Construct Human Fibroblasts in Polyglactin Scaffold

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Construct Human Fibroblasts in Polyglactin Scaffold is a living monolayer skin substitute derived from neonatal foreskins. It is composed of fibroblasts, an extracellular matrix, and a bioabsorbable scaffold. Human fibroblasts are seeded onto a bioabsorbable, nonantigenic and nonpyrogenic mesh scaffold composed of polyglactin, a copolymer of glycolide and lactide. The fibroblast-cell banks, from which Construct Human Fibroblasts in Polyglactin Scaffold are derived have passed applicable donor eligibility requirements for relevant communicable diseases. Maternal blood sera are tested for evidence of infection with human immunodeficiency virus types 1 and 2, hepatitis B and C viruses, syphilis, and human T-lymphotropic virus type 1 and are found negative for the purpose of donor selection. Construct Human Fibroblasts in Polyglactin Scaffold is manufactured with sterile components under aseptic conditions within the final package. Reagents used in the manufacture of Construct Human Fibroblasts in Polyglactin Scaffold are tested and found free of viruses, retroviruses, endotoxins, and mycoplasma before use. All materials derived from bovine sources originate from countries free of bovine spongiform encephalopathy. During subsequent screening of the fibroblast cell strain at various stages in the manufacturing process, testing for these same viruses, as well as Epstein-Barr virus and human T-lymphotropic virus type 2, is carried out and found to be negative. The final product is inspected and tested to ensure that the product meets specifications.

2 SPECIFIC TESTS

2.1 HISTOLOGICAL EVALUATION

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

Preparation of tissue for staining: Cut Construct Human Fibroblasts in Polyglactin Scaffold into 3 - mm x 3 - mm sections. Place three sections into suitable histological tissue cassettes,² and insert the cassettes into suitable histological cassette basket(s). ³At a temperature of 40°, sequentially immerse the histological cassette basket(s) in separate solutions of Buffered formalin (2 h), two changes of 80% alcohol (30 min/step), alcohol (30 min), three changes of dehydrated alcohol (30 min/step), suitable histological xylene substitute (30 min), ⁴and two changes of suitable xylene substitute (30 min/step). Immerse the histological cassette basket(s) into molten paraffin⁵ that is at a temperature of 60° for 30 min. Remove the cassette basket(s), and immerse in a fresh container of molten paraffin at 60° for 60 min. Remove the histological tissue cassette from the container and basket, and disassemble. Fill preheated embedding molds with molten paraffin heated to 56°-60°, and place on top of a preheated warming platform that is designed for histology work. Transfer Construct Human Fibroblasts in Polyglactin Scaffold 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 15-30 min. 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, ⁶and heat to 5º 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-µm 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 6-10 sections of Construct Human Fibroblasts in Polyglactin Scaffold. Pick up the ribbon with forceps, and stretch it across the tissue flotation water bath. Separate 2-3 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 Construct Human Fibroblasts in Polyglactin Scaffold 3-mm x 3-mm sections. Allow the mounted sections to air-dry completely, or dry the slide in a 60° oven for 1 h.

2.1.1 Hematoxylin-eosin staining

Hematoxylin-alcohol solution: Dissolve 2.5 g of hematoxylin in 25.0 mL of dehydrated alcohol with heating.

Potassium alum solution: Dissolve 50.0 g of potassium alum in 500 mL of water with heating.

Hematoxylin staining solution: Mix Hematoxylin-alcohol solution and Potassium alum solution. Bring to a boil as rapidly as possible with constant stirring. Do not heat for more than 1 min. Slowly add 0.185 g of sodium iodate. Reheat to a simmer until the solution becomes a deep purple. Remove from heat, and quickly cool. Filter daily before use.

10% Acid alcohol: Add 5.0 mL of hydrochloric acid to 495 mL of 70% alcohol.

Eosin solution: Dissolve 1 g of eosin Y in 100 mL of alcohol. Filter daily before use.

Analysis: Sequentially immerse the microscope slide with affixed tissue, as prepared in Preparation of tissue for staining, in three changes of a suitable histological, aliphatic xylene substitute (2 min/step), three changes of dehydrated alcohol (1 min/step), alcohol (20 s), running tap water rinse (1 min), Hematoxylin staining solution (4-5 min), running tap water rinse (1 min), 10% Acid alcohol (15 s), running tap water rinse (1 min), a suitable bluing reagent ⁷(20-30 s), running tap water rinse (1 min), alcohol (20 s), Eosin solution (10-20 s, until a reddish-brown color is obtained in the tissue), three changes of dehydrated alcohol (10 s/step), and three changes of a suitable histological xylene substitute (10 s/step). Adjust the above immersion times as needed to suitably stain the tissue. Remove the slide from the last histological xylene substitute wash, and blot dry the back of the slide. Do not allow the tissue to dry. Affix a coverslip over the tissue using a coverslip mountant.

