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Box 2 | ISoLATIoN oF FIBRoBLASTS (IN ADDITIoN To KERATINoCYTES) FRoM
$ F' Z9 ?8 U0 x3 s1 @5 X3 XTHE SAME SAMPLE ● tIMInG 2–4 wEEKS
( j+ p$ g: X8 N) x8 z6 ?) PIn biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,9 ]: I U+ d7 \. r0 f! \
which may be cultured in addition to keratinocytes.
0 [( X0 M* k2 C: N/ pprocedure# \! ^8 Z5 _" u, X+ o
1. After overnight digestion with dispase and removal of the epidermis (Steps 1–4), the dermis is placed in DMEM culture medium and5 m- i3 U! S W& d2 D3 ~9 \7 f( O
kept at 4 °C until use.9 b* ]. L! P( u |
pause poInt Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain- p% B# f+ u( c, g3 H
cells up to several days later.
+ H' B8 Z7 j& f$ p3 k3 }' Q" D2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces.; C, M, Q* p$ E
3. Using tweezers, dip each piece in DMEM culture medium and place directly in tissue culture plates. Place ~10 to 15 pieces in each- z* j* E3 s. w' u6 ]; |8 }
100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample.
9 f9 c" N% J* W2 o! k, J4. Place 1–2 drops of complete DMEM medium onto each piece of tissue.
: o" ^+ w0 f! t* E5. Incubate in a 37 °C, 5% CO
5 [7 E9 Q# i |2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum).( {4 J" [# y4 Z. o+ F8 Y; o
crItIcal step Do not allow pieces to dry out completely.* V0 v5 Y& \! u7 n
6. Gently add 7–8 ml of complete DMEM medium to each 100-mm plate.
1 y: Z( o7 C# L7 \4 X( [" Z crItIcal step It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish3 g& ], y, c7 J
following the two steps above./ q4 {+ ~: w& i$ {4 b1 n) ?" {
? trouBlesHootInG1 T7 B) }# \% m( L$ k
7. Carefully return the plate to the incubator.
) ]0 p( _5 s- E8 H, D* `+ @8. Replace with fresh medium every 3–4 d and remove any tissue pieces that are floating.8 W4 q$ X7 V4 O8 |! [5 P6 j# F
9. Within 7–10 d, outgrowths of fibroblast should appear.
$ N0 O( w$ m- R10. 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
% L) |% D: G/ n+ T! Gfor 4–5 min.
/ P; z& E9 x% w J$ [0 W m) H3 Y) p11. 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, N J% s+ q+ Q! A! Nand then immediately add 10 ml of complete DMEM medium.1 B& Z E5 g0 }9 H( r+ a8 v
12. Centrifuge at 200g for 5 min.
6 H$ |0 e% ?8 K2 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( q& b9 G9 A2 P7 t5 L
100% confluence (typically once every week). |
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发表于 2015-7-17 13:18
