Primary Cell Culture FAQs

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.

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.

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. More specific instructions are included in each cell line’s protocol.

4. How many passages can I obtain with primary cells?

For this purpose, the term "population doubling" is used 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. Our primary cells are guaranteed for a specified number of population doublings (as indicated within the product descriptions) when the corresponding cell-specific growth media are used for the culturing procedures.

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:

Total Cell #	=	cells	=	Surface Area (cm2)
Seeding Cell Density		cells/cm2

Surface Area (cm2)	=	Number of Tissue Culture Vessels that can be Seeded
Growth Area of selected TC Ware

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

6. Do I need to add any components into cell-specific growth media?

No, our complete growth media are fully supplemented and ready-to-use for your convenience. Each complete medium contains all the essential components for optimized cell growth.

7. Can I obtain basal media separate from the growth supplements?

Yes, we offer the basal media and growth supplements packaged individually.

8. What are the storage conditions and shelf life of the media and growth supplements?

• The complete, fully supplemented, growth media is stable for 6 months at 4 °C.
• The basal media is stable for 1 year at 4 °C.
• The growth supplements are stable for 4 days at 4 °C or are stable for 1 year at -20 °C.

For optimal cell growth please follow the handling instructions below.

• Do not expose to light due to light-labile vitamins in the media.
• Do not freeze the growth media.
• Do not warm up the growth media to 37 °C prior to use.
• Preferably, take the growth media out of the refrigerator and warm it at room temperature in the dark (in the cabinet) for a couple of hours prior to use.

9. 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. For specific instructions, please see the respective cell product web pages to access the subculturing protocols.

10. 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.)
• Do not expose the cryovial to ambient temperature. Always keep the cryovials in dry ice for transfer.