Acceptance criteria: Using USP Construct Human Fibroblasts in Polyglactin Scaffold Reference Photomicrograph 1⁸ for comparison, the test article shows a polyglactin scaffold mesh and a secreted collagen-based matrix; the tissue contains normal human fibroblast distributed throughout the secreted matrix and resembles normal human papillary dermis. Fibroblasts appear elongated and spindle-shaped. The tissue, which is 100-300 µm thick, contains 106 cells/cm².

2.1.2 Collagen staining

Bouins's solution: Mix 75 mL of 1.22% picric acid solution, 25 L of dimethoxymethane, and 5.0 L of acetic acid.

Weigert's iron hematoxylin solution A: Dissolve 1 g of hematoxylin in 100 mL of alcohol.

Weigert's iron hematoxylin solution B: Mix 4.0 mL of 29% ferric chloride, 95.0 mL of water, and 1.0 mL of hydrochloric acid.

Weigert's iron hematoxylin working solution: Mix equal volumes of Weigert's iron hematoxylin solution A and Weigert's iron hematoxylin solution B. Pass the solution through a filter of 0.45-µm pore size. Prepare fresh as needed.

Gomori's trichrome solution: Mix 1.0 mL of acetic acid and 100 mL of water. Dissolve 0.6 g of chromotrope 2R, 0.3 g of Fast Green FCF, and 0.6 g of phosphotungstic acid.

1% Acetic acid: Dilute 1 mL of glacial acetic acid with water to make 100 mL of solution.

Analysis: The microscope slide with affixed tissue, as prepared in Preparation of tissue for staining, is sequentially immersed in three changes of a suitable histological, aliphatic xylene substitute (2 min/step), three changes of dehydrated alcohol (1 min/step), alcohol (20 s), and running tap water rinse (1 min). Immerse the slide in Bouins's solution, and place in a 42° water bath for 1 h. Rinse the slide in water for 10 s. Sequentially immerse the slide in Weigert's iron hematoxylin working solution (10 min) and running tap water rinse (10 min). Rinse the slide in water for 10 s, and immerse in Gomori's trichrome solution (3-5 min). Rinse the slide in 1% acetic acid for 20 s. Sequentially immerse the slide in three changes of alcohol (10 s/step) and three changes of a suitable histological, aliphatic xylene substitute (10 s/step). Affix a coverslip over the tissue using a suitable coverslip mountant. Nuclei will stain black; cytoplasm, keratin, and muscle fibers will stain red; and collagen and mucin will stain blue.

Acceptance criteria: Using USP Construct Human Fibroblasts in Polyglactin Scaffold Reference Photomicrograph 2 for comparison, the tissue contains normal human fibroblast distributed throughout the secreted matrix and resembles normal human papillary, dermis, muscle fibers, and keratin are absent.

2.1.3 Distribution of fibronectin

Tris-saline buffer: Prepare a solution containing 0.1 M tris-(hydroxymethyl)aminomethane hydrochloride and 0.15 M sodium chloride, adjusted to a pH of 7.8.

3% Hydrogen peroxide: Dilute 30 mL of Hydrogen peroxide with water or methanol

Diaminobenzidine solution: Use a suitable solution.⁹

Hematoxylin staining solution: Prepare as directed for Hematoxylin-eosin staining.

