protocol-Organ Culture invitro

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In vitro culture of embryonic lungs
8 f6 H3 l# z  M( k/ N/ cfrom Hogan Lab 5 `+ ]- s; q/ |/ [) m3 h/ n
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Isolation of Lung Bud Endoderm
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What you need:
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E11-12 mouse embryos ; I: M4 t! _9 s. y+ O
DMEM with 5% fetal bovine serum " j6 b: }$ k7 }
petri dishes for dissections and washes
1 e% u& G7 M3 L: d$ WTyrode-Ringer's solution, pH 7.6-7.7 (recipe below)
4 n. M6 V+ `2 a) Z( rpancreatin-trypsin solution (recipe below) 3 l) V' P$ g: I
1. Dissect E11-12 lungs in DMEM and 5% serum on ice. Young lungs (E11 to 11.5) are best. 3 h5 V9 Q! q0 `$ l; Z4 f. d
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2. Wash the lungs 3 x 5 minutes on ice in Tyrode-Ringer's Solution, pH 7.6-7.7 to optimize physiological conditions for pancreatin-trypsin activity. , I# F- C$ I+ R6 I
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3. Incubate whole lungs for appoximately 5 minutes in pancreatin-trypsin solution on ice. The time required will vary depending on the batch of PT. 9 ^( ^% ~! `" z

& E8 {" I: `: z  \% z4 n4. Wash lungs 3 x 5 minutes in DMEM + serum to saturate pancreatin-trypsin activity. . \4 t. G' F! Z$ ^( z
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5. Using tungsten needles, dissect off the mesenchyme and cut off the buds. Alternatively, cut off the buds first and remove the mesenchyme surrounding them. ) o9 Q% g3 m, y3 z3 A1 r
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6. Place into matrigel matrix and culture for three to five days (see accompanying protocol). Isolated endoderm will not survive without growth factor added to the culture medium.
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Solutions
9 H2 V. u' K7 \& N3 `2 @; D( yTyrode-Ringer's Solution, pH 7.6-7.7 # d8 k* B3 Y* a7 [, N& E9 h

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' j4 H7 t" `) l7 @& H# q0 Q$ PNaCl 8.0 & l$ j  m; L; C: \
KCl 0.3 % l( ?: m( M* @8 O$ h' b2 c$ G& o! u' `
NaH2PO4.5H2O 0.093
( r0 |6 k; Z* K5 _# h; `6 dKH2PO4 0.025 : T# \3 M7 E* K- t3 m/ g/ z" i$ p
NaHCO3 1.0 * t& U; w/ q- p8 {+ I4 h
Glucose 2.0 / g) v+ ~% q8 N4 w& }$ }$ Y' L
Pancreatin/Trypsin Solution   e9 M6 i$ f& W+ _3 z- X

/ U& y  T* h3 ]6 m5 scomponent g/20 ml final concentration 4 S3 t' F6 O3 l# {/ e# p

0 i7 j1 |+ r3 z- S! wpancreatin 0.50 2.5%
. I+ e% `* D" T/ s4 ttrypsin 0.10 0.5% ! I9 h/ E% W1 O  {/ `/ O
polyvinylpyrrolidone(PVP) 0.10 0.5%
! l5 A$ ~5 @" Q+ P& WTyrode-Ringer's Solution 20 ml
. a! q) D7 [& a" o$ C2 |8 OSuspend by mixing for about 30 minutes. Centrifuge at 3000Xg in the Sorvall hanging bucket rotor for 5 minutes. Filter sterilize the supernatant using .8 micron Millipore filter(s). Store in aliquots at -80 C. This process is a big pain and time consuming, mainly because the supernatant does not filter efficiently and the Millipore filters must be switched often. 8 D$ J+ B( b" s% B" F; t3 O

) P2 O: k) c# B, BCollagen Gels
# y" O' A) \& I8 k% k1 VWhat you need: , W/ c: M5 i' `' n4 e* W

% I" C& Z  @, ~" CCollagen, type I rat tail (cat no 40236, Collaborative Biomedical Products) . `, H' p5 J" z2 K4 `
10X DMEM, low glucose (cat no D6921, Sigma Cell Culture) , i4 ?( w/ Y+ _% X. B4 g: i
0 .8M NaHCO3 in sterile water
# {% ?( w  g0 H/ c+ lfour-well (1.5cm diameter/well) Nunc tissue culture dishes ( cat no 176740, Nalge Nunc International) 6 E5 C5 f2 t/ M4 E0 p* i+ K
culture medium (for lung buds) 6 y: m; @4 f# \. C$ e! i. g
tissue culture incubator
3 m+ E2 E# }* M% iTissues to be cultured are placed into 50 ul drops of collagen in four-well Nunc dishes. For each treatment, make a separate 50 ul aliquot of collagen gel as described in the protocol below and store on ice. Directly before adding samples, add the NaHCO3 and mix well. It is necessary to prepare each collagen gel separately because addition of NaHCO3 makes the collagen polymerize quickly.
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. F3 p  g% H3 a+ R8 Y1. Place collagen, 10X DMEM and on ice. Also chill the four-well dishes in the rfridge before use. 9 {1 k' u1 w! m+ a# y9 l: c/ F
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2. Mix 45 ul collagen and 5 ul 10X DMEM. Mix well on ice. The collagen will not gel if kept cold. 5 O2 O# Y( ^& N' T& {3 ^; N

