On this page you can read the answers to the frequently asked questions. Can’t find the answer you are looking for? Please contact us and we will help you!
- Questions about the application of ROKEPIE
- Benefits of ROKEPIE
- Product characteristics
- Questions about regulations
- Questions about results
- Safety questions
- Storage and shelf life
- Testing
- Usage of ROKEPIE
- Video material
Questions about the application of ROKEPIE
Can ROKEPIE® be used to store cells in suspension? Yes, it can. Successful storage at 2-8°C has been achieved in experiments in which adherent cell lines were stored in ROKEPIE® enriched medium immediately after trypsinization. These cells remained detached during storage and could be seeded in 6-well plates straight out of the refrigerator. Please download the protocol: Trypsinized cells transport at 2-8 °C protocol with data
What dilution should I use for my cells? We advise that you test ROKEPIE® on your cell line of choice with at least three concentrations (e.g. 1:20, 1:50, 1:100). We have found that some cell lines require less protection to endure 7 days of storage at 2-8°C than others.
Are cells permanently altered by the use of cold-storage and ROKEPIE®? The effect of ROKEPIE® on epigenetics or differentiation of the cell is unknown. However, ROKEPIE® is not designed to actively alter any of these characteristics. This is the reason we advise users to test cells on their cell line of choice, critical changes in cell behavior can be different for each cell line.
How can the use of ROKEPIE® in my daily process influence a production process? ROKEPIE® could alter your production process in the following ways:
- A change in timing for Quality Control, e.g. delay analysis.
- Increase time windows of crucial steps in the process, e.g. trypsinization, inoculation, allowing you to increase batch sizes.
Has ROKEPIE® been tested on any human primary cells? Yes, we have performed tests on human umbilical vein endothelial cells (HUVEC). These cells were stored in 96-wells plates successfully for at least 3 days.
Does ROKEPIE® or 2 – 8 °C storage affect stem cell differentiation? The results of the differentiation of the stem cells after ROKEPIE® facilitated 2 – 8°C storage is not yet available.
When shipping, will movement of container disrupt sheets of cells? When transporting cells in a cell culture flask, the movement of medium will cause stress to the monolayer of cells. A solution to this is to top off the flask with cell culture medium so no air is present. We are currently looking into using ROKEPIE® in a (thermos) reversible hydrogel that sets at 4°C and becomes liquid at 37°C.
Benefits of ROKEPIE
How can the use of ROKEPIE® in my daily process influence a production process? ROKEPIE® could alter your production process in the following ways:
- A change in timing for Quality Control, e.g. delay analysis.
- Increase time windows of crucial steps in the process, e.g. trypsinization, inoculation, allowing you to increase batch sizes.
Can any media formulation be used with ROKEPIE®? ROKEPIE® can be used with regular media such as DMEM or Ham’s F12. ROKEPIE® has also been tested in specific serum free media with cells adapted to this medium. We have also tested ROKEPIE® on cells that were cultured in serum containing medium but stored in serum-free medium. These cells were protected by ROKEPIE®.
Product characteristics
Does ROKEPIE® contain any animal components? No, ROKEPIE® is not derived from animal sources and does not contain any animal components.
Is ROKEPIE® toxic to my cells if I expose them for too long? According to preliminary cytotoxicity studies based on ISO regulations (10993-5:2009), ROKEPIE® only exhibits cytotoxicity at a concentration 5 times higher than the advised working concentration.
What is ROKEPIE® made of? ROKEPIE® is a fully synthetic well- characterized small molecule in a pyrogen free 0,9% NaCl solution.
Is ROKEPIE® sterile and ready for use in cell culture? Yes. ROKEPIE® is prepared in sterile pyrogen free 0,9% NaCl and filtered over a 0,2 µm filter.
What is the shelf life of ROKEPIE®? The shelf life of the freeze-dried (lyophilized) ROKEPIE is approx. 3 years when stored at 2-8°C and protected from direct light (hence the dark brown vial). The best before date is also printed on the label of the bottle. When the freeze-dried ROKEPIE is dissolved into stock solution the shel life of the solution is approx. 6 months.
How long does a vial of ROKEPIE® last? This depends of course on the frequency of use and the volumes used in each vessel. The following table shows an indication of the number of flasks/wells can be treated with ROKEPIE® supplemented cell culture medium.
