Guidelines for construction of collagen I (1.5 mg/mL) based dermal layer.
|Constituents||Calculated vol. of each constituent (µL)
||Calculated vol. of each constituent (µL)
|Total vol. of acellular layer = 200 µL per insert||Total vol. of cellular layer = 400 µL per insert|
|10× M199 (Sigma)||20 µL (10% of final vol. of 200 µL results in 1× M199)||40 µL (10% of final vol. of 400 µL results in 1× M199)|
|200 mM Glutamine (Biosciences)||0.68 µL||1.36 µL|
|Formula: M1·V1 = M2·V2 (200 mM × V1 = 0.68 × 200 µL) (This 0.68 µL vol. results in 0.68 mM Glutamine in final 200 µL vol. of acellular layer)||Formula: M1·V1 = M2·V2 (200 mM × V1 = 0.68 × 400 µL) (This 1.36 µL vol. results in 0.68 mM Glutamine in final 400 µL vol. of cellular layer)|
|ddH2O||113.32 µL (vol. calculated in the end)||176.64 µL (vol. calculated in the end)|
|7.5% of NaHCO3 (Sigma)||6 µL (This volume is 10% of the volume of collagen I, that results in a final mixture pH of 7.2–7.4 – optimized)||12 µL (This is an optimized volume as 10% of the volume of collagen I, that results in a final mixture pH of 7.2–7.4)|
|5 mg/mL Collagen I (Ibidi)||60 µL (This volume makes final conc. of 1.5 mg/mL in final volume)||120 µL (This volume makes final conc. of 1.5 mg/mL in final volume)|
|NHDF (PromoCell)||N/A||50 µL (this volume should contain 5 × 104 NHDF per insert)|
|Total vol.||200 µL||400 µL|
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