Time Needed: ~2.5 days
Day 1: Clean P2322 inserts.
Assemble materials (dissolve collagen).
Make first casting + drying.
Day 2: Make second casting + drying.
Fix and rinse.
Third cast with Vitrogen.
Day 3: Sterilize and store.
Materials Needed:
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P2322 inserts (polycarbonate).
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Rat tail collagen Type I, Sigma #C-7661 (can see cells microscopically)
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Calfskin collagen Type III, Sigma #C-3511 (cheap and works for bioelectrics).
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0.2% Acetic acid
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2.5% Gluteraldehyde; Sigma (buy full strength stock).
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Vitrogen 100 from Collagen Corporation (Palo Alto, CA 94303)
1. Start with clean, dry P2322 inserts placed bottom up on a rack or on inverted Snapwell hangers.
Tips for Cleaning: Place inserts in Clorox diluted about 1:4 with DI H2O and soak until collagen from previous experiments is removed (24 hours minimum). Rinse vigorously with water (tap then DI H2O) to remove Clorox completely. Place bottom up on a rack to dry.
2. Dissolve collagen in 0.2% acetic acid at 15 mg/ml (100 mg collagen/6.7 ml acetic acid).
Tips for Dissolving: Place collagen/acetic acid mixture in a 50 ml centrifuge tube and cap tightly. Place tube in hot water. Shake the tube vigorously every few minutes until all of the collagen dissolves. Centrifuge (500 g for 5 minutes) to eliminate bubbles and debris. Can be stored in the refrigerator.
3. Mix the first cast of collagen: 1 ml collagen / 3 drops of 2.5% gluteraldehyde. (Use a glass pipette for the drops of gluteraldehyde.
Tips: Mix only amount to be used within 1 hour (1 ml coats about 20 P2322 inserts).
Try to avoid bubbles when mixing and spreading the collagen.
4. First Cast: Cast the bottom of the P2322 insert.
a. With a Pasteur pipette, place a line of collagen/gluteraldehyde (approx. 30-35 ml) on the bottom of the P2322 insert. Draw the collagen/gluteraldehyde across the hole with the side of the pipette. Extend the collagen on the P2322 bottom well beyond the edge of the hole.
b. Let dry completely (about 3 hours or overnight).
5. Second Cast: Coat the aperture from inside of each P2322 insert.
a. Coat bottom of insert with dissolved collagen (no gluteraldehyde) as in step 4.
b. Immediately cast the inside orifice of the same insert with the same solution. One small drop should be sufficient.
c. Tap the bottom of the P2322 insert against the table to spread the inner drop.
d. Dry overnight (or at least 3 hours) with the apical (inside) side up.
6. Crosslink the collagen.
a. Fix the back of the insert with 2-3 drops of 2.5% gluteraldehyde for 5-10 minutes.
b. Rinse well with distilled water.
c. Dry P2322 inserts bottom side up on a rack.
7. Third Cast: Vitrogen Coating
a. Dilute Vitrogen 1:9 (v:v)with sterile H2O.
b. Coat inside of P2322 well with 1 drop of Vitrogen. (1 drop should enough to cover the entire orifice).
c. DRY overnight in the hood.
8. Sterilize inserts:
a. Immerse inserts in 70%EtOH for 20 minutes or expose to UV light for 30 minutes.
b. Rinse with sterile F12 solution and store in F12 in sterile specimen cup or on Snapwell hanger in Snapwell culture dish.
c. Store inserts in incubator and check media inside cup for contamination prior to use.
Porous Membrane Protocol:
Materials Needed:
1. Clean inserts: Start with clean, dry P2322 inserts bottom up on a rack or on inverted Snapwell hangers.
Tips for Cleaning: Using a straight edge razor blade, scrape bottom of P2322 to remove any attached membrane from previous experiments. Sand bottom of P2322 lightly with #600 wet/dry sandpaper to remove traces of cyanoacrylate adhesive. Place inserts in Clorox bleach diluted about 1:4 with DI H2O and soak until collagen is removed (24 hours minimum). Rinse vigorously with water (tap then DI H2O) to removeClorox completely. Place bottom up on a rack to dry.
2. Prepare membranes for attachment.
Using a sharp razor or scalpel blade carefully cut the membrane from the bottom of a 24mm diameter commercial culture dishes (e.g., Costar Transwell, Nunc Anapore, Millicell, etc). Alternatively, it should be possible to use standard filter membranes (e.g., Poretic PCTE polycarbonate membrane available from Fisher Scientific). Cut the filter into squares sufficient to cover the aperture on the P2322 insert.
3. Mounting the Membrane on the P2322 Insert:
Place a tiny drop of cyanoacrylate adhesive on the bottom of a clean insert and use a toothpick to paint the adhesive around the perimeter of the aperture (Note: a thin coating is best. Thick coatings will cause problems you don�t want.). Using forceps place a square of filter material over the aperture and press into the adhesive using a flat surface such as a microscope slide
4. Coat the membrane with collagen if required (see step 5 on Casting protocol above).
5. Sterilize as in step 8 above.
