Test solution— Thaw Cryopreserved Human Fibroblast-Derived Dermal Substitute by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to a temperature of 34 to 37 for 2 to 3 minutes. The minimum amount of water in the water bath is 2 L per Cryopreserved Human Fibroblast-Derived Dermal Substitute unit. Perform the test on 20 mL of the cryopreservative.

Buffered formalin— Prepare a solution containing 10% (w/v) formaldehyde solution and 1.0% to 1.5% methanol in a suitable buffer, adjusted to a pH of 6.8 to 7.2.1

Preparation of tissue for staining— Cut Cryopreserved Human Fibroblast-Derived Dermal Substitute into 3-mm × 3-mm sections. Place three sections into suitable histological tissue cassettes,2 and insert the cassettes into suitable histological cassette basket(s).3 At a temperature of 40, sequentially immerse the histological cassette basket(s) in separate solutions of Buffered formalin (2 hours), two changes of 80% alcohol (30 minutes per step), alcohol (30 minutes), three changes of dehydrated alcohol (30 minutes per step), suitable histological xylene substitute (30 minutes),4 and two changes of suitable xylene substitute (30 minutes per step). Immerse the histological cassette basket(s) into molten paraffin5 that is at a temperature of 60 for 30 minutes. Remove the cassette basket(s), and immerse in a fresh container of molten paraffin (60) for 60 minutes. Remove the histological tissue cassette from the container and basket, and disassemble. Fill preheated embedding molds with molten paraffin heated to 56 to 60, and place on top of a preheated warming platform that is designed for histology work. Transfer Cryopreserved Human Fibroblast-Derived Dermal Substitute specimens from the cassettes using forceps, and place specimens into individual molds. Orient the specimens in molds so as to cut cross-sections. Cool the paraffin by sliding the mold down the platform to its cool side until the paraffin has solidified. Maintain specimen orientation with forceps during cooling, removing the forceps when the paraffin becomes translucent. Slide the paraffin block onto a histological cold plate to rapidly cool the block. Trim the paraffin block with a new single-edged razor blade to form a rectangle or slight trapezoid to within 5 mm of the tissue mass, if necessary. Cool the block at 4 for 15 to 30 minutes. Clamp the tissue block into the block holder of the microtome. Fill a histological tissue flotation water bath with fresh water, add an appropriate amount of a suitable histological adhesive,6 and heat to 5 less than the melting point of the paraffin. Properly mount and adjust the tissue and paraffin block into a microtome. Set the microtome to make cuts 5 microns thick with a blade angle of 5 ± 2. Insert a sharp stainless steel microtome knife that has been properly honed or a new disposable microtome knife into the knife holder. Cut a ribbon that contains 6 to 10 sections of Cryopreserved Human Fibroblast-Derived Dermal Substitute. Pick up the ribbon with forceps, and stretch it across the tissue flotation water bath. Separate 2 to 3 adjacent sections from the ribbon on the water bath. The selected sections should not be compressed, wrinkled, or scratched. Pick up the selected sections by dipping a microscope slide into the water bath under the floating sections, and gently lift the slide out of the water. For each staining procedure, prepare three slides from each of the three starting Cryopreserved Human Fibroblast-Derived Dermal Substitute 3-mm × 3-mm sections. Allow the mounted sections to air-dry completely, or dry the slide in a 60 oven for 1 hour.

HEMATOXYLIN–EOSIN STAINING—

COLLAGEN STAINING—

DISTRIBUTION OF FIBRONECTIN—

DPBS solution A— Dissolve 1.32 g of calcium chloride and 1.21 g of magnesium sulfate heptahydrate in 2 L of water.

DPBS solution B— Dissolve 80.0 g of sodium chloride; 2.0 g of potassium chloride; 11.5 g of dibasic sodium phosphate; 2.0 g of monobasic potassium phosphate; 10.0 g of glucose; 0.36 g of sodium phosphate; 0.5 g of streptomycin sulfate; and 1,000,000 USP Units of penicillin G sodium in 8 L water.

DPBS working solution— Mix DPBS solution B with DPBS solution A (8:2). Pass the solution through a filter having a 0.22-µm porosity.

Dulbecco's modified Eagle's tissue culture medium— Prepare a solution that contains the following components:

L-Glutamine solution— Prepare a 100-mL solution containing 2.92 g of L-glutamine.

