Keratinocyte Culture

[William D. Graziadei]

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.

>Kathy, asked....
>
>
>I would like to have a good recipe for setting up keratinocyte cultures
> human fetal skin. Any recipes gratefully received.
>Kathy Cheah
>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
skin.

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


PROCEDURES

              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
     scissors.

3.   Make a longitudinal dorsal incision through the skin from tail
     to snout.


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
     dermis.

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      
     cells.

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      
     clumps.

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
     freezing.

6.   Seeding density  in MEM-l  should be around 8 x 10^6 cells per
     100 mm dish (10^5/cm2).


                         Cryopreservation


Cells to  be frozen  should be  healthy  semiconfluent  monolayers.
Primary cultures  prepared as  above are  ready  in  3-4  days  for
freezing.

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.


                        ADDITIONAL EXERCISE


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.

                        MATERIALS REQUIRED

ANIMALS

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
          be substituted.

     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

     SOLUTIONS

     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

     3.   Antibiotics

     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


DATE:__________PREPARED BY:_____________VOLUME:_____________

OSMOLARITY:______________pH:_________STORAGE:_______________




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


ANTIBIOTICS:

ALternatively one can purchase keratinocyte medium from GIBCO.


   Regards,

      Bill 

   Question: What is remedial mitosis?

   Signature and answer follows:


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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