The number of cell culture vessels you can treat with a 2 mL vial of ROKEPIE®:
| Dilution of ROKEPIE® used | |||
| 1:10 | 1:20 | 1:50 | |
| No. T-25 flask | 4 (500 µL per flask) | 8 | 20 |
| No. wells on a 6-well plate (9,6 cm2) | 13 (150 µL per well) | 26 | 65 |
| No. wells on a 96-well plate | 192 (10 µL per well) | 384 | 960 |
Can ROKEPIE® be used to store cells in suspension? Yes, it can. Successful storage at 2-8°C has been achieved in experiments in which adherent cell lines were stored in ROKEPIE® enriched medium immediately after trypsinization. These cells remained detached during storage and could be seeded in 6-well plates straight out of the refrigerator. Please download the protocol: Trypsinized cells transport at 2-8 °C protocol with data
Should I add ROKEPIE® to fresh culture medium, or straight to my cells? ROKEPIE® performs best when it is added straight to your cells prior to storage at 2-8°C. This means there is no need to add ROKEPIE® to fresh culture medium and subsequently adding it to the cells prior to storage.
Does ROKEPIE® or growth storage alter the functionality or growth characeristics of my cells? Growth characteristics, as indicated by the growth rate (doubling time) after a period of storage at 2-8°C are unaffected for the cell lines we tested (i.e. HEK293 and 3T3-L1).
Are cells permanently altered by the use of cold-storage and ROKEPIE®? The effect of ROKEPIE® on epigenetics or differentiation of the cell is unknown. However, ROKEPIE® is not designed to actively alter any of these characteristics. This is the reason we advise users to test cells on their cell line of choice, critical changes in cell behavior can be different for each cell line.
Questions about regulations
Will ROKEPIE® be GMP produced in the near future? Plans are made to produce ROKEPIE® under GMP conditions. The implementation of these plans is planned for Q4 2015.
Is ROKEPIE® GMP certified ? No, at this moment the active ingredient of ROKEPIE®-FD01 is not GMP produced and not tested conform medical device as additive component. A part of the GMP production is the Drug Master File(DMF), this DMF is in progress. Toxicology of the compound is performed by cell based assays and we have data of animal study with our compound.
Questions about results
I see cell death after storage, should this happen? Depending on the duration of storage, some cell death can be expected. For a storage duration of 7 days, early passage HEK293 cells stored in ROKEPIE® have to exhibit a viability of >65% in order for the batch of ROKEPIE® to pass our quality checks.
Is the growth rate of my cells in any way affected by cold storage? Growth rates after storage have been determined for HEK293 cells and 3T3-L1 cells. After respectively 3 and 7 days of storage at 2-8°C no difference in growth rate (doubling time) was observed when compared to an equal number of viable cells originating from continuous 37°C cultures.
How can the use of ROKEPIE® in my daily process influence a production process? ROKEPIE® could alter your production process in the following ways:
- A change in timing for Quality Control, e.g. delay analysis.
- Increase time windows of crucial steps in the process, e.g. trypsinization, inoculation, allowing you to increase batch sizes.
Has ROKEPIE® been tested on any human primary cells? Yes, we have performed tests on human umbilical vein endothelial cells (HUVEC). These cells were stored in 96-wells plates successfully for at least 3 days.
Does ROKEPIE® or 2 – 8 °C storage affect stem cell differentiation? The results of the differentiation of the stem cells after ROKEPIE® facilitated 2 – 8°C storage is not yet available.
How should I determine viability of my cells after cold-storage with ROKEPIE®? Use of a classical viability determination, such as the Trypan Blue exclusion method should suffice. However, please not that not only the %viability but also the total cell number is of great importance when comparing ROKEPIE® with the control.
Do you have experience with any type of stem cells? Yes, ROKEPIE® has been tested on human adipose derived stromal cells (hADSC) by Dr. Guido Krenning (University Medical Center Groningen ). ROKEPIE® works just as good as N2 preservation, but the tests for long term storage have not yet been completed.
My control cells are equally viable as ROKEPIE® treated cells. How is this possible? Some cell lines have a high innate tolerance to hypothermia. This can result in survival after several hours to days at 2 – 8°C. ROKEPIE® is designed to extend this maximum storage duration. Please use our suggested test-protocols to determine the maximum storage duration at 2 – 8°C with and without ROKEPIE® for your cell line of choice.
Will ROKEPIE® work with cells in suspension as well as attached cells? Yes, we have experienced this with the tested cell lines and the information we have received from users.
Cells with ROKEPIE® in the video appear to detach in large sheets rather than individual fragments, is that correct? This is a correct observation, large intact pieces of the monolayer detached and re-attached upon placing back at 37°C.
Safety questions
Is ROKEPIE® toxic to my cells if I expose them for too long? According to preliminary cytotoxicity studies based on ISO regulations (10993-5:2009), ROKEPIE® only exhibits cytotoxicity at a concentration 5 times higher than the advised working concentration.
Is ROKEPIE® safe for me to use? ROKEPIE® is a for-research-only product and should be used with all necessary safety precautions associated with these products. Use ROKEPIE® only in sterile environments and wear appropriate protection like gloves, facial protection and protective clothes.