Sodium pyruvate solution— Prepare 100 mL of a solution containing 1.10 g of sodium pyruvate.

Antibiotic-antimycotic solution— Prepare 100 mL of a solution containing 0.85 g of sodium chloride, 10,000 USP Units of penicillin G sodium, 10,000 µg of streptomycin (base), and 25 µg of amphotericin B.

Assay stock medium— Mix 1000 mL of Dulbecco's modified Eagle's tissue culture medium, 10 mL of L-Glutamine solution, 10 mL of Sodium pyruvate solution, 10 mL of Antibiotic-antimycotic solution, and 20 mL of fetal bovine serum.14

MTT-assay solution— Dissolve 0.50 g of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide in 1 L of Assay stock medium, using constant stirring. Sterilize the solution by passing it through a suitable filter having a 0.2-µm porosity.

MTT formazan stock solution— Prepare a solution containing 100 µg of 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan per mL of isopropyl alcohol.

MTT formazan calibration solutions— Prepare solutions containing the following five MTT formazan concentrations: 15 µg per mL, 30 µg per mL, 45 µg per mL, 60 µg per mL, and 75 µg per mL of MTT formazan, using MTT formazan stock solution and diluting with isopropyl alcohol.

Procedure— Thaw Cryopreserved Human Fibroblast-Derived Dermal Substitute by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to between 34 and 37 for 2 to 3 minutes. The minimum amount of water in the water bath is 2 L per Cryopreserved Human Fibroblast-Derived Dermal Substitute unit. Cut three 11-mm × 11-mm sections of Cryopreserved Human Fibroblast-Derived Dermal Substitute, and immerse the sections into separate, 3.0-mL portions of MTT-assay solution. Incubate for 2 hours at 37 ± 2 in a 3% to 7% CO2–air environment with shaking on an orbital shaker at 150 to 200 rpm. After incubation remove from the 37, 3% to 7% CO2–air environment. Remove the MTT-assay solution, and rinse twice with DPBS working solution. Immerse the Cryopreserved Human Fibroblast-Derived Dermal Substitute in 2 mL of isopropyl alcohol, and incubate at ambient temperature for 1 hour with shaking on an orbital shaker at approximately 125 rpm. Transfer 200-µL aliquots of the five MTT formazan calibration solutions, in triplicate, and 200-µL aliquots of the three isopropyl alcohol extracts of Cryopreserved Human Fibroblast-Derived Dermal Substitute to a suitable 96-well flat-bottom plate. Read the absorbance of each aliquot at 540 nm, using 200 µL of isopropyl alcohol as the blank. Plot the responses of the MTT formazan calibration solutions versus concentration, in µg of MTT formazan per mL, and calculate the regression line using the least-squares method; the test is considered valid if the regression line has a square of the correlation coefficient not less than 0.95: the absorbance value of individual, thawed, Cryopreserved Human Fibroblast-Derived Dermal Substitute sections is between 0.30 and 0.86.

Cell culture water— Use sterile water containing not more than 0.005 USP Endotoxin Unit per mL.

DNA extraction buffer— Transfer about 850 mL of Cell culture water to a sterile, 1-L graduated container. Dissolve 12.110 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 3.802 g of edetate disodium, 23.380 g of sodium chloride, and 0.080 g of sodium dodecyl sulfate, with stirring. Adjust with 1 N hydrochloric acid or 1 N sodium hydroxide to a pH of 7.0. Dilute with Cell culture water to 1 L.

Proteinase K solution— Prepare a solution of Tritirachium album proteinase K in 10 mM of 2-amino-2-hydroxymethyl-1,3-propanediol, adjusted to a pH of 7.5, having an activity of 600 units per mL.15

Working DNA extraction buffer— Add 1.22 mL of Proteinase K solution to 38.78 mL of DNA extraction buffer, and mix.

Dilution buffer— Transfer 850 mL of Cell culture water to a sterile, 1-L graduated container. Add 1.211 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 3.802 g of edetate disodium, and 5.844 g of sodium chloride, with stirring. Adjust with 1 N hydrochloric acid or 1 N sodium hydroxide to a pH of 7.0. Dilute with Cell culture water to 1 L.

