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Box 2 | ISoLATIoN oF FIBRoBLASTS (IN ADDITIoN To KERATINoCYTES) FRoM
$ |, m8 N6 N, T" p. o7 D* BTHE SAME SAMPLE ● tIMInG 2–4 wEEKS
' o2 R2 S2 G- eIn biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,
: y7 P- H ^2 F, C# p7 Cwhich may be cultured in addition to keratinocytes.
& s* U, X) F/ D4 A9 Tprocedure, C5 R# u; y% w2 W
1. After overnight digestion with dispase and removal of the epidermis (Steps 1–4), the dermis is placed in DMEM culture medium and
" ]) G+ L* F6 b; _2 G5 x0 J" Gkept at 4 °C until use.
- A: @1 X8 r5 Y) k' }/ b pause poInt Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain
7 i1 F/ w& u/ i# @$ W5 q$ Dcells up to several days later.+ U/ N1 A; C' o# X
2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces. p4 }* |4 l$ `4 |2 q
3. Using tweezers, dip each piece in DMEM culture medium and place directly in tissue culture plates. Place ~10 to 15 pieces in each1 ?4 @1 ~) W; e3 N5 j, }
100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample.
+ }7 f# p* k/ R4 ?9 {# e) ?0 q4. Place 1–2 drops of complete DMEM medium onto each piece of tissue.5 E! t, \: a2 r( \4 v
5. Incubate in a 37 °C, 5% CO
2 y4 X/ {1 T+ c5 z2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum).
* t$ M6 q2 L) r crItIcal step Do not allow pieces to dry out completely.! p- Z2 w$ v$ C8 h; m7 U
6. Gently add 7–8 ml of complete DMEM medium to each 100-mm plate.
, ~. @# K- u8 l- W( p7 f8 I crItIcal step It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish. x1 `; I) X# Z6 @
following the two steps above.
) b" x( C2 h; X- ~9 M, u? trouBlesHootInG! u8 I8 t$ o/ ~2 k7 r/ x
7. Carefully return the plate to the incubator.4 y5 ?, B" ^+ D8 M8 ?. m6 Z" a
8. Replace with fresh medium every 3–4 d and remove any tissue pieces that are floating.
! t- k6 d$ U) b+ l3 M8 c3 g9 i4 R9. Within 7–10 d, outgrowths of fibroblast should appear.
3 v: U3 n) |) i6 r* {4 \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
9 l1 V; {9 W. M2 y0 Qfor 4–5 min.
6 q @: Z2 j& u$ u t( m11. Once fibroblasts have rounded up and some have detached, tap the tissue culture dish on the side to detach the rest of the cells6 u# C+ M C% ^. P& Q# V) N
and then immediately add 10 ml of complete DMEM medium.
. w8 R! h: Q) G/ ]) o5 p# b( Q) D12. Centrifuge at 200g for 5 min.: a* w1 I2 @) T. q
13. 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" w2 Z( U5 ]: s: T7 E! w- }
100% confluence (typically once every week). |
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发表于 2015-7-29 16:56
