Tissue Engineering and Cell-Populated Collagen Matrices - Methods in Molecular Biology: Extracellular Matrix Protocols - page 275

3. 8.75 m M Acetic Acid: Add 12.5 mL of glacial acetic acid to 25 L of purified

water. Mix well and filter the solution through a 0.22- µ m filter into sterile,

screwcap storage vessel(s). Store the solution at 2-8 C before Use (it is not rec-

ommended to store this solution. Large volumes are required and maintenance of
sterility can be an issue).

°

2.2. Production of Fibroblasts
All solutions must be sterilized by passing through an appropriate sterile,
0.22-

µ m filter before use.

1. Antimicrobial solution: PBS containing 100 µ g/mL Gentamicin sulphate; 250 µ g/mL

amphotericin B (store at -20 ° C, stable for 1 yr).

2. Enzyme solution: PBS containing: 2 mg/mL trypsin, 5 mg/mL collagenase, 50 µ g/mL

gentamicin sulphate, and 1.25 meq/mL of amphotericin B (store at -20 ° C, stable

for 1 yr).

3. Trypsin/versene: 500 µ g/mL trypsin; 200 µ g/mL versene (store at -20 ° C, stable

for 1 yr).

4. Sterile filtered aqueous solution of 95% (v/v) ethanol.

5. Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn calf

serum (NBCS). Store at 2-8 ° C (stable for 1 mo).

2.3. Production of Cell-Populated Collagen Matrix
All solutions must be sterilized by passing through an appropriate sterile,
0.22- µ m filter before use.

1. 71.2 mg/mL sodium bicarbonate. Once filter sterilized, the solution can be stored

in 50-mL aliquots at -20

° C before use.

2. 7.25 m M L-glutamine. Once filter sterilized, the solution can be stored in 15-mL

aliquots at -20 ° C before use.

3. 50 g/mL gentamicin sulphate. Once filter sterilized, the solution can be stored

in 15-mL aliquots at -20

µ

° C before use.

4. Premix: Add the following in sequence, to a 15-mL conical tube. 2.2 mL of 10X

MEM, 0.2 mL of L-glutamine, 0.025 mL of gentamicin sulphate, 0.7 mL of
sodium bicarbonate, 2.5 mL of newborn calf serum. The premix must be stored
on ice and used on the day of preparation.

3. Methods
3.1. Production of Collagen Solution
The mechanical properties of cell-contracted collagen gels is markedly
superior if the type I collagen used contains intact telopeptides. Atelopeptide
collagen in which the telopeptides have been enzymatically digested contracts
more rapidly and extensively and the resulting lattice has inferior tensile
strength (7) ( see Note 1 ). Bovine digital extensor tendons provide a suitable
yield of high-purity acid-extracted collagen (7) . This solution is stable for a