Storage and shelf life
Does it matter in what density I store my cells? Post-storage viability is affected by the density in which you seed and store your cells. Therefore, we advise to store your cells when they are in a (near) confluent state. Somehow storing cells in lower density results in a decrease in viability after storage. ROKEPIE® still offers protection at lower cell densities but hypothermic storage will result in relatively lower viable cell yields.
I see cell death after storage, should this happen? Depending on the duration of storage, some cell death can be expected. For a storage duration of 7 days, early passage HEK293 cells stored in ROKEPIE® have to exhibit a viability of >65% in order for the batch of ROKEPIE® to pass our quality checks.
Is the growth rate of my cells in any way affected by cold storage? Growth rates after storage have been determined for HEK293 cells and 3T3-L1 cells. After respectively 3 and 7 days of storage at 2-8°C no difference in growth rate (doubling time) was observed when compared to an equal number of viable cells originating from continuous 37°C cultures.
Why should I close my caps or seal my plates in airtight bags? Most buffers used in cell culture are based upon a bicarbonate buffer, which requires the presence of CO2 enriched air. This enriched air is present in most humidified incubators, therefore cells can be cultured in plates, in flasks with loosened caps or in flasks with filtered caps. The lack of CO2 enriched air in refrigerators means the bicarbonate buffer will not be able to fully function, resulting in a sharp increase in pH (phenol red will turn purple). Additionaly, the drop in temperature will result in different solubility of O2 and CO2, also affecting the pH. Closing the vessels or storing plates in airtight bags allows for a portion of the CO2 enriched air to remain present in the vicinity of the bicarbonate buffer, resulting in a much more stable pH. The use of a HEPES buffer or CO2 independent medium is also possible. Please download our Datasheet Prevent pH change
What is the optimum storage temperature for cells with ROKEPIE®? ROKEPIE® is purely designed to facilitate storage of cells at 2-8°C. Our product is designed to allow cells to enter a hibernation-like state once they are stored at 2-8°C and it is uncertain if this hypometabolic state also occurs at higher temperatures.
How should I store ROKEPIE®? ROKEPIE® should be protected from direct light (hence the dark brown vial) and stored at 2-8°C. The period you can store the cells at 2-8°C can differ per cell type. We advise users to determine the maximum storage duration for their specific cell line themselves.
How long does it take cells to cool down to 2 – 8 °C? Temperature of cell culture medium reaches a temperature of 2 – 8 °C about 40 minutes after the vessel is placed in a cold storage room (For 96-wells plates, Tt = 36,881 x e-0,05 t).
My control cells are equally viable as ROKEPIE® treated cells. How is this possible? Some cell lines have a high innate tolerance to hypothermia. This can result in survival after several hours to days at 2 – 8°C. ROKEPIE® is designed to extend this maximum storage duration. Please use our suggested test-protocols to determine the maximum storage duration at 2 – 8°C with and without ROKEPIE® for your cell line of choice.
What is the shelf life of ROKEPIE®? The shelf life of the freeze-dried (lyophilized) ROKEPIE is approx. 3 years when stored at 2-8°C and protected from direct light (hence the dark brown vial). The best before date is also printed on the label of the bottle. When the freeze-dried ROKEPIE is dissolved into stock solution the shel life of the solution is approx. 6 months.
Testing
Has ROKEPIE® been tested on any human primary cells? Yes, we have performed tests on human umbilical vein endothelial cells (HUVEC). These cells were stored in 96-wells plates successfully for at least 3 days.
Does ROKEPIE® or 2 – 8 °C storage affect stem cell differentiation? The results of the differentiation of the stem cells after ROKEPIE® facilitated 2 – 8°C storage is not yet available.
Should ROKEPIE® needs be individually tested for each cell line? Yes.
How should I determine viability of my cells after cold-storage with ROKEPIE®? Use of a classical viability determination, such as the Trypan Blue exclusion method should suffice. However, please not that not only the %viability but also the total cell number is of great importance when comparing ROKEPIE® with the control.
Do you have experience with any type of stem cells? Yes, ROKEPIE® has been tested on human adipose derived stromal cells (hADSC) by Dr. Guido Krenning (University Medical Center Groningen ). ROKEPIE® works just as good as N2 preservation, but the tests for long term storage have not yet been completed.
My control cells are equally viable as ROKEPIE® treated cells. How is this possible? Some cell lines have a high innate tolerance to hypothermia. This can result in survival after several hours to days at 2 – 8°C. ROKEPIE® is designed to extend this maximum storage duration. Please use our suggested test-protocols to determine the maximum storage duration at 2 – 8°C with and without ROKEPIE® for your cell line of choice.
What is the low end of the time frame for the storage of cells? Please take a look at our website to check out the different time frames of the different cell types we have tested.
