FAQs

FREQUENTLY ASKED QUESTIONS

What is the difference between primary cells and cell line?

What is the difference between population doubling and passage number?

How should I handle cryopreserved cells upon receiving?

How much experience do I need for starting with normal human cell cultures?

How do I establish a culture from cryopreserved cells?

Do I need to spin the cells to remove DMSO when I first plate cryopreserved cells?

How often should I change the medium?

Can I expand and refreeze ScienCell’s normal human cells?

Can I freeze ScienCell’s growth media for long-term storage?

How much volume of medium should I add to the flasks?

Do I need additional medium supplements to work with your specialty media?

What is the shelf-life time of supplemented complete growth media?

Can I use my own medium or other commercially available media for culturing ScienCell’s normal human cells?

How many passages can I perform with normal human cells?

What is the recommended split ratio for normal human cells?

Is it possible to subject normal human cells to an experimental starvation?

Is it possible to transfect normal human cells with plasmid DNA?

If your questions have not been answered by this list, please contact our cell culture specialist at 877-602-8549 or techsupport@sciencellonline.com

 

What is the difference between primary cells and cell line?

Cell cultures may be derived from primary explants or dispersed cell suspensions. Because cell proliferation is often found in such cultures, a confluent monolayer or a dense cell suspension is formed. According to the traditional definition, the first harvesting and subculturing of this cell population is called primary cells [Freshney, R.I. (1987). Culture of Animal Cells. A Manual of Basic Technique. (New York, Alan R. Liss, Inc.)]. This type of cell has a finite lifespan, during which cells with the highest growth capacity will predominate, resulting in a DEGREE of genotypic and phenotypic uniformity in the population.

A cell line is a population of cells that grow and replicate continuously and has undergone a genetic transformation, resulting in indefinite growth potential. Cell lines have been maintained in vitro for medical and/or research purposes. In practice, continuous cell lines can be cultured through a very high number of subcultures and further genotypic/phenotypic changes may occur at very high passage numbers.

It should note that the definitions of these terms can vary between research groups. Many researchers do not use the term “cell line” to refer to any cell population unless it has undergone a genetic transformation.

What is the difference between population doubling and passage number?

A population doubling is a two-fold increase in the total number of cells in a culture, and is most commonly referred to during the exponential, or “log”, phase of growth. The term passage number refers to the number of times that a cell population has been removed from the culture vessel and undergone a subculture (passage) process, in order to keep the cells at a sufficiently low density to stimulate further growth.

How should I handle cryopreserved cells upon receiving?

Upon receiving the package, immediately transfer the cryopreserved cells from the dry ice SHIPPING CONTAINER to a liquid nitrogen freezer. The interval between transfer from the SHIPPING CONTAINER to the liquid nitrogen freezer should be as short as possible to prevent warming. Do not store the cells at -80°C as this causes irreversible damage to the cells.

How much experience do I need for starting with normal human cell cultures?

Experience working under sterile conditions with a LAMINAR FLOW HOOD is highly recommended, and it is of advantage to have experience with other cell types. If you are a beginner in cell culture or would like to establish a cell culture lab, we can assist you. Please contact our Cell Culture Specialist.

How do I establish a culture from cryopreserved cells?

  1. Remove a vial of cells from liquid nitrogen freezer, taking care to protect hands and eyes.
  2. Loosen the cap on the vial 1/4 turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
  3. Place the vial in a 37°C waterbath, hold and rotate the vial gently until the contents are completely thawed.
  4. Remove the vial from the waterbath immediately, wipe it dry, and transfer it to a sterile field.
  5. Rinse the vial with 70% ethanol, and then wipe to remove excess.
  6. Remove the cap, being careful not to touch the interior threads with fingers. Using 1 ml eppendorf pipette gently resuspend the contents of the vial.
  7. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture vessels (a T-75 flask for most of cells). A seeding density of 5,000 cells/cm2 for most cell type is recommended.
  8. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen caps if necessary to permit gas exchange.
  9. Return the culture vessels to the incubator (37°C, 5% CO2/95% air).
  10. Change the growth medium 6 -16 hours after plating to remove the residual DMSO and unattached dead cells.

