Keratinocyte Products Selection Guide

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Keratinocyte Products Selection Guide
Cascade Biologics offers a wide array of products
for keratinocyte culture. This year, we are
adding a new set of keratinocyte products that
are free of any animal-derived components such as
bovine pituitary extract (BPE), serum, or any
other components that are typically purified from
animal sources
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A complete line of products for the Animal
Product-Free culture of Normal Human Epidermal
Keratinocytes! We refer to these new products as
being APF (Animal Product-Free) and use this
logo to identify them.
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Frequently Asked Questions What is the
difference between "Cell line," "Cell strain,"
and "Cell type?" What does "normal" mean in the
designation "normal human cell type? What is the
difference between a "population doubling" and a
"passage number?" How do I determine the
concentration of cells in a suspension? How do I
establish a culture from cryopreserved cells? Do
I need to spin the cells out of the
cryopreservation medium to plate them? Can I
expand your cells and re-freeze them? If so, how?
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. 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 Cascade Biologics, 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. Q How do
I determine the concentration of cells in a
suspension? A Please refer to the following
link for a detailed protocol for counting cells
using a hemacytometer (PDF) 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 Sigma Chemical
Companys cat. T8154).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 Cascade Biologics).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 Cascade Biologics 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
• Q Can I expand your cells and re-freeze them?
  If so, how?
• A When either cryopreserved or proliferating
  cultures are purchased from Cascade Biologics,
  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-Freze, a defined, protein-free
  cryopreservation medium available from Cascade.
• Please note Due to differences in
  cryopreservation equipment and individual
  techniques, Cascade Biologics 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. 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.
  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.
• 2. 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. Pellet the cells by centrifugation.
  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.
• 3. Distribute the cell suspension in an
  appropriate number of cryopreservation vials.4.
  Cool the vials of cells to 4 C as quickly as
  possible.
• 5. 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. If using a cell
  cryopreservation container Prepare the container
  according to the manufacturers instructions.
• 6. 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 -80C.
• 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
  Cascade Biologics, Inc. Mr Frosty is a trademark
  of Nalge Nunc International.