Analysis: The microscope slide with affixed tissue as prepared in Preparation of tissue for staining is dried either overnight at 37° or for 1 h at 60". The microscope slide with affixed tissue as prepared in Preparation of tissue for staining is sequentially immersed in three changes of a suitable histological, aliphatic xylene substitute (2 min/step), three changes of dehydrated alcohol (1 min/step), alcohol (20 s), and running tap water rinse (1 min). Sequentially immerse the slide in Tris-saline buffer (10 min), 3% hydrogen peroxide (30 min), three changes of Tris-saline buffer (1 min/step), a suitable normal rabbit serum ¹⁰(30 min), water (5 min), and three changes of Tris-saline buffer (1 min/step). Incubate the slide with a suitable solution of rabbit anti-human fibronectin antibody, ¹¹diluted using a suitable antibody diluent¹² to an antibody concentration of 21.0 ± 2.1 mg/L for 1 h. Sequentially immerse the slide in water (5 min) and three changes of Tris-saline buffer (1 min/step). Place enough drops of a biotinylated goat anti-rabbit antibody solution¹³ to cover the tissue section, and incubate for 30 min. Sequentially immerse the slide in water (5 min) and three changes of Tris-saline buffer (1 min/step). Place enough drops of a streptavidin conjugated horseradish Peroxidase solution¹⁴ to cover the tissue section, and incubate for 30 min. Sequentially immerse the slide in water (5 min) and three changes of Tris-saline buffer (1 min/step). Incubate the slide with Diaminobenzidine solution for 1-5 min, until a suitable difference in staining is seen by comparison with a control in which the fibronectin (primary) antibody is omitted. Sequentially immerse the slide in water (1 min), Hematoxylin staining solution (4-5 min), and water (1 min). Do not allow the tissue to dry. Affix a coverslip over the tissue using a low-viscosity, aqueous, synthetic-resin coverslip mountant.

Acceptance criteria: Using USP Construct Human Fibroblasts in Polyglactin Scaffold Reference Photomicrograph 3 for comparison, fibronectin is found colocalizing with the collagen fibers. The intensity of staining may vary from region to region of the slide.

2.2 METABOLIC ACTIVITY ASSESSMENT

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 of 0.22-um pore size.

Dulbecco's modified Eagle's tissue culture medium: Prepare a solution that contains the components listed in Table 1.

   Table 1 

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. ¹⁵

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

MTT formazan stock solution: 100 µg/mL of 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan in isopropyl alcohol

MTT formazan calibration solutions: Prepare calibration solutions of 15, 30, 45, 60, and 75 µg/mL of MTT formazan, using MTT formazan stock solution and diluting with isopropyl alcohol.

Analysis: Thaw Construct Human Fibroblasts in Polyglactin Scaffold by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to between 34° and 37° for 2-3 min. The minimum amount of water in the water bath is 2 L/Construct Human Fibroblasts in Polyglactin Scaffold unit. Cut three 11-mm x 11-mm sections of Construct Human Fibroblasts in Polyglactin Scaffold, and immerse the sections into separate, 3.0 mL portions of MTT-assay solution. Incubate for 2 h at 37 ± 2° in a 3%-7% CO₂-air environment with shaking on an orbital shaker at 150-200 rpm. After incubation remove from the 37°, 3%-7% CO₂-air environment. Remove the MTT-assay solution, and rinse twice with DPBS working solution. Immerse the Construct Human Fibroblasts in Polyglactin Scaffold in 2 ml. of isopropyl alcohol, and incubate at ambient temperature for 1 h 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 Construct Human Fibroblasts in Polyglactin Scaffold 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/mL of MTT formazan, and calculate the regression line using the least-squares method.

System suitability: The test is considered valid if the regression line has a square of the correlation coefficient NLT 0.95.

Acceptance criteria: The absorbance value of individual, thawed Construct Human Fibroblasts in Polyglactin Scaffold sections at 540 nm is between 0.30 and 0.86.

2.3 DNA CONTENT

Cell culture water: Sterile water containing NMT 0.005 USP Endotoxin Unit/mL

DNA extraction buffer: Transfer 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/mL.¹⁶

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/L of sodium chloride, 1.15 g/L of dibasic sodium phosphate (anhydrous), 0.20 g/L of potassium chloride, and 0.20 g/L of monobasic potassium phosphate in water.

Calf thymus DNA solution: Prepare a solution containing 1 mg/L of calf thymus DNA in DPBS without Ca⁺⁺, Mg⁺⁺ solution, mixing thoroughly for 12-24 h at ambient temperature. Dilute the resulting solution with DPBS without Ca⁺⁺, Mg⁺⁺ solution to prepare a solution containing 50 µg/mL of calf thymus DNA, mixing thoroughly on a vortex mixer for 10 min.

Calf thymus DNA calibration solutions: Prepare four calibration solutions containing 5, 10, 15, and 20 µg/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/mL of Dilution buffer. Store in low-actinic glassware.

