N sheet was prepared, as described earlier [16]. In brief, 1.0 ml of

N sheet was prepared, as described earlier [16]. In brief, 1.0 ml of fibrin composite, as described earlier, was mixed with 1.0 ml (5 IU) thrombin to 3-Bromo-5-chloro-2-fluoroaniline make a clot in a culture well (about 3.3 cm diameter) and was lyophilized to form a stable sheet. The PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 fibrin sheet was suspended in the medium for 5 to 10 minutes before using for cell culture.Isolation of peripheral blood mononuclear cellsincubated for 1 hour in a humidified incubator under 5 CO2 at 37 . After 1 hour, the medium was removed gently, and fresh medium was added. After 48 hours, the medium was removed, and nonadherent but settled cells were flushed with complete keratinocyte culture medium, and the cells attached to the bare polystyrene surface were discarded. The plastic-nonadherent cells were seeded onto matrix-coated dishes and were incubated in a humidified incubator under 5 CO2 at 37 . The medium was replaced with fresh medium each day until 72 hours; afterward, the medium was changed on alternate days. The morphology of cultured cells was observed with a phasecontrast microscope (Leica Microsystems, DMIRB Wetzlar, Germany). Fibroblasts were isolated from human foreskin from a local hospital (collected with informed consent) by using a standard protocol [17]. Cells were subcultured, and either the third- or the fourth-passage monolayer was used for seeding KPCs. The protocol for co-culture was to seed PBMNCs in an uncoated dish for 48 hours, transfer to a coated dish, and after 4 days on biomimetic matrix, KPCs were flushed out and seeded onto either a fibroblast monolayer or a fibrin sheet. Culture was continued in the same medium composition as described earlier.Immunostaining for fluorescence microscopyIsolation of PBMNCs from the buffy coat (discards from Blood Bank collected with informed consent) was done with Histopaque-1077 density gradient centrifugation. As per the standard operating procedure s12998-016-0127-6 of Institutional Ethics Committee, Sree Chitra Tirunal Institute for Medical Sciences and Technology (IEC-SCTIMST), the collection PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 of discarded buffy coat is exempted from review if the donor identity is not publicized. In brief, red blood cells (RBCs) were settled by centrifugation of the buffy coat at 1,200 g for 15 minutes in 15-ml centrifuge tubes (Hareus Stratos, Hanau, Germany). Superficial plasma was discarded; PBMNCs at the interface were collected and diluted with equal volumes of Hank Balanced Salt Solution (HBSS), layered over Histopaque-1077 in centrifuge tubes (Nunc, Roskilder, Denmark), and centrifuged at 400 g for 30 minutes at 25 . The layer containing PBMNCs was carefully separated from the plasma-Histopaque interface and was washed with serum-free DMEM: F12 by centrifugation at 150 g at 4 for 10 minutes. The washed PBMNCs were suspended in complete medium.Cell cultureCells cultured on FC-coated 1.75-cm2 wells for a specific period of time were rinsed with phosphate-buffered saline (PBS), fixed with 3.7 formaldehyde in PBS, washed with PBS, and permeated with Triton X-100 (0.1 ) in PBS. Cells tert-Butyl (7-bromoheptyl)carbamate were then incubated with primary antibodies against p63 (1:50), FITC-conjugated cytokeratin 14 antibody (1:50), or with phycoerythrin (PE)-conjugated cytokeratin 5 (1:50), antiinvolucrin (1:100), and antifilaggrin (1:100). Conjugated secondary antibody was used at 1:200 dilution, and cultures were viewed by using a fluorescence microscope (Leica Microsystems, DMIRB, Wetzlar, Germany). Actin staining was done by using phalloidin conjugated with Texas Red (Molecula.