Box 2 | ISoLATIoN oF FIBRoBLASTS (IN ADDITIoN To KERATINoCYTES) FRoM, a% ]) y- @' Z4 M7 w
THE SAME SAMPLE ● tIMInG 2–4 wEEKS 7 ^; K& G) }* T+ e& X; }In biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,8 k0 k v/ ~9 s/ z
which may be cultured in addition to keratinocytes. ; R8 ?0 Y2 y# v6 g" cprocedure" [: q9 `4 R; ?! ?8 g; N
1. After overnight digestion with dispase and removal of the epidermis (Steps 1–4), the dermis is placed in DMEM culture medium and , L. Z# ~! E$ e: c Fkept at 4 °C until use. . u: ]$ }( x$ m8 B pause poInt Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain - B! p% Q% o0 @7 z' f* c, gcells up to several days later. 5 Q; F, i1 g0 t" E& S/ w2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces. ~+ K/ ^! q6 V6 N
3. Using tweezers, dip each piece in DMEM culture medium and place directly in tissue culture plates. Place ~10 to 15 pieces in each/ J* {8 ? E! P n0 q, C
100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample. % x0 E$ v0 j8 V# W( m! g4. Place 1–2 drops of complete DMEM medium onto each piece of tissue. & d! S+ T+ F2 m" d2 F1 {( m5. Incubate in a 37 °C, 5% CO. b3 W" H% } p
2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum). 9 j* c' |1 I$ `; i$ ?+ M$ W crItIcal step Do not allow pieces to dry out completely. / Q, R0 D4 i# K" X& ^- w6. Gently add 7–8 ml of complete DMEM medium to each 100-mm plate. 7 ^8 ?6 X. E, y) B( g3 e crItIcal step It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish 9 q W5 L6 T0 F+ S1 Pfollowing the two steps above.4 r2 y% _# f2 o" x
? trouBlesHootInG 0 l: z2 `1 v# b$ Z7. Carefully return the plate to the incubator. 5 w! k- @3 {9 b, X( X8. Replace with fresh medium every 3–4 d and remove any tissue pieces that are floating.! b4 K& \, F" I5 s/ Y1 R, D
9. Within 7–10 d, outgrowths of fibroblast should appear.6 J$ x" {5 S3 T* C6 x
10. After 14–21 d, aspirate the medium and wash twice with PBS. Add 4–5 ml of a 0.05% Trypsin/EDTA solution and incubate at 37 °C # ^" Q- E; D; d" N9 S9 kfor 4–5 min. * x" Z3 }. q5 O L" u" z, u11. Once fibroblasts have rounded up and some have detached, tap the tissue culture dish on the side to detach the rest of the cells/ D% x$ W/ C S* f* R% c6 |; M; Q ` W
and then immediately add 10 ml of complete DMEM medium.8 E* B( ~4 F0 F
12. Centrifuge at 200g for 5 min. [! ~5 P/ l/ t$ j! N9 P13. 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! i/ e, I( r6 |0 K3 \4 C
100% confluence (typically once every week).