka in a verticalposition, squeeze it until the level o f media reaches the air exit & thenrelease until the air input stops.
Then the inside/outside pressure will
be balanced.
5. Incubate at 37ºC, in a vertical position,
at least for 24 hours before cell
SUBCULTURE

PetakaG3

TMpH adjustment ofgrowing cultures
pH ADJUSTMENT WITH
SODIUM BICARBONATE
1. Disinfect port with 90% Ethanol & aflame
2. Withdraw 1ml of Medium
3. Whilst holding the Petaka in a verticalposition, squeeze it until the levelof media reaches the air exit & thenrelease until the air input stops

PetakaG3

Then the inside/outside pressure will bebalanced
4. Inject 1ml of 7.5% Sodium Bicarbonate
sterile solution
5. Shake gently for 10-15 seconds.
6. Return the Petakas to the incubator

TMMedia change
MEDIA CHANGE
1. Disinfect port with 90% Ethanol & aflame
2. Slowly withdraw all the medium
completely
3. Inject the same volume of NEW
medium with the same additives
4. Whilst holding the Petaka in a vertical
position, squeeze it until the levelof media reaches the air exit & thenrelease until the air input stops.

Then the inside/outside pressure willbe balanced.
5. Incubate at 37ºC, in a vertical position,
at least for 24 hours before cell
SUBCULTURE
6. Return Petaka to the incubator

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TMFreezing cells
First follow the harvesting cells
protocol up to the pellet formationand extraction.
1. Disperse the cell pellet in FREEZING
MEDIUM at a final concentration:
1,000,000 cells per ml of medium
2. Transfer 1.5 ml of cell suspension into
each Cryotube.
3. Proceed freezing the Cryotubes(stepwise) up to -120ºC & store inLIQUID NITROGEN
In order to remove the media whilst
keeping the cells within the Petaka
we need to gather the cells in thebottom left hand corner, therefore forcentrifuging the air filter must be in
the uppermost corner of the Petaka
4. Place the Petaka in the centrifuge, set
speed & time, then begin
5. Remove Petaka from the centrifuge
6. Pellet cells will have gathered in thebottom left hand corner

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7. Whilst holding the Petaka upright withthe pellet cells in the bottom corner,
slowly remove the media, careful notto disturb the pellet cells
8. Repeat centrifuge 2 more times
ensuring the port is disinfected eachtime
Then to collect washed pellet cells,inject new medium, place the Petaka
in the centrifuge rotor pocket with theport being in the upper most corner.
9. Set the centrifuge speed & time &begin
10.Remove the Petaka from thecentrifuge
11. Note the pellet cells in the bottom
corner under the port
12. Remove the pellet cells using amicro pipette
13. Transfer into a conical tube
containing freezing media
14. Transfer this into a cryotube
15. Place in the freezer at least -80ºC orin liquid nitrogen

TMThawing frozen cells
1. PREPARE “READY TO HOST”
PETAKA WITH ONLY 15ml OF MEDIA
2. Disinfect the port with 90% Ethanol& a flame
3. Warm the RTHP in an incubator,
at 37ºC for 10-20 minutes in a
VERTICAL position.
4. Inject 3ml of Serum (*)
5. Remove the cells from the freezer
6. Thaw the cryotube in a disinfectedwater bath at 38ºC for 1-3 minutes.
7. Be sure the ice has melted
8. Extract the cell suspension from thecryotube with a 1ml tip.

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9. Transfer the cell suspension into aPetaka(RTHP)
10. Whilst holding the Petakain avertical position, squeeze it until thelevel of media reaches the air exit &then release until the air input stops.
Then the inside/outside pressure will
be balanced.
11.Shake gently for 10-15 seconds
12. Incubate the Petaka in a horizontal
position according to the Cell type(see INCUBATION POSITIONS)TMIn Situ monitoring
adherent cells viability
TRYPAN BLUE METHOD
1. Disinfect port with 90% Ethanol & aflame
2. Withdraw 1ml of Medium
3. Inject 1ml of Trypan Blue 0.4%
“sterile” solution
4. Shake gently for 5-10 seconds
5. Leave the Petaka in a vertical position
for 10 minutes at room temperature

6. Count proportion of “blue-stained”cells (dead cells) versus unstained perfield (living cells)
7. Disinfect port with 90% Ethanol & aflame
8. Slowly withdraw the medium
9. Inject the same volume of NEWmedium
10. Incubate in the required position

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Shipping cells in Petaka
PREPARING ADHERENTCELLS FOR SHIPMENTRECOVERING CELLS AFTERSHIPMENT

1. Withdraw the Petakas from theincubator when cells become around
75% confluent
2. Protect Petaka with Polyspan Shield
3. Introduce Petaka/s in a mailer
envelope
4. Ensure that during the trip Petaka
will not be exposed to temperatures
below 10ºC or over 39ºC (optimaltemperature 20-22ºC)
5. Optimal traveling time is up to 7-10days.

1. Disinfect port with 90% Ethanol & aflame
2. Slowly withdraw the medium
3. Inject the same volume of NEWmedium
4. Whilst holding the Petaka in a vertical
position, squeeze it until the levelof media reaches the air exit & thenrelease until the air input stops. Thenthe inside/outside pressure will bebalanced.
5. Incubate at 37ºC, in vertical position,
at least for 6-12 hours before cell
SUBCULTURE

Intercontinental delivery times havebeen successfully up to 14 to 21 days;
however, depending on the cell type,final viability at destination could be as
low as 30-50%

PetakaG3

TMTissue cell isolationand primary culture
1. Inside the laminar flow hood or in
the operating room, excise a tissue
sample and place into a 10 cm tissue
culture dish containing approximately
10 ml of Hanks Balanced Salt
Solution (HBSS).
2. Inside the laminar flow hood, remove
any fat and necrotic areas fromtissue sample. Maintain a sterile
environment all times.
3. In a minimal amount of HBSS,
thoroughly mince the tissue sample
into a fine slurry of 1mm3 or smaller
particles
4. Transfer the minced tissue sample
into a 50 ml conical tube and spin
down at 2000 rpm for 3 minutes in arefrigerated centrifuge
5. Fill the 50 ml conical containing thecollagenase digest up to 40 ml withHBSS and Spin at 3000 rpm for 5minutes. Discard the supernatant.

6. Add warm DMEM with 10% FBS
and 1% antibiotic, up to 10 Ml anddisperse the cell pellet.
7. Suck up the mixture into a 10ml
plastic pipette and eject it, repeating
both operations 10X in order torelease free cells.
8. Pass the mixture through a 100μm
filter (cell strainer) into a new 50 ml
conical tube.
9. Count cell concentration and seed
two million cells in 1-5 ml suspension
per Petaka.
10. Add media with 10% FBS and 2%
Penicillin/Streptomycin solution up
to 20 ml per Petaka.
11. Incubate in the adequate position