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Express procedure of shipping cells door to door at environment normal temperature in Petaka G3 bioreactors.
Many people are exchanging cell cultures without cryopreservation using the Petaka G3 LOT bioreactor with cells in in-vitro dormancy, first time users of this procedure ask a broad variety of questions that prompted us to write this explanatory post.
Specialists use this cell shipping technology for several reasons:
The method is ideal and simple for attached cell cultures because in Petaka G3 LOT there is no need for the long, risky and expensive procedures such as: cell detachment, cell concentration by centrifugation, cell dispersion in cryopreserving media, progressive freezing and shipping in bulky packages containing pounds of dry ice.
Growing cells are transferred from one incubator to another incubator, miles apart, without any other requirement than an adequate envelope.
In Petaka G3 cells are virtually hermetic, protected without risk of contamination
Inside the bioreactor there is negligible dehydration even after 30 days at normal environmental temperature and humidity.
The almost absence of CO2 in the environmental atmosphere does not influence the pH of the Petaka culture chamber
The bioreactor can be turned and shacked accidentally in any direction and force without cell damage caused by media displacement and shear forces. The structure of the bioreactor quenches turbulences and media displacement as long as it has been properly prepared before shipping
In Petaka cells in in-vitro dormancy last viable at environmental temperature and normal environment CO2 concentration for many days (minimal 10 and maximal up to 30 days, depending on the cell type, media and culture conditions).
The cultures can be shipped directly from the incubator without any complicated protocol.
The shape of the bioreactor is so thin that fits in a simple well protected envelope, and can be mailed following the shipping rules for each country.
Expanding the cells after shipment has no risk, is direct and easy.
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The first thing to do before shipping the cell culture in Petaka G3 LOT, is to grow healthy cells well attached to the bioreactor surface.
The culture will be ready to be shipped when:
· The cell monolayer is approximately 75% confluent at least in one of either surfaces of Petaka G3 LOT
· The media pH is neutral or slightly acid (yellowish phenol red indicator)
· Petaka G3 LOT is full of media (24ml)
After receiving the cells in Petaka check under the microscope for accidental cell detachment from the bioreactor walls and in such case make sure that the culture was properly shipped.
It is prudent to live the culture rest for 2 hours or even overnight in horizontal position with the main cell culture surface below, so any detached cell will re-attach to the bioreactor wall.
After the resting period the shipping media should be replaced by new fresh media. See video
Now the culture is ready for expansion.
Occasionally, if the journey has been rough, the cell monolayer may have many detached cells and debris on top of the well attached and extended cells, this should not be a problem, after the first media change most will disappear.
Once the cells are comfortable attached and growing they can be harvested and distributed in new devices of any type (T flasks, multi-well dishes, Petri dishes or any kind of bioreactor).
WARNING: Do not send IN SUSPENSION anchorage dependent cells!
Cultures of suspended cells such as lymphocytic cells suspended CHO or others, can also be shipped in Petaka following the same procedure with some protocol differences.
Here we include two simple protocols to follow
(A) SHIPPING CELL MONOLAYER CULTURES IN PETAKA G3
STEP A (BEFORE SHIPPING)
1. Be sure that the cells were properly prepared for shipment (ATTACHED)
2. Be sure that the cells were 75% confluent immediately before shipping
3. Be sure that the pH of the media was neutral or slightly acidic (orange or yellow the media Phenol read indicator)
4. Avoid, if possible, traveling times longer than 15 days
5. Be sure that the shipment didn’t suffer disastrous accidents
6. At arrival check under the microscope the cell conditions
7. Live the Petaka resting in horizontal position for at least 2 hours or even overnight
8. Check under the microscope the cell conditions
9. Withdraw the shipping media completely
10. Check under the microscope the cell conditions
11. Inject carefully 24 ml of new media with additives
12. Incubate in HORIZONTAL POSITION until the cells become confluent
13. (If both sides are cultured flip the petaka every 12 hours)
14. Detach the cells with 3 ml of the detaching solution and incubate following the specific protocol
15. Check cell detachment under the microscope
16. Inject in Petaka G3 7 ml of new media with the appropriate additives according with the new cell culture protocol
17. Softly shake the Petaka in order to disperse evenly the cells in the media
18. With a Pipette and a 200 microL tip get a sample of the cell suspension and estimate the cell concentration and cell viability
19. Withdraw all media with cells and transfer them dosing the suspension to new cell culture devices, Petaka G3 or any other device
20. Add new media up to the normal level according with the cell culture device and protocol.
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SHIPPING SUSPENDED CELL CULTURES IN PETAKA G3
STEP A (BEFORE SHIPPING)
1. Use preferable Petaka G3 FLAT
2. Be sure that the cells are properly prepared for shipment (24 ml of total media)
3. Be sure that the cells are viable immediately before shipping
4. Be sure that the pH of the media is neutral or slightly acidic (orange or yellowish media Phenol read indicator)
5. Avoid if possible traveling times longer than 15 days
6. Be sure that the shipment didn’t suffer disastrous accidents
7. At arrival check under the microscope the cell conditions (pick through the port 200 microL sample and check cell concentration and viability)
8. Softly shake the Petaka in order to disperse evenly the cells in the media
9. With a Pipette-pipettor withdraw and transfer X ml of media with cells to new Petakas G3 (preferable FLAT) or other device
10. Add to the ne Petakas (or other devices) new media up to 24 ml (or the normal level according with the cell culture device and protocol).
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