Analysis: Thaw Construct Human Fibroblasts in Polyglactin Scaffold by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to between 34° and 37° for 2-3 min. The minimum amount of water in the water bath is 2 L/Construct Human Fibroblasts in Polyglactin Scaffold unit. Cut three 11-mm x 11-mm sections of Construct Human Fibroblasts in Polyglactin Scaffold. To each of three microcentrifuge tubes add 1 mL of DPBS without Ca⁺⁺, Mg⁺⁺ solution. Immerse a single Construct Human Fibroblasts in Polyglactin Scaffold 11-mm x 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 Construct Human Fibroblasts in Polyglactin Scaffold is completely submerged in the extraction buffer. Incubate the samples in a 56-60° water bath for 4-18 h. Sonicate for 10-15 s 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 12,000-15,000 x 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 30-45 min at 15°-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/mL of calf thymus DNA, and calculate the regression line using the least-squares method. From the regression line so obtained, determine the amount of DNA in µg/11-mm x 11-mm sample.

System suitability requirements: 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 NLT 0.990.

Acceptance criteria: The amount of DNA of an individual Construct Human Fibroblasts in Polyglactin Scaffold 11-mm x 11-mm section is between 6 and 15 µg.

2.4 TOTAL COLLAGEN CONTENT

DPBS without Ca⁺⁺, Mg⁺⁺ solution: Prepare as directed for DNA Content.

DPBS with Ca, Mg solution: 8.00 mg/mL of sodium chloride, 1.15 mg/mL of dibasic sodium phosphate (anhydrous), 0.20 mg/mL of potassium chloride, and 0.20 mg/mL of monobasic potassium phosphate, 0.10 mg/mL of magnesium chloride hexahydrate, and 0.10 mg/mL of calcium chloride (anhydrous) in water

Collagenase extraction solution: At least 250 Units/mL of Clostridium histolyticum collagenase, type 2, in DPBS with Ca⁺⁺, Mg⁺⁺ solution

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

Collagen standard stock solution: 2 mg/mL of collagen, type I, in 2% Acetic acid solution

Collagen calibration standards: Cut polyglactin mesh¹⁷ into 17 11-mm x 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 220 mm² and a volume of 3.5 mL. In quadruplicate, prepare wells containing 0.050, 0.100, 0.200, and 0.400 mg of collagen by adding 25, 50, 100, and 200 µL, 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: 1 mg/mL of Direct Red 80 in 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.

Analysis: Thaw Construct Human Fibroblasts in Polyglactin Scaffold by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to between 34° and 37° for 2-3 min. The minimum amount of water in the water bath is 2 L/Construct Human Fibroblasts in Polyglactin Scaffold unit. Cut three 11-mm x 11-mm sections of Construct Human Fibroblasts in Polyglactin Scaffold. 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 100-150 rpm for 60-70 min 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 100-150 rpm for 60 min 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 min. 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 150 rpm for 90 min at 37°. Transfer 200 µL from each well, to a suitable 96-well, flat-bottom plate. Read the absorbance of each aliquot at 540 nm. Dilute the Construct Human Fibroblasts in Polyglactin Scaffold 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. Determine the mg of collagen/Construct Human Fibroblasts in Polyglactin Scaffold section from the regression line and using the following equation:

                Result = D x A x SC_SR

D = dilution factor (normally 4, unless the sample had to be further diluted)

A = absorbance at 540 nm A

SC_SR = slope of the regression line of the standards calculated above

System suitability requirements: The test is considered valid if the slope of the regression line is between 3.00 and 5.00 and the square of the correlation coefficient is greater than or equal to 0.950.

Acceptance criteria: The amount of collagen in individual Construct Human Fibroblasts in Polyglactin Scaffold 11-mm x 11-mm samples is 0.40-2.0 mg.

2.5 STERILITY TESTS (71)

Sample solution: Thaw Construct Human Fibroblasts in Polyglactin Scaffold by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to a temperature of 34"-37" for 2-3 min. The minimum amount of water in the water bath is 2 L/Construct Human Fibroblasts in Polyglactin Scaffold unit.

Analysis: Perform the test on 20 mL of the cryopreservative.

Acceptance criteria: Meets the requirements

2.6 BACTERIAL ENDOTOXINS TEST (85)

Sample: Thaw Construct Human Fibroblasts in Polyglactin Scaffold by placing the tissue, still in its ethyl vinyl acetate bag, in a water bath heated to a temperature of 34°-37° for 2-3 min. The minimum amount of water in the water bath is 2 L/Construct Human Fibroblasts in Polyglactin Scaffold unit. Remove the unit from the ethyl vinyl acetate bag, and immerse in 25 mL of LAL Reagent Water.