& P" {: i. ]% c6 f# y6 R3. Add 1.9 ul of NaHCO3. Mix well. This step begins polymerization of the collagen gel. ( T  c. j; p$ A8 m! P# c% Q

9 {) p! s+ l7 K' b, C2 d( z4. Add the 50 ul collagen mixture to a chilled four-well dish.
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5. First prepare a bed of collagen and allow it to set at 37C. After it has polymerized, add your sample to the bed of collagen and add cold unpolymerized collagen in a layer over the top of the sample. 9 t. Y. x6 I* G: c

: p+ p  j7 y1 a6. Place at 37 C for approximately 10 minutes to allow complete polymerization.
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Add culture medium to cover the bead of collagen and tissue and incubate the sample at 37 C.
% _, D" f6 Q1 K7 b: qReplace media daily.
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FROM:http://www.mshri.on.ca/nagy/Protocols/lung.htm

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ORGANOTYPIC LIMB CULTURES . N' j7 F5 J1 u5 [) w

3 y1 O2 `2 c  D% P1 u, R/ J-embryos are dissected from timed-pregnant mice from 10.5 - 11.5 d.p.c.
! G) i4 M/ Y  X  h-limb buds are microdissected and placed in holding medium (L15 medium supplemented with 1 x MITO+ serum extender) (all media is supplemented with 100 U penicillin/ml and 0.1 mg streptomycin/ml) & s1 F' O; S% f2 n$ |; v- ^
-limbs are allowed to sit in holding medium anywhere from 20 min to 2 hours " G  G+ [5 K) L- r/ u) ?
-0.2 ml of serum-free explant media (DMEM-F12 medium supplemented with 5 x MITO+ with or without 10% fetal bovine serum) is added to each well of a six-well tissue culture plate containing Cyclopore (polyethylene terphthalate; Falcon Labware) inserts
5 r! ~) g9 N3 _" c5 G; [-limbs are transferred from the holding media onto the surface of the Cyclopore membranes (for transfer, the end of a 200 祃 pipette tip is removed with a sterile razor blade (to enlarge the hole), and limbs are taken up in 5 – 10 祃 of holding media) ( N' G8 D+ N+ h5 ?  f
-limbs are grown on the tissue culture inserts in a 37oC incubator under an atmosphere of 5% CO2
  f5 q" c( r5 l6 Z* ?# F-explant media is replaced every 48 hours + K: z; H5 F6 ~& g  ^+ l( l9 I( C
Limbs can be successfully grown for 3– 4 days
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FROM:http://www.mshri.on.ca/nagy/Protocols/limb.htm

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ORGANOTYPIC KIDNEY CULTURES
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-embryos are dissected from timed-pregnant mice from 11.5 d.p.c. to 13.5 d.p.c.
8 p# j2 y# t/ a& J, l5 k* ]-metanephroi and associated ureteric buds are microdissected and placed in holding medium (L15 medium supplemented with 1 x MITO+ serum extender) (all media is supplemented with 100 U penicillin/ml and 0.1 mg streptomycin/ml) * S# K6 B& |; m: O! a
-metanephroi are allowed to sit in holding medium anywhere from 20 min to 2 hours
7 ^0 n+ w0 S2 A5 m/ {* C4 m0 P-0.2 ml of serum-free explant media (DMEM-F12 medium supplemented with 5 x MITO+ with or without 10% fetal bovine serum) is added to each well of a six-well tissue culture plate containing Cyclopore (polyethylene terphthalate; Falcon Labware) inserts % w9 Z8 w  S1 ~1 v; G' {7 r# t
-metanephroi are transferred from the holding media onto the surface of the Cyclopore membranes (for transfer, the end of a 200 祃 pipette tip is removed with a sterile razor blade (to enlarge the hole), and metanephroi are taken up in 5 – 10 祃 of holding media) - t& }+ G* O4 ]- B1 t' h
-metanephroi are grown on the tissue culture inserts in a 37oC incubator under an atmosphere of 5% CO2
3 N5 e6 P4 b" i-explant media is replaced every 48 hours
* a: [4 O; k; D3 `0 wMetanephroi can be successfully grown for 7 – 10 days

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Lung Organ Culture