Primary Cell Culture Support—Troubleshooting

Primary cells are not immortalized and undergo a limited number of population doublings. Each cell type has a different maximum population doubling number that is found on the certificate of analysis for the assigned lot number. For information on “Population Doubling” vs. “Passage Number” refer to the “Getting Started”  page.

Be sure to use the Coating Matrix Kit if you are using the Animal Origin–Free (AOF) supplementation for your culture. There are no attachment factors in the AOF supplements, and Coating Matrix Kit is required for cells to adhere properly.

Yes, we strongly recommended that you contact our Molecular Biology scientists at techsupport@lifetech.com to help you choose the best transfection reagent option, as primary cells tend to be very sensitive.

It is important to store the cells, once received, in the vapor phase of liquid nitrogen. The vials are not leak proof, and submerging them into the liquid phase can allow liquid nitrogen to leak into the vial and affect the viability of the cells.

These are senescent cells, and this is normal for adult keratinocyte culture. You always have a population of cells that are old and no longer proliferate. However younger cells, which are small in size should keep proliferating. When a culture gets older, you see more and more large cells, and the culture will eventually stop growing. With the right care an attention, the culture should yield at least 25 population doublings. 

It is very important to do a viability count prior to plating and to follow the recommended seeding density. The flask number mentioned in some of the protocols is a guideline so that you are aware of how many vessels should be anticipated from each vial. Cell counts on the vial are the minimum guaranteed, but there are usually more supplied to make sure that you receive the amount of cells that we promise. That being said, it is important to count the cells to make sure you have the correct seeding density and so that you can monitor the number of population doublings that your cells go through. 

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

Please see the following causes and recommendations:

There are two possible causes for this issue:

• It is more likely that the matrix coated on the 96-well plate dried because the time interval between removal of coating solution and addition of cells was too long. Once the coating matrix is dry, the cells likely lose the attachment ability. We recommend shortening the interval between removal of coating solution and addition of cells, and work with only a few wells at a time.
• It takes more time to dispense cells in a 96-well plate so that the remaining cells in the tube could form clumps. We recommend that you resuspend the cells well before dispensing them.

These cells are very fragile. We recommend that you follow the procedure in the manual and use the correct medium. Fast thawing is the key for healthy culture. There are several critical points to consider:

• Pre-rinse all materials with medium before use. Do not use PBS, DPBS, or HBSS to rinse because they do not contain proteins.
• Thaw cells quickly and do not expose cells to air. 
• Transfer cells to a pre-rinsed tube first, then add slowly add medium in a drop-wise manner. Do not add the full amount of medium to the cells at once because this may lead to decreased cell viability due to osmotic shock.
• Use pre-warmed complete growth medium and correct seeding density. 
• Matrix coating is required for some cell cultures.
• For primary neuron cells, do not centrifuge the cells as they are extremely fragile upon recovery from cryopreservation.

There are several possibilities causing the failure of neural induction:

• High quality of hPSC cells is critical to the success of neural induction. Remove differentiated and partially differentiated hPSC cells before neural induction.
• Before plating hPSCs for induction, cell counting is recommended because too low or too high cell confluency will reduce induction efficiency. The recommended plating density for induction is 2–2.5 x 10E4cells/cm².
• Cell clumps but not single cell suspension should be plated for induction.
• To increase induction efficiency, overnight treatment with 10 µM ROCK Inhibitor Y27632 at the time hPSC splitting can be used to prevent extensive cell death.

• Check if the correct B-27™ Supplement was used to culture the cells.
• Check expiration date of B-27™ Supplement to see if it has expired.
• Check if the B-27™ supplemented medium used was fresh. The supplemented medium is stable for only 2 weeks at 4°C. 
• Check if the B-27™ Supplement was exposed to excessive heat. Thawed B-27™ Supplement should not be exposed to RT or higher for more than 30 mins. 
• Check if the B-27™ supplement was thawed and refrozen multiple times. Thawed B-27™ Supplement at 4°C should be used within 1 week.
• Check appearance of the B-27™ Supplement. It should be a transparent yellow liquid. Green color appearance indicates that the supplement is not good.