Q: What is the difference between "Cell line," "Cell strain," and "Cell type?"

A:  When cells are isolated from a tissue to form a primary culture, assuming that the cells proliferate in vitro, a confluent monolayer or a dense cell suspension is formed.  According to the traditional definition, the first harvesting and subculture of this cell population results in the formation of a cell line [Freshney, R.I. (1987). Culture of Animal Cells. A Manual of Basic Technique. (New York, Alan R. Liss, Inc.)].  This type of cell line 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.

Using this nomenclature system, a continuous cell line is a population of cells that has undergone a genetic transformation, resulting in indefinite growth potential.  Continuous cell lines are usually aneuploid. In practice, continuous cell lines can be cultured through a very high number of subcultures, although some further genotypic, and therefore phenotypic, changes may occur at very high passage numbers.  Immortalization can occur spontaneously, or may be virally- or chemically- induced.  It should be remembered that the working definitions of these terms can vary between research groups.  Many researchers do not use the term “cell line” to refer to any population unless it has undergone a genetic transformation. 

A cell strain is a subpopulation of a cell line that has been positively selected from the culture, by cloning or some other method.  A cell strain will often have undergone additional genetic changes since the initiation of the parent line. Individual cell strains may, for example, have become more or less tumorigenic than the established line, or they may be designated as a separate strain following transfection procedures.

The term cell type refers to all cells with a common phenotype, eg keratinocyte, melanocyte.  Therefore keratinocytes isolated from a number of different donors are all the same cell type.

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Q: What does "normal" mean in the designation "normal human cell type?"

A:  "Normal" means that the cells in question were isolated into primary culture from normal healthy tissue, rather than from diseased tissue.  "Normal" also refers to a cell population that constitutes a cell line as opposed to a continuous cell line, according to the traditional definitions given above, since the cells have not been genetically altered and do not have indefinite growth potential.

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Q: What is the difference between a population doubling and a passage number?

A:  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.  At Invitrogen™, the first culture following the isolation of cells from tissue is termed the primary culture.  Following the first subculture, the cells are described as a secondary culture (or passage 1).  After the second subculture, the cells become a tertiary culture (or passage 2), and so on.

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Q: How do I determine the concentration of cells in a suspension?

A: See our detailed protocol for counting cells using a hemacytometer.

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Q: How do I establish a culture from cryopreserved cells?

A:  The procedure given below is a sample protocol for establishing cultures from the contents of one vial.

  1. Prepare a beaker of water at 37° C.

  2. Remove a vial of cells from liquid nitrogen storage, taking care to protect hands and eyes.

  3. 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.

  4. Dip the lower half of the vial into the 37° C water to thaw.

  5. When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood.

  6. Open the vial and pipette the suspension up and down with a 1 ml pipette to disperse the cells.

  7. Remove 20 ml from the vial and dilute the cell suspension in 20 ml of trypan blue solution (for example: Invitrogen™ Cat. # 15250-061).

  8. Use a hemacytometer to determine the number of viable cells per ml.

  9. Dilute the contents of the vial (1 ml) to the concentration recommended by the product instructions (for example 1.25 x 104 viable cells/ml for neonatal human epidermal keratinocytes from GIBCO® Cell Culture).

  10. Add 5 ml of cell suspension to each 25 cm² culture flask or 15 ml of cell suspension to each 75 cm² culture flask.

  11. Following inoculation, swirl the medium in the flasks to distribute the cells.  Many cell types attach to culture surfaces quickly, and if the medium is not distributed immediately following inoculation, the cells may grow in uneven patterns.

  12. Incubate the cultures in a 37° C, 5% CO²/95% air, humidified cell culture incubator.  For best results, do not disturb the culture for at least 24 hours after the culture has been initiated.

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Q: Do I need to spin the cells out of the cryopreservation medium to plate them?

A:  We do not recommend spinning cells out of cryopreservation medium prior to plating.  Centrifugation can be harmful to cells, particularly if inappropriately high speeds are used.  Experience in the cell culture laboratory at Invitrogen™ has shown that cells do not suffer deleterious effects if the DMSO concentration is sufficiently low.  Therefore, our product instructions include a detailed protocol which involves diluting the cells into culture medium such that the final DMSO concentration is less than 0.4% (v/v) at the recommended seeding density and volume of medium.

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Q: Can I expand your cells and re-freeze them? If so, how?

A:  When either cryopreserved or proliferating cultures are purchased from Invitrogen™ GIBCO® Cell Culture, they may be expanded and cryopreserved again.  However, the cryopreservation process may result in altered growth performance of the cells.  The following protocol provides a basic guideline for the cryopreservation of cells using Synth-a-Freeze®, a defined, protein-free cryopreservation medium available from Invitrogen™. 

Please note: Due to differences in cryopreservation equipment and individual techniques, Invitrogen™ cannot guarantee that cells cryopreserved using this protocol will be viable upon recovery from cryopreservation, and does not provide a warranty for cells cryopreserved in an investigator's laboratory.

  1. Thaw Synth-a-Freeze® in a 37° C water bath or overnight at 4° C.

  2. If thawed in a water bath, do not exceed 37° C and do not leave the product at 37° C for an extended period of time.

  3. Synth-a-Freeze® should be equilibrated to 4° C prior to use. For optimal results, the use of a controlled-rate freezer is recommended. In the absence of a controlled-rate freezer, a cell cryopreservation container (such as Mr Frosty®, available from Nalge Nunc International) may be useful.

  4. If enzymatic agents are used to remove the cells from a culture surface, resuspend the cells in a solution that will neutralize the effects of the enzyme.

  5. Pellet the cells by centrifugation.

  6. After removing the supernatant, resuspend the cell pellet in cold Synth-a-Freeze® medium at a concentration of 5 x 105 to 3 x 106 cells/ml.

  7. Distribute the cell suspension in an appropriate number of cryopreservation vials.

  8. Cool the vials of cells to 4° C as quickly as possible.

  9. If using a controlled-rate freezer: Freeze the material by reducing the temperature 1° C per minute until the temperature reaches -40° C.  Then reduce the temperature at a rate of 2° C per minute until the temperature reaches approximately -90° C.

  10. If using a cell cryopreservation container:  Prepare the container according to the manufacturer’s instructions.

For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facility as soon as possible after the cells have reached -80°C.

As a substitute for Synth-a-Freeze®, the recommended basal medium for the cell type being cryopreserved, supplemented with 10% fetal bovine serum (FBS) and 10% DMSO, may be used.  Please note that Synth-a-Freeze® is NOT recommended for the cryopreservation of human epidermal melanocytes.  Synth-a-Freeze® is a trademark of Invitrogen™ Corp. Mr Frosty® is a trademark of Nalge Nunc International.

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Q: How can I calculate the correct rotor speed (RPM) to use for pelleting my cells by centrifugation?

A:  Cells should be pelleted by centrifugation at 180 x g (relative centrifugal force, RCF).  The correct rotor speed can be calculated by measuring the maximum radius of your rotor and entering the information into the table found at this website which can be reached via this link: www.eppendorfna.com/support/online-calculator.asp.  For example, to centrifuge cells at 180 x g (RCF) in a rotor with a radius of 15 cm, you would spin the cells at 1036 rpm.

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