DPBS without Ca++, Mg++ solution— Prepare a solution containing 8.00 g of sodium chloride, 1.15 g of dibasic sodium phosphate (anhydrous), 0.20 g of potassium chloride, and 0.20 g of monobasic potassium phosphate per L.

Calf thymus DNA solution— Prepare a solution containing 1 mg of calf thymus DNA per mL of DPBS without Ca ++, Mg++ solution, mixing thoroughly for 12 to 24 hours at ambient temperature. Dilute the resulting solution with DPBS without Ca ++, Mg++ solution to prepare a solution containing 50 µg of calf thymus DNA per mL, mixing thoroughly on a vortex mixer for 10 minutes.

Calf thymus DNA calibration solutions— Prepare four calibration solutions containing 5 µg per mL, 10 µg per mL, 15 µg per mL, and 20 µg per mL of calf thymus DNA, using Calf thymus DNA solution and diluting with DPBS without Ca ++, Mg++ solution.

DNA staining solution— Prepare a solution containing 0.5 µg of 2¢-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5¢-bi-1H-benzimidazole trihydrochloride pentahydrate per mL of Dilution buffer. Store in low-actinic glassware.

Procedure— Thaw Cryopreserved Human Fibroblast-Derived Dermal Substitute by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to between 34 and 37 for 2 to 3 minutes. The minimum amount of water in the water bath is 2 L per Cryopreserved Human Fibroblast-Derived Dermal Substitute unit. Cut three 11-mm × 11-mm sections of Cryopreserved Human Fibroblast-Derived Dermal Substitute. To each of three microcentrifuge tubes add 1 mL of DPBS without Ca ++, Mg++ solution. Immerse a single Cryopreserved Human Fibroblast-Derived Dermal Substitute 11-mm × 11-mm section into each microcentrifuge tube to remove the cryopreservative. Aspirate the DPBS without Ca ++, Mg++ solution from each tube, and replace with 1 mL of Working DNA extraction buffer, making sure that each Cryopreserved Human Fibroblast-Derived Dermal Substitute is completely submerged in the extraction buffer. Incubate the samples in a 56 to 60 water bath for 4 to 18 hours. Sonicate for 10 to 15 seconds using an ultrasonic cell disrupter to achieve complete cellular disruption of the tissue and to mix the contents of the tube. Centrifuge the microcentrifuge tubes at 12,000 to 15,000 × g to pellet non-DNA material. Transfer three 50-µL aliquots of each sample supernatant to individual wells of a 96-well black plate suitable for performing fluorescent analysis. Transfer triplicate 50-µL aliquots of each of the Calf thymus DNA calibration solutions to the 96-well plate, as well as a 50-µL aliquot of DPBS working solution for the blank. Add 150 µL of DNA staining solution to all wells containing the tissue samples, Calf thymus DNA calibration solutions, and the blank. Cover with aluminum foil, and place in a dark cabinet for 30 to 45 minutes at 15 to 30. Read the fluorescence of each well, using an excitation wavelength of 355 nm and an emission wavelength of 460 nm, blanking against the DPBS without Ca ++, Mg++ solution well. Plot the responses of the Calf thymus DNA calibration solutions versus concentration, in µg of calf thymus DNA per mL, and calculate the regression line using the least-squares method. The test is considered valid if the %CV of the replicate values is less than 15%, the slope is between 4.48 and 6.27, the y-intercept is between –2.04 and 3.65, and the square of the correlation coefficient is not less than 0.990. From the regression line so obtained, determine the amount of DNA, in µg per 11-mm × 11-mm sample: the amount of DNA of individual Cryopreserved Human Fibroblast-Derived Dermal Substitute 11-mm × 11-mm section is between 6 and 15 µg.

DPBS without Ca++, Mg++ solution— Proceed as directed for DNA content.

DPBS with Ca++, Mg++ solution— Prepare a solution containing 8.00 g of sodium chloride, 1.15 g of dibasic sodium phosphate (anhydrous), 0.20 g of potassium chloride, 0.20 g of monobasic potassium phosphate, 0.10 g of magnesium chloride hexahydrate, and 0.10 g of calcium chloride (anhydrous) per L.

Collagenase extraction solution— Prepare a solution of Clostridium histolyticum collagenase, type 2, in DPBS with Ca++, Mg++ solution having an activity of at least 250 units per mL.