What type of cells should ROKEPIE® not be used with? Up until today we have had only one disappointing result with a certain cell line. However, this information was passed onto us by a user and we are not at liberty to disclose which cell line this is.
What is the cell number to ROKEPIE® volume ratio (#/v)? That is depended on the cell line of your choosing. We advise you to test this under your own circumstances with your specific cell culture medium.
Usage of ROKEPIE
What does “standard cell culture medium” mean? ROKEPIE® is tested in Dubelco’s Modified Eagle Medium containing high glucose (4,5 g/L), serum and antibiotics. This cell culture medium was used in CaCo-2 cells, Skov-3, MEF, MS5, HEK293, 3T3-L1, N2A cells etc. ROKEPIE® also performed well in Ham’s F12K. These media are regarded by us as “standard cell culture medium”.
How is ROKEPIE® removed from the cells at end of storage? It is not necessary to remove ROKEPIE® from your medium before rewarming. But if you do want to remove ROKEPIE®, the product is removed by replacing the medium after storage and washes with PBS are optional. This can be done once cells have regained their morphology after cold storage.
Are there any additives to cell culture medium that can affect storage at 2-8°C? We have tested various concentrations of additives of which a negative effect on viability after storage at 2-8°C was expected (serum and pyruvate). Serum concentrations of 0 – 20% did not alter the viability after storage at 2-8°C. However, addition of pyruvate (>500 µM) to the cell culture medium negatively affected the viability after 7 days of storage. Therefore we do not advise the use of high concentrations of pyruvate in your cell culture medium during storage at 2 – 8°C.
What is the cell number to ROKEPIE® volume ratio (#/v)? That is depended on the cell line of your choosing. We advise you to test this under your own circumstances with your specific cell culture medium.
Why should I close my caps or seal my plates in airtight bags? Most buffers used in cell culture are based upon a bicarbonate buffer, which requires the presence of CO2 enriched air. This enriched air is present in most humidified incubators, therefore cells can be cultured in plates, in flasks with loosened caps or in flasks with filtered caps. The lack of CO2 enriched air in refrigerators means the bicarbonate buffer will not be able to fully function, resulting in a sharp increase in pH (phenol red will turn purple). Additionaly, the drop in temperature will result in different solubility of O2 and CO2, also affecting the pH. Closing the vessels or storing plates in airtight bags allows for a portion of the CO2 enriched air to remain present in the vicinity of the bicarbonate buffer, resulting in a much more stable pH. The use of a HEPES buffer or CO2 independent medium is also possible. Please download our Datasheet Prevent pH change
Can any media formulation be used with ROKEPIE®? ROKEPIE® can be used with regular media such as DMEM or Ham’s F12. ROKEPIE® has also been tested in specific serum free media with cells adapted to this medium. We have also tested ROKEPIE® on cells that were cultured in serum containing medium but stored in serum-free medium. These cells were protected by ROKEPIE®.
Is ROKEPIE® sterile and ready for use in cell culture? Yes. ROKEPIE® is prepared in sterile pyrogen free 0,9% NaCl and filtered over a 0,2 µm filter.
What is the shelf life of ROKEPIE®? The shelf life of the freeze-dried (lyophilized) ROKEPIE is approx. 3 years when stored at 2-8°C and protected from direct light (hence the dark brown vial). The best before date is also printed on the label of the bottle. When the freeze-dried ROKEPIE is dissolved into stock solution the shel life of the solution is approx. 6 months.
How long does a vial of ROKEPIE® last? This depends of course on the frequency of use and the volumes used in each vessel. The following table shows an indication of the number of flasks/wells can be treated with ROKEPIE® supplemented cell culture medium.
The number of cell culture vessels you can treat with a 2 mL vial of ROKEPIE®:
| Dilution of ROKEPIE® used | |||
| 1:10 | 1:20 | 1:50 | |
| No. T-25 flask | 4 (500 µL per flask) | 8 | 20 |
| No. wells on a 6-well plate (9,6 cm2) | 13 (150 µL per well) | 26 | 65 |
| No. wells on a 96-well plate | 192 (10 µL per well) | 384 | 960 |
Should I add ROKEPIE® to fresh culture medium, or straight to my cells? ROKEPIE® performs best when it is added straight to your cells prior to storage at 2-8°C. This means there is no need to add ROKEPIE® to fresh culture medium and subsequently adding it to the cells prior to storage.
Video material
Cells with ROKEPIE® in the video appear to detach in large sheets rather than individual fragments, is that correct? This is a correct observation, large intact pieces of the monolayer detached and re-attached upon placing back at 37°C.
Which video’s of ROKEPIE® are available? You can find all our video’s on the ROKEPIE® product page or on our YouTube channel.