Analysis: Extract for 60 min at 37° with shaking on an orbital shaker set at 125 revolutions/min. Remove a 4-mL aliquot of the extract for testing.

Acceptance criteria: It contains NMT 0.5 USP Endotoxin Unit/mL.

3 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Construct Human Fibroblasts in Polyglactin Scaffold is aseptically packaged and supplied frozen in a clear ethyl vinyl acetate bag. The solution within the bag is a saline-based cryoprotectant supplemented with 10% Dimethyl sulfoxide and bovine serum to facilitate long-term storage. An aluminum-plastic foil envelope surrounds the bag for its protection. Construct Human Fibroblasts in Polyglactin Scaffold should be stored at -75 ± 10° for no longer than 6 months.

LABELING: The label indicates the dimensions of the Construct Human Fibroblasts in Polyglactin Scaffold material enclosed. It contains the expiry date, the required storage conditions, and the lot number. The label cautions that Construct Human Fibroblasts in Polyglactin Scaffold is not to be used if the package shows signs of damage. Additional labeling requirements include instructions on the proper thawing and handling of Construct Human Fibroblasts in Polyglactin Scaffold, the time frame for use after package opening, and a statement that cytotoxic agents are not to be used.

Change to read:

USP REFERENCE STANDARDS (11).

USP Authentic Visual References RSA _{▲(CN 1-Aug-2020)}

USP Construct Human Fibroblasts in Polyglactin Scaffold Reference Photomicrographs

[NOTE-These three photomicrographs represent examples of passing units. They are specified to assist in ascertaining histological quality.]

¹ A suitable Buffered formalin can be obtained from VWR International, 1310 Goshen Pkwy., West Chester, PA 19380.

² A suitable histological tissue cassette can be obtained from Sakura Finetek U.S.A., Inc., 1750 West 214th St., Torrance, CA 90501.

³ A suitable histological tissue cassette basket can be obtained from Sakura Finetek U.S.A., Inc., 1750 West 214th St., Torrance, CA 90501.

⁴ A suitable histological xylene substitute is Citrosolve® Clearing Agent, available from Fisher Scientific, 200 Park Ln., Pittsburgh, PA 15275.

⁵ A suitable paraffin for use is Tissue Prep* 2 Embedding Media, available from Fisher Scientific, 200 Park Ln., Pittsburgh, PA 15275.

⁶ A suitable histological adhesive for use is Histoslide® Adhesive, which can be obtained from Poly Scientific Research Corp., 70 Cleveland Ave., Bay Shore, NY 11706-1282.

⁷ A suitable bluing reagent can be obtained from Sigma-Aldrich Corp., P.O. Box 14508, St. Louis, MO 63178.

⁸ These photomicrographs are available as a CD from the USP Reference Standards collection, available to the user through USP Customer Services. To order these and other Reference Standards, call 1-800-227-8772 (U.S. and Canada), +1-301-881-0666 or 00-800-4875-5555 (select Europe); or go online to www.usp.org. Order item number 1535868.

⁹ A suitable Diaminobenzidine solution can be obtained from Sigma-Aldrich Corp., P.O. Box 14508, St. Louis, MO 63178; catalog number D-6815.

¹⁰ A suitable normal rabbit serum can be obtained from Dako Corp., 6392 Via Real, Carpinteria, CA 93013.

¹¹ Suitable rabbit anti-human fibronectin antibodies can be obtained from Dako Corp., 6392 Via Real, Carpinteria, CA 93013.

¹² Suitable antibody diluent can be obtained from Dako Corp., 6392 Via Real, Carpinteria, CA 93013.

¹³ Suitable biotinylated goat anti-rabbit antibody solution can be obtained from BioGenex, 4600 Norris Canyon Rd., San Ramon, CA 94583.

¹⁴ A suitable streptavidin conjugated horseradish peroxidase solution can be obtained from BioGenex, 4600 Norris Canyon Rd., San Ramon, CA 94583.

¹⁵ A suitable fetal bovine serum can be obtained from HyClone, 925 West 1800 South, Logan, UT 84321; catalog number SH30070.03.

¹⁶ A suitable Proteinase K solution can be obtained from Roche Diagnostics Corp., Roche Applied Sciences, P.O. Box 50414, 9115 Hague Rd., Indianapolis, IN 46250-0414.

¹⁷ A suitable polyglactin mesh can be obtained from Ethicon Co., Johnson & Johnson Corp., 425 Hoes Ln., P.O. Box 6800, Piscataway, NJ 08855.