Do I need to spin the cells to remove DMSO when I first plate cryopreserved cells?

It is not recommend to spin cells to remove DMSO residue when you first plate the cryopreserved cells. The centrifugation procedure can be MORE harmful to cells than the DMSO residue, particularly if inappropriately high speeds are used. We experience that cells do not suffer deleterious effects if the DMSO concentration is sufficiently low. If you plate the whole cryovial (one ml) of cells in a T-75 flask with the recommended 15 ml of medium, the DMSO concentration will be less than 0.67% (v/v) and no adverse effect were found. Practically, if you plate cells at 5000 – 10,000 cells/cm2, the concentration of DMSO is even lower.

How often should I change the medium?

The medium should be changed every 2 – 3 days depends on how vigorously the cells grow. Most cell culture laboratories routinely feed cells every Monday, Wednesday, and Friday.
Note: After thawing and plating of cryopreserved cells, the first medium change should be done within 6 – 16 hours to remove the DMSO residual and dead cells.

Can I expand and refreeze ScienCell’s normal human cells?

It depends on which cell type. Some cell types such as neurons, microglia and some slow growing epithelial cells are not recommended for expansion and refreezing. Other cell types such as fibroblasts, stellate cells, mesangial cells, astrocytes and so on are can be expanded and refrozen. However, one should note that the refreezing process may result in altered growth performance in the cells. If you want to refreeze the cells, please consult our TECH SUPPORT personal and use ScienCell SF-CFM, a defined serum-free freezing medium, for the best result.

Can I freeze ScienCell’s growth media for long-term storage?

It is not recommend to freeze growth media. The freezing of high nutrient medium will lead to irreversible precipitation of medium components.

How much volume of medium should I add to the flasks?

It is recommend to add 5 ml of medium to a T-25 flask, 15 ml of medium to a T-75 flask, and 30 ml of medium to a T-150 flask.

Do I need additional medium supplements to work with your specialty media?

ScienCell’s growth medium is specially designed for each cell type. After adding the associated components (growth supplement, antibiotics, and fetal bovine serum) to the basal medium, you have the complete growth medium. No further addition is required.

What is the shelf-life time of supplemented complete growth media?

After adding all the supplements to the basal medium, the medium becomes a complete growth medium. If the complete growth medium is stored at 4°C and the duration for which the medium is kept at room temperature or 37°C is as short as possible for each use, the shelf-life time for the complete growth medium is 6 weeks.

Can I use my own medium or other commercially available media for culturing ScienCell’s normal human cells?

ScienCell’s normal human cells have been cultured and tested in our complete growth medium and have adapted to this formula. Using other media for culturing ScienCell’s normal human cells may yield unsatisfactory growth results due to the adaptation process. We recommend the use of our special media for culturing the normal human cells.

How many passages can I perform with normal human cells?

ScienCell does not use the term “passage” for describing the growth potential because each customer uses a different split ratio. ScienCell Research Laboratories guarantees 15 population doublings for normal human cells (unless otherwise indicated on the cell specification sheet) when using ScienCell’s cell culture media and reagents for the culturing procedures.

What is the recommended split ratio for normal human cells?

ScienCell does not recommend a split ratio for normal human cells because the yield of cells after trypsinization varies. We recommend counting the cells after trypsinization and seeding the cells at 5,000 to 10,000 cells per cm² (find the recommended seeding density on the corresponding product information sheet).

Is it possible to subject normal human cells to an experimental starvation?

Yes, it is possible to perform an experimental starvation phase with ScienCll’s normal human cells. Cells can be kept for some time in basal medium without supplements, but the cells must be in a good condition. After this period, the experiment should be terminated because prolonged periods lead to cell death. It is advisable to test prior to the experiment how long cells will survive a starvation period; this duration will depend on many factors.

Is it possible to transfect normal human cells with plasmid DNA?

Yes, we have successfully transfect many cell types such as dermal fibroblasts, lung fibroblasts, dermal microvascular endothelial cells and others.

If your questions have not been answered by this list, please contact our cell culture specialist at 877-602-8549 or techsupport@sciencellonline.com

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