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Box 2 | ISoLATIoN oF FIBRoBLASTS (IN ADDITIoN To KERATINoCYTES) FRoM$ a u4 ]" u* L p+ b$ p
THE SAME SAMPLE ● tIMInG 2–4 wEEKS
% d" r( f1 n) P. a/ rIn biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,( Y2 {8 w3 s: @/ E
which may be cultured in addition to keratinocytes.6 b2 `! ] y# ?, i B( c1 X
procedure
; [3 o8 O2 Z1 ]4 e+ r5 \1. After overnight digestion with dispase and removal of the epidermis (Steps 1–4), the dermis is placed in DMEM culture medium and' S, A _( Q0 X; w
kept at 4 °C until use.
2 A T* t7 @' i pause poInt Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain
% x9 }% R* f1 l& Y" u, \cells up to several days later., d0 X. B6 w* v; y. N2 ]2 X
2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces.4 S4 f% {# @. 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
2 s6 U1 s: Q: o6 u7 g0 p9 T$ x0 {100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample., h2 H( U1 R6 R* T/ c# Z. D
4. Place 1–2 drops of complete DMEM medium onto each piece of tissue.4 z" N& D, T' c4 ?6 `/ k
5. Incubate in a 37 °C, 5% CO5 {; _4 o5 Q' _! o: L+ L
2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum).0 I' `. d# v) _2 H7 L6 m5 T0 \4 A ?
crItIcal step Do not allow pieces to dry out completely.
/ S+ ?5 \* @# t7 d6. Gently add 7–8 ml of complete DMEM medium to each 100-mm plate.
* P5 N- W* j$ j# y+ V4 t crItIcal step It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish
3 `4 a' E' F# G3 v6 H3 U& Q1 \2 \following the two steps above.
) {9 C u, D4 M) V; x? trouBlesHootInG
- C+ E V' e" U+ F& }% Y7. Carefully return the plate to the incubator.$ j! i/ L; Q2 G7 q
8. Replace with fresh medium every 3–4 d and remove any tissue pieces that are floating.. v3 w% {9 ^ Y9 e4 T t# j
9. Within 7–10 d, outgrowths of fibroblast should appear.
, S @2 o6 a: \' e) ^. Y. [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. D/ m; a# r: \. H" [- }, ]
for 4–5 min.2 i: v* u+ x+ a$ ^3 k
11. Once fibroblasts have rounded up and some have detached, tap the tissue culture dish on the side to detach the rest of the cells
* z! v0 I" ~5 wand then immediately add 10 ml of complete DMEM medium.
0 ^% y/ N7 Y5 a' \. f- _12. Centrifuge at 200g for 5 min.
4 r" [( i2 q$ j; M13. 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
8 ~" {7 E J' a0 I% ^8 N100% confluence (typically once every week). |
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发表于 2015-7-29 16:56
