Primary Cell Culture: Frequently Asked Questions (FAQs)
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1. How should I store cryopreserved primary cells?
- Upon receiving the package, cryopreserved cells should be immediately transferred from the dry ice shipping container to a liquid nitrogen storage tank.
- In the unlikely event that no dry ice is left in the package upon receipt, thaw and use the cell immediately.
- Please note that storing the cells at -80°C can cause irreversible damage to them.
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2. I've heard that properly thawing cryopreserved cells is an essential step in the culturing process. Could you give me some pointers?
- Thaw the cryovial of cells in a 37°C water bath. It usually takes about 1-2 minutes.
- Take the cryovial out of the water bath when there are still some small ice crystals remaining in the vial.
- Prolonged exposure to heat will damage the cells, causing them not to plate.
- Do not spin down the cells to remove DMSO after thawing because the centrifugation process can cause even more cell damage than DMSO. Once the cells are plated in the recommended amount of media, the DMSO should be sufficiently diluted to prevent any immediate harm to the cells.
- Do not thaw the cells on the bench top at room temperature.
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3. How often should I change the media?
After thawing and plating the cryopreserved cells, the first medium change should be done after 24 hours or overnight, so that both residual DMSO and any dead cells are removed. Thereafter, the medium should be changed every 48 hours until the cells are ready to be passaged.
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4. How many passages can I obtain with Cell Applications' primary cells?
Cell Applications uses the term "population doubling" instead of "passage" for describing the growth potential of the cells. A population doubling is a two-fold increase in the total number of cells in culture. A passage is the propagation of a cell population by subculturing from one vessel to 3 or 4 vessels. Because different researchers use different split ratios when they subculture, it is difficult to predict how many passages can be obtained with a particular primary cell. Most of Cell Applications primary cells are guaranteed for at least 15 population doublings (unless indicated otherwise in the product literature) when Cell Applications' cell-specific growth media are used for the culturing procedures.
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5. How do I calculate the number of flasks or plates I should set up for culturing the cells?
The following equations can be used to determine the maximum number of culture vessels that can be set up:
The effective growth areas for common tissue culture ware are listed below:
| Culture Flasks |
T-25 |
T-75 |
T-150 |
T-250 |
| Growth Area |
25 cm2 |
75 cm2 |
150 cm2 |
250 cm2 |
|
| Culture Dishes |
35 mm |
60 mm |
100 mm |
150 mm |
| Growth Area |
9.6 cm2 |
20.4 cm2 |
57 cm2 |
143 cm2 |
|
| Multiwell Plates |
6 well |
12 well |
24 well |
96 well |
| Growth Area |
9.6 cm2 |
3.5 cm2 |
1.9 cm2 |
0.33 cm2 |
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6. Do I need to add any supplements into Cell Applications' cell-specific growth media?
No, our growth media are fully supplemented for your convenience. Just open the bottle and use. All the required supplements are pre-added in the media, including: growth factors, antibiotics, and fetal bovine serum.
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7. Can I obtain basal media separate from the growth supplements?
Yes. We also offer growth media in the format of growth medium kits, which consist of basal media and growth supplements packaged separately. These kits are provided with instructions for preparing growth medium and storing any remaining basal medium at 4 °C in the dark and growth supplements at -20 °C for long term storage. Growth supplements are stable for only 2 days at 4 °C upon receipt.
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8. Can I request for some specific component removed from the growth media?
Yes, we routinely provide growth media without antibiotics, without FBS, or without certain growth factors per your request to facilitate your research needs.
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9. What is the shelf life of the cell-specific growth media?
Each lot of cell-specific growth media is made fresh and supplemented with everything needed for cell growth. It is stable for 6 months from the date of receipt when stored at 4°C in the dark. In order to protect the efficiency of growth media, please follow the instructions below:
- Do not expose to light due to light-labile vitamins in the media.
- Do not freeze the growth media.
- There is no need to warm up the growth media to 37 °C prior to use.
We suggest taking the growth media out of the refrigerator and warming it at room temperature in the dark (in the cabinet) for a couple of hours prior to use.
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10. Can I perform transfections with Cell Applications primary cells?
Yes, Cell Applications' Cytofect™ Transfection Kits deliver DNA to primary endothelial cells, primary epithelial cells, primary fibroblasts, and primary keratinocytes, as well as cell lines. Researchers have found that this cost-effective, uniquely formulated transfection reagent yields superior effiiency and lower toxicity when compared to transfection reagents from other manufacturers. For your convenience, Cytofect™ Transfection Kits include Cytofect™ tranfection reagents, enhancers, transfection media, and growth media.
For more information about these transfection kits, click on the appropriate Cytofect™ link in the table below.
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11: Are there any special substrates or coating materials that I need to use for culturing Cell Applications' primary cells?
Most cells we offer can be plated on standard tissue culture grade ware. However, some cell types require coating material. Please see the respective cell product web pages below for information on substrate reagents offered by Cell Applications:
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12: When subculturing primary cells, what are the most important considerations to keep in mind?
- All reagents should be at room temperature. Do Not Warm Any Reagents to 37 °C.
- Do not over-trypsinize. Strictly follow the trypsinization instructions in our protocol.
- Watch cells under the microscope during the entire trypsinization process.
- When the cells become rounded but are still attached, hit the side of the flask against your palm until the cells detach. (When rounded cells detach by themselves without hitting, it means the cells are over-trypsinized.)
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