Development_of_full_thickness_human_skin_equivalent.pdf

Development of Full Thickness Human Skin ® Model Using Alvetex Scaffold Technology Introduction: ® This protocol describes the ability and advantages of using Alvetex Scaffold technology to support dermal fibroblast growth within its structure, and the co-culture of primary human keratinocytes to form a terminally differentiated, cornified human skin equivalent. Method ® 1) 12-well Alvetex Scaffold inserts (AVP005) were used in 6-well plates (Figure 1). The inserts were washed twice with media. Please refer to website protocols for preparation of Alvetex®Scaffold for cell culture /alvetex/workflow. ® Figure 1 Alvetex Scaffold in 12-well insert format used in a 6-well plate. NB. Refer to Table 1 for all media formulations 2) Primary fibroblasts isolated from human foreskin (P5) were incubated on the insert for 1 week. 1 million fibroblasts were seeded per insert in 100 µl of culture medium 1 and incubated for 1 hr at 37 ˚C with 5 % CO . After 1 hr the insert was submerged in 2 culture medium 1 (10.5 ml) and the fibroblasts were cultured for 1 week changing culture medium 1 every other day. (Figure 2A). 3) After 1 week of fibroblast culture, culture medium 1 was removed and primary human keratinocytes (500,000 cells) isolated from foreskin (P4) were seeded on to the insert in 100 µl of culture medium 2 and incubated for 1hr at 37 ˚C with 5% CO2 . 4)