2% Acetic acid solution— Mix 10 mL of acetic acid with 490 mL of water.

Collagen standard stock solution— Prepare a solution having a concentration of 2 mg of collagen, type I, per mL of 2% Acetic acid solution.

Collagen calibration standards— Cut polyglactin mesh16 into seventeen 11-mm × 11-mm squares, and place one square into 17 individual wells of a 24-well plate. Each well of the 24-well plate has a surface area of about 220 mm2 and a volume of about 3.5 mL. In quadruplicate, prepare wells containing 0.050 mg, 0.100 mg, 0.200 mg, and 0.400 mg of collagen by adding 25 mL, 50 mL, 100 mL, and 200 mL, respectively, of the Collagen standard stock solution. The remaining well to which no Collagen standard stock solution has been added is used as the blank. Allow the wells to air dry.

Sirius red solution— Dissolve 0.5 g of Direct Red 80 in 500 mL of saturated picric acid.

1% (p-tert-Octylphenoxy)polyethoxyethanol solution— Mix 10 mL of (p-tert-octylphenoxy)polyethoxyethanol in 990 mL of DPBS with Ca++, Mg++ solution.

Procedure— Thaw Cryopreserved Human Fibroblast-Derived Dermal Substitute by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to between 34 and 37 for 2 to 3 minutes. The minimum amount of water in the water bath is 2 L per Cryopreserved Human Fibroblast-Derived Dermal Substitute unit. Cut three 11-mm × 11-mm sections of Cryopreserved Human Fibroblast-Derived Dermal Substitute. Place each test section into separate wells of a 24-well plate. Add 200 µL of 1% (p-tert-Octylphenoxy)polyethoxyethanol solution to each sample. Shake on a rotating platform shaker at 100 to 150 rpm for 60 to 70 minutes at room temperature. Aspirate off the 1% (p-tert-Octylphenoxy)polyethoxyethanol solution, and rinse three times with 2 mL of DPBS without Ca++, Mg++ solution. Transfer each of the collagen standards to individual wells of the 24-well plate. Add 0.5 mL of Sirius red solution to each test sample and collagen standards. Shake on a rotating platform shaker at 100 to 150 rpm for 60 minutes at room temperature. Aspirate off the Sirius red solution from each well. Rinse each well twice with 2 mL of DPBS without Ca++, Mg++ solution. Add an additional 2 mL of DPBS without Ca++, Mg++ solution to each well, and allow to stand for 2 minutes. Aspirate off the DPBS without Ca++, Mg++ solution, and rinse twice more with 2 mL of DPBS without Ca++, Mg++ solution. Aspirate off all traces of DPBS without Ca++, Mg++ solution. Add 0.5 mL of Collagenase extraction solution to each well containing the Collagen calibration standards. Add 2.0 mL of Collagenase extraction solution to each well containing test samples. Rotate the plate on an orbital rotator at about 150 rpm for 90 minutes at 37. Transfer 200 µL from each well, and transfer to a suitable 96-well flat-bottom plate. Read the absorbance of each aliquot at 540 nm. Dilute the Cryopreserved Human Fibroblast-Derived Dermal Substitute sample preparation further with DPBS without Ca++, Mg++ solution if the absorbance is greater than the absorbance of the highest of the Collagen calibration standards. Plot the responses of the Collagen calibration standards versus the amount, in mg of collagen, and calculate the regression line using the least-squares method. The test is considered valid if the slope is between 3.00 and 5.00 and the square of the correlation coefficient is greater than or equal to 0.950. Determine the mg of collagen per Cryopreserved Human Fibroblast-Derived Dermal Substitute section from the regression line and using the following equation: in which CCSR is the collagen content of the piece, in mg; Df is the dilution factor (normally 4, unless the sample had to be further diluted); A is the absorbance at 540 nm; and SCSR is the slope of the regression line of the standards calculated above. The test is considered valid if the slope of the regression line is between 3.00 and 5.00 and has a square of the correlation coefficient greater than 0.950: the amount of collagen in individual Cryopreserved Human Fibroblast-Derived Dermal Substitute 11-mm × 11-mm samples is between 0.40 and 2.0 mg.

(Official January 1, 2007)