|
- 积分
- 14
- 威望
- 14
- 包包
- 124
|
Box 2 | ISoLATIoN oF FIBRoBLASTS (IN ADDITIoN To KERATINoCYTES) FRoM0 }5 l2 X* r6 C8 F! i4 I8 {
THE SAME SAMPLE ● tIMInG 2–4 wEEKS6 E. f0 E0 J5 R3 I2 R/ W. }' {
In biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,
' g6 l! w/ g7 l3 X' mwhich may be cultured in addition to keratinocytes.6 C! O; W1 H3 n: ]
procedure
, A: H" J- l0 y1. After overnight digestion with dispase and removal of the epidermis (Steps 1–4), the dermis is placed in DMEM culture medium and
4 g* m/ S0 ]5 W9 S) d+ z5 Y9 lkept at 4 °C until use.: ]. \0 L3 O& c4 d. k8 b0 ?$ ^4 Z
pause poInt Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain* n v8 R3 S+ Y7 P6 c
cells up to several days later.
) a" k8 ]) i3 W* \ i. g2 E2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces.
6 G7 t7 ]3 S8 T7 C# j1 _3. Using tweezers, dip each piece in DMEM culture medium and place directly in tissue culture plates. Place ~10 to 15 pieces in each
! s$ b8 V+ X: g8 d" g' ?" u: [7 i100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample. ~( K/ R8 K: J) v+ c
4. Place 1–2 drops of complete DMEM medium onto each piece of tissue.+ h8 S9 `; {2 s2 G5 q) Y8 A
5. Incubate in a 37 °C, 5% CO
8 X5 {( l+ [3 N& o9 B4 d8 k2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum).
: g2 V' X' \. \+ @* K: m" I% C crItIcal step Do not allow pieces to dry out completely.! K% C+ n9 p# t; x2 Z
6. Gently add 7–8 ml of complete DMEM medium to each 100-mm plate. Y$ L" {% E, p/ z
crItIcal step It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish
! S/ i; i$ b1 |following the two steps above.
T3 R' `/ L7 P% m6 \; h8 v? trouBlesHootInG
0 V; q" M* \3 H9 E& @( P7. Carefully return the plate to the incubator.- U6 L3 m. r7 H( _$ o' n
8. Replace with fresh medium every 3–4 d and remove any tissue pieces that are floating.
- }0 G% N2 e- a1 U& z" M- j0 e9. Within 7–10 d, outgrowths of fibroblast should appear.2 I0 |) s( s3 e+ k# 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
1 l" P2 j& g6 `7 o& x$ Lfor 4–5 min., n/ E, L# b4 l3 Z. [
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; l; J! ^$ d- ?. R/ v1 _
and then immediately add 10 ml of complete DMEM medium.
0 o% E& m- J) p- i8 y- [12. Centrifuge at 200g for 5 min.& U+ `2 L' N: U J' C* n: N: Z% {
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 reaching0 |& T! x9 i' t
100% confluence (typically once every week). |
-
总评分: 威望 + 2
包包 + 10
查看全部评分
|
发表于 2015-7-29 16:56
