Having difficulties with your experiment?

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View the relevant questions below:

General

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. 

Hepatocytes

Please see the following causes and recommendations:

Possible cause

Recommendation

Improper thawing technique

Sub-optimal thawing medium

Rough handling of hepatocytes during counting

Improper counting technique

Cells left out too long

Review thawing, plating, and counting protocols

Thaw cells <2 mins at 37°C

Use Gibco CHRM™ Medium during thawing to remove cryoprotectant

Mix slowly; use wide-bore pipette tips

Ensure a homogenous cell mixture prior to counting

Count cells on 2 of the 4 grid lines

Do not let cells sit in trypan blue mixture for more than 1 min prior to loading

Plate cells immediately after counting

Please see the following causes and recommendations:

Possible cause

Recommendation

Improper thawing technique 

Sub-optimal thawing medium 

Incorrect centrifugation speed 

Rough handling of hepatocytes during counting 

Improper counting technique

Review thawing, plating, and counting protocols 

Thaw cells <2 min at 37°C 

Use CHRM Medium during thawing to remove cryoprotectant 

Check thawing protocol for proper centrifugation speed and time (varies by species; human is 100 x g for 10 min at RT) 

Mix slowly; use wide-bore pipette tips 

Ensure a homogenous cell mixture prior to counting 

Count cells on 2 of the 4 grid lines 

Do not let cells sit in trypan blue mixture for more than 1 min prior to loading

Please see the following causes and recommendations:

Possible cause

Recommendation

Not enough time for cells to attach 

Poor-quality substratum 

Hepatocyte lot not characterized as plateable

Compare your cultures to pictures on the lot-specific characterization specification sheet (human cells) that accompanied the cells

Wait before overlaying with Gibco™ Geltrex™ matrix to see if attachment increases 

Use Gibco™ Collagen I–Coated Plates 

Review thawing, plating, and counting protocols (see above section for additional protocol suggestions) 

Check lot specifications to ensure it is qualified for plating

Please see the following causes and recommendations:

Possible cause

Recommendation

Seeding density too low 

Insufficient dispersion of hepatocytes during plating 

Insufficient plating volume used for well format 

Low attachment efficiency (see above) 

Some animal lots are not >80% confluent

Check lot-specific characterization specification sheet for appropriate seeding density (human cells) 

Observe cells under microscope for appropriate seeding prior to incubation 

Disperse cells evenly by moving plate slowly in a figure-eight and back & forth pattern in incubator 

Refer to literature or technical support for suggested plating volumes

Please see the following causes and recommendations:

Possible cause

Recommendation

Seeding density too high 

Insufficient dispersion of cells during plating 

Improper plating volume used for well format

Check lot-specific characterization specification sheet for appropriate seeding density (human cells) 

Observe cells under microscope for appropriate seeding prior to incubation 

Disperse cells evenly by moving plate slowly in a figure-eight and back & forth pattern in incubator 

Shake plate and wash cell monolayers prior to applying Geltrex™ Matrix overlay 

Refer to literature or technical support for suggested plating volumes

Please see the following causes and recommendations:

Possible cause

Recommendation

Hepatocyte lot not characterized as plateable 

Sub-optimal culture medium 

Cells were cultured for too long

Check lot specifications to ensure it is qualified for plating 

Use Gibco™ William's E Medium with Gibco™ Plating and Incubation Supplement Packs 

Refer to our plating protocol 

In general, plateable cryopreserved hepatocytes should not be cultured for more than five days

Please see the following causes and recommendations:

Possible cause

Recommendation

Hepatocyte lot not transporter-qualified 

Sub-optimal culture medium 

Not enough time for bile canaliculi to form

Check lot specifications to ensure it is transporter-qualified 

Use Gibco™  William's E Medium with Gibco™ Plating and Incubation Supplement Packs 

Refer to our plating protocol 

In general, at least 4–5 days in culture is required for bile canalicular network formation

Please see the following causes and recommendations:

Possible cause

Recommendation

Sub-optimal monolayer confluency (see above) 

Poor monolayer integrity (see above) 

Inappropriate positive control 

Incorrect concentration of positive control

Compare results to those reported on the lot-specific characterization specification sheet (human cells) shipped with the cells

Refer to our enzyme induction protocol 

Check positive control to ensure suitability

Please see the following causes and recommendations:

Possible cause

Recommendation

Toxicity of test compound 

Sub-optimal culture medium 

Hepatocyte lot not characterized as plateable 

Cells were cultured for too long

Compare cell morphology of treated and non-treated cells 

Refer to our plating protocol 

Check lot specifications to ensure it is qualified for plating 

In general, plateable cryopreserved hepatocytes should not be cultured for more than five days

Neural Cells

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 10E4 cells/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.