Frequently Asked Questions About Primary Cells

What is the difference between “primary cells” and “cell lines”?

    Primary cells are cells extracted directly from living tissue and cultured in vitro. Primary cells have a limited lifespan and are widely used because they retain many of the markers observed in vivo. Primary cells from different species allow you to highlight potential differences between humans and preclinical test species. Using primary cells prior to in vivo studies can help refine dosages and reduce the number of animals required for preclinical toxicology. Human primary cells can be used to determine the accuracy of human data inferred from animal models.

    A cell line is a population of cells that has been genetically modified to confer the potential for indefinite growth. In practice, cell lines can be passaged a very large number of times, although some further genotypic and phenotypic changes may occur at very high passage numbers.

    Generally, cell lines lack many of the markers observed in vivo and exhibit marker profiles that are very different from those of primary cells.

How should cryopreserved cells be handled upon receipt?

    Upon receipt of the package, cryopreserved cells should be immediately transferred from the dry ice shipping container to a liquid nitrogen storage tank to prevent thawing.

If you are unsure and there is no dry ice in the package upon receipt, thaw the cells immediately and use them.

Do not store the cells at -20°C or -80°C, as this will cause irreversible damage to the cells.

How do I establish a culture from cryopreserved cells?

Note: Please refer to the cell product manual for detailed instructions.

1) Remove a vial of cells from the liquid nitrogen, taking care to protect your hands and eyes.

2) Loosen the cap on the vial, rotate it a quarter turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then retighten the cap.

3) Place the frozen vial in a 37°C water bath. Gently hold and rotate the vial until the contents are completely thawed.

4) Quickly remove the vial from the water bath, wipe it with 70% ethanol, and transfer it to a sterile area.

5) Carefully remove the cap, taking care not to touch the internal threads. Gently resuspend the contents.

6) Draw 20 μL from the vial and dilute the cell suspension with 20 μL of pancrezine solution.

7) Use a hemocytometer to determine the number of live cells per milliliter.

8) Dilute the contents of the vial (1 mL) to the concentration recommended in the product instructions.

9) Add 5 mL of cell suspension to each 25 cm² culture flask, or 15 mL of cell suspension to each 75 cm² culture flask. Gently swirl the container to distribute the cells evenly.

10) Return the culture flasks to the incubator (37°C, 5% CO₂/95% air).

11) For best results, do not disturb the culture for at least 24 hours after initiation. On the second day, refresh the medium to remove residual DMSO and unadhered cells.

How often should the culture medium for primary cells be changed?

    After thawing and seeding cryopreserved cells, the first medium change should be performed 24 hours later or overnight to remove residual DMSO and any dead cells. Typically, the medium should be changed every 2–3 days, depending on the confluence of the cells, until the cells are ready for passaging.

How many passages can primary cells undergo?

    We use the term “population doubling” rather than “passage” to describe a cell’s growth potential. A population doubling refers to a twofold increase in the total number of cells in culture. The term “number of passages” refers to the number of times a cell population is removed from the culture vessel and subjected to the passaging process. Since different researchers use varying split ratios during passaging, it is difficult to predict how many passages a specific primary cell line can undergo. Our primary cells are guaranteed to undergo the number of population doublings specified in the cell product table.

Can primary cells be expanded and refrozen?

We do not recommend that our customers refreeze primary cells. The cryopreservation process may alter the cells’ growth performance.

There are visible flocs in the FBS—is this normal?

    The presence of flocs in FBS is due to lipoprotein precipitation—these are normal and harmless to the cells. Customers may briefly centrifuge at 400g and remove the supernatant to eliminate the precipitates, but this is not necessary.

Do I need to centrifuge the cells out of the cryopreservation medium before seeding?

    We do not recommend separating the cells from the cryopreservation medium before seeding. Centrifugation may be harmful to the cells, especially at improperly high speeds. When you seed the cells, the DMSO will be sufficiently diluted in the medium. For example, if you seed an entire cryotube (1 mL) of cells from a T-75 flask with 15 mL of medium, the DMSO concentration will be less than 0.67% (v/v). Therefore, our product instructions include diluting the cells in culture medium at the recommended seeding density and medium volume.

    KMD Bioscience specializes in providing customized primary cell services designed to meet the demand for high-quality primary cells in the field of life sciences research. Our services include, but are not limited to, the isolation of primary cells from various tissues sourced from healthy or disease models, as well as the effective culture, expansion, and rigorous characterization of these cells. Our professional team utilizes advanced technologies and extensive experience to ensure that the primary cells we provide possess high viability and stability, capable of supporting a wide range of research projects, including drug screening, disease model studies, and cell therapy research. In addition, KMD Bioscience offers customized processing services for primary cells, such as immunoselection with specific markers, cell gene editing, and cell culture under special conditions, to meet clients’ specific research needs. We are committed to ensuring that, through our customized primary cell services, clients gain access to cell models that most closely resemble physiological conditions, thereby providing robust support for scientific research and new drug development and accelerating the process of scientific discovery and clinical application.