William D. Graziadei
GRAZIAWD at SPLAVA.CC.PLATTSBURGH.EDU
Mon Mar 30 23:18:00 EST 1992
About a week ago I reponded to Kathy's message but inadvertantly typed
Genba*m*K instead of Genba*n*K in the address. So, the mail *bounced*.
Hence, I'm reposting and apologize for the delay. Hope this helps.
>I would like to have a good recipe for setting up keratinocyte cultures
> human fetal skin. Any recipes gratefully received.
>HRMBDKC at HKUCC.BITNET
Here is a receipe that we use for mouse keratinocytes. Perhaps you
could modify portions that would meet your needs for human fetal
IVCBB Laboratory Protocol
In addition to elimination of serum, a key modification of the
basal medium involves reduction of the Ca2+ concentration.
Application of these two approaches, i.e., replacement of serum by
hormones and growth factors and reduction in the Ca2+ concentration
has made possible reproducible subculture as well as primary
culture of mouse keratinocytes.
The following demonstrates several techniques:
1. Dissection of mouse epidermis
2. Separation of dermis from epidermis
3. Dissociation of the epidermis
4. Primary culture in a selective medium
5. Subculture of primary cultures
6. Cryopreservation of keratinocytes
7. Terminal Differentiation
Dissection of skin and primary culture.
l. Anesthesize mice with CO2. Wash the newborn mice with 20%
Betadine Solution, then with 70% ethanol and place them in a
sterile plastic dish on top of chopped ice.
2. Amputate all four limbs at midjoint and the tail with
3. Make a longitudinal dorsal incision through the skin from tail
4. Remove the skin with forceps as demonstrated.
5. Stretch the skins on dry plastic dishes.
6. Add trypsin/EDTA to plastic dishes so that when the dishes are
slanted at an angle the solution covers only half of the dish.
7. With two pair of forceps grasp the skin at each side and slide
in onto the surface of the liquid dermis side down so that it
floats. Cover the dishes and leave them for 16 hours at 4oC.
8. Transfer the skins to dry dishes dermis up. With forceps, you
can easily remove the dermis from the epidermis. Discard the
9. Mince the epidermis in a small beaker and add medium
containing 5% fetal bovine serum to inactivate the trypsin.
10. Add a stirring bar and stir the epidermal fragments for 30
minutes at room temperature.
ll. Filter the suspension through nylon gauze (157 mesh) to remove
the stratum corneum and count the cells a hemocytometer. Each
skin should yield about 10^7 cells.
12. Collect the cells by gentle centrifugation (500g, 5 min) and
resuspend them in serum-free medium (MEM-l).
13. Seed the cells at 4 x 10^6 in 60 mm dishes containing 4.0 ml
of medium (MEM-l).
14. Replace the medium after 5 hours to remove unattached cells.
15. Feed the cultures daily for three days. The cultures are
incubated in C02 incubator at 95% relative humidity, at 37oC
and 9.5% C02 in air.
Subculture of keratinocytes.
l. Wash a subconfluent monolayer with HBS to remove unattached
2. Add a solution of trypsin, 0.25% and EDTA, 0.02% in HBS and
incubate for 15 min at 4oC.
3. Add 1/10th volume of deoxyribonuclease, 4 mg/ml to disperse
4. After a minute, add cold medium containing 5% serum to
inactivate the trypsin.
5. Sediment the cells and resuspend them in MEM-l for counting or
6. Seeding density in MEM-l should be around 8 x 10^6 cells per
100 mm dish (10^5/cm2).
Cells to be frozen should be healthy semiconfluent monolayers.
Primary cultures prepared as above are ready in 3-4 days for
The cells are suspended as above, counted and suspended in medium
containing 5% serum and 7.5% dimethylsulfoxide. Sufficient medium
is added to adjust the cell number to 3-4 x 10^6 cells/ml. This
suspension is distributed in 1 ml aliquots in ampoules and frozen
in liquid nitrogen vapor or at -70oC. Cells are stored in liquid
nitrogen and have been found to be at least 80% viable.
This exercise is designed to illustrate why conventional media
containing 1-2mM Ca2+ or serum are inappropriate for the growth of
epidermal keratinocytes and other kinds of cells that have the
ability to keratinize. Terminal differentiation is accompanied by
cell enlargement, a sharp decrease in dividing cells and detachment
of differentiated cells from the dish.
The procedure is simple. Take 3 secondary cultures one to two days
after seeding. Discard the medium (MEM-l). Label the plates A, B
and C. Add the following media:
Plate A: MEM-l + 5% FBS
Plate B: MEM-l + 1.0 mM CaCl2
Plate C: MEM-l as a control
Examine each plate before you start and daily thereafter. Primary
cultures can also be used for this demonstration. Keratinocytes
retain the ability to terminally differentiate even after extensive
multiplication. If you like, you might want to try other media used
in this course.
Newborn mice (1-3 days old of either sex), 5/person. The Balb/c
strain is used in our lab.
STERILE SUPPLIES (PER PERSON)
1. Blunt forceps, 4 inch (4)
2. Sharp pointed scissors, curved or straight, 4.5 in.(2)
3. Nytex filters, 157 mesh, 4x4 in.(2). Gauze (4 layers) can
4. Pyrex funnels, 2.5 in. (2)
5. Beaker, 100 ml + stirring bar, covered with Al foil
6. Freezing ampoules, 1.2 ml
7. Centrifuge tubes, 40 & 15 ml
8. Plastic dishes, 60, 100 mm (10@)
9. Plastic pipets, various sizes
1. 20%, Betadine solution (surgical scrub)
2. 70% ethanol
3. Trypsin, 0.25%/ EDTA, 0.02% in HBS. A 10x conc. is
diluted for use.
4. Deoxyribonuclease, 4mg/ml in HBS
MEDIUM (MEM-l), 5 liters
1. Eagle's MEM + nonessential amino acids- bottles in 200 ml
aliquots (without glutamine).
2. Glutamine, 100x
4. Fetal bovine serum, 20 ml
5. Epidermal growth factor (EGF), 5ug/m1 (1000x)
6. Insulin (INS), 1 mg/ml (200x)
7. Transferrin (TF), 5 mg/ml (1000x)
8. Hydrocortisone (HC), 5 x 10^-4M (1000x)
9. Phosphoethanolamine (PEA), 5 x 10^-2M (1000x)
10. Ethanolamine (EA), 5 x 10^-2M (1000x)
11. Bovine pituitary extract (BPE, used at 150ug protein/ml)
MOUSE KERATINOCYTE MEDIUM, MEM-l
COMPONENT DATE or LOT# AMOUNT
MEM ____________ 200 ml
L-GLUTAMINE ____________ 2.0 ml
EGF, 5ug/ml ____________ 0.2 ml
INS, 1mg/ml ____________ 1.0 ml
TF, 10mg/ml ____________ 0.1 ml
HC, 5 E-4 M ____________ 0.2 ml
PEA, 5 E-2 M ____________ 0.2 ml
EA, 5 E-2 M ____________ 0.2 ml
BPE, whole(l% final) ____________ 2.0 ml
ALternatively one can purchase keratinocyte medium from GIBCO.
Question: What is remedial mitosis?
Signature and answer follows:
/ William D. Graziadei, Ph.D. / In Vitro Cell Biology & Biotechnology/|
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/ TEL (518) 846-7144 / GRAZIAWD at SPLAVA.CC.PLATTSBURGH.EDU / /
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"Remedial mitosis is a math course for cells that can't multiply or divide."
Instructor: Dr. G. Factor; Sections: G1, G0 (Remedial), S, G2, M; Time: Varies
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