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Box 2 | ISoLATIoN oF FIBRoBLASTS (IN ADDITIoN To KERATINoCYTES) FRoM
/ b, X4 D5 P: e+ [* j# aTHE SAME SAMPLE ¡ñ tIMInG 2¨C4 wEEKS
8 [, `$ i& S  |6 X; x5 B$ j$ O4 h: eIn biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,6 j; ~" L( j  u$ W
which may be cultured in addition to keratinocytes.
. a. G6 I* |: l2 e- Yprocedure" i- N3 e7 [7 _6 L
1. After overnight digestion with dispase and removal of the epidermis (Steps 1¨C4), the dermis is placed in DMEM culture medium and
4 _- J) S) n8 z0 ]kept at 4 ¡ãC until use.
# H* @" u6 {8 Y; h' R7 O! e- \# k«­ pause poInt Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain
- t5 k1 v- X: `+ Z0 J/ Ncells up to several days later.
& _6 y( u4 G% i2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces.0 t) K" e7 E8 p4 }6 n" p
3. Using tweezers, dip each piece in DMEM culture medium and place directly in tissue culture plates. Place ~10 to 15 pieces in each
9 Q4 O5 a# J3 v100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample.7 g: |! i- C4 f1 z4 P" ~5 G
4. Place 1¨C2 drops of complete DMEM medium onto each piece of tissue.
9 p! z/ B! t0 N, c% f8 l% i. k% }5. Incubate in a 37 ¡ãC, 5% CO
6 r# X* ?1 D! H1 K2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum)." n; \2 [& F' a! l7 l; B
«° crItIcal step Do not allow pieces to dry out completely.3 }7 h& @4 b+ H' y
6. Gently add 7¨C8 ml of complete DMEM medium to each 100-mm plate.' j: T  H& u0 e- e. K
«° crItIcal step It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish# j! A  s" V( s
following the two steps above.
6 Z4 g; T) O* ~? trouBlesHootInG# E$ T/ {8 l5 n8 Z' D* s3 S
7. Carefully return the plate to the incubator.! R9 S3 p8 h# j8 w
8. Replace with fresh medium every 3¨C4 d and remove any tissue pieces that are floating.+ H* p9 m' r! R& y3 d$ ?
9. Within 7¨C10 d, outgrowths of fibroblast should appear.! B+ H0 E6 R2 v* l8 X1 K8 e
10. After 14¨C21 d, aspirate the medium and wash twice with PBS. Add 4¨C5 ml of a 0.05% Trypsin/EDTA solution and incubate at 37 ¡ãC
# W+ \" J# E2 o6 \% N( S( \for 4¨C5 min.
! }$ q# t' t/ k( `1 }  H2 D11. Once fibroblasts have rounded up and some have detached, tap the tissue culture dish on the side to detach the rest of the cells
1 E8 i4 ^" l) g% Y7 kand then immediately add 10 ml of complete DMEM medium.
0 g4 h$ p5 i% B) `12. Centrifuge at 200g for 5 min.
: m: Y4 T9 D) q13. Passage at a ratio of 1:4 in 150-mm tissue culture dishes by changing the medium every 3 d and splitting cells before reaching
4 z. R! J. G( T( i, U; \100% confluence (typically once every week).