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Please You look at the following articles:

Article Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fib...

Maintenance of cell lines

Human dermal fibroblast cells were obtained from PromoCells (Heidelberg, Germany) and established cell line human keratinocyte (HaCaT) cells were obtained from animal cell culture repository National Centre for Cell Science (NCCS), Pune, India. Fibroblasts and keratinocytes were cultured in complete DMEM and in DMEM: DMEM F12 (3:1 ratio) medium respectively supplemented with 10% fetal bovine serum (FBS), 200 mm l-glutamine, 2 mg/ml sodium bicarbonate, and 100 µg/ml penicillin/streptomycin. The cells were cultured in tissue culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Once the cells reached 80–90% confluent state, they were split using 0.05% trypsin/EDTA solution. The medium was replaced after every three days.

Co-culture on fabricated matrices

Human dermal fibroblast cells and human keratinocyte (HaCaT) cells were cultured in media and trypsinised when reached 80–90% confluent state (Figure 1B (a and b)). Fibroblasts-keratinocytes co-culture was done on sericin constructs by seeding 1 × 105/matrice (6 mm x 2mm) fibroblasts on sericin matrices pre-soaked with DMEM medium at day 0. On day 2, keratinocytes (1 × 106) were directly seeded onto the surface of sericin matrices containing fibroblasts to create co-culture model. The construct were cultured further for 7 days in DMEM: DMEM F12 (3:1) medium supplemented with 10% FBS. After 7 days the co-culture matrices were raised to the air-liquid (A/L) interface to allow stratification of the keratinocytes on the matrices. All co-culture constructs were maintained at 37 °C with 5% CO2 and 100% relative humidity. Similarly fibroblasts-keratinocytes co-culture was done on chitosan matrices as control. Schematic representation of matrices preparation and fibroblasts-keratinocytes co-culture on construct is shown in Figure 1A.

Immunohistochemistry of matrice sections

After 7, 14 and 21 days of cultures at the air–liquid interface, matrices were fixed and embedded in paraffin. Sections of 10 µm of the matrices were, incubated for 12 h at 4°C with mouse anti-bovine type IV collagen IgG (Col IV), mouse monoclonal anti-involucrin and mouse monoclonal anti fibroblast surface protein IgG (Sigma-Aldrich), (diluted 1:100) and washed with PBS (pH 7.4) (3 times, each for 5 min). Subsequently, the sections were incubated for 30 min at 37 °C with HRP conjugated secondary antibodies followed by the chromogen detection using DAB as substrate.

Article Design and Fabrication of Human Skin by 3D Bioprinting.

Cell culture and hydrogel preparation

Dear Shyam

I found an article that might be of interest to you

Choi HR, Kim SK, Kwon SB, Park KC. The fixation of living skin equivalents. Appl Immunohistochem Mol Morphol. 2006 Mar;14(1):122-5. doi: 10.1097/01.pai.0000142159.75540.e0. PMID: 16540743.

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