Generation of Human Induced Pluripotent Stem Cell-derived Planar Hair-bearing Skin Organoids Using an Air-Liquid Interface Culture System

The scope of my research is to try and develop a UVB-induced damage model so I can study the pathogenicity of a rare disease, xeroderma pigmentosum, or more precisely, XPA. One of the challenge of these in vitro model is that it takes quite a long time to culture, approximately four months, and since it's still a pretty young field, we still need to characterize them a bit better. The advantages of this model, so the hiPSC-derived 3D model, the culture that the air-liquid interface, recapitulate human skin architecture and has a skin appendage such as sebaceous gland and hair follicles.

To begin, prepare a one-molar solution by adding sodium hydroxide pellets to an empty 15-milliliter tube. Add distilled water to the pellets and vortex the tube to dissolve the pellets. Filter sterilize 0.1 molar sodium hydroxide solution using a 0.2-micrometer pore-size membrane filter.

On ice, dilute collagen type I solution with 10 times PBS, five-millimolar sodium hydroxide, and distilled water to adjust the final volume. Distribute 150 microliters of this collagen solution into each insert, placed in a standard 12-well plate. Incubate the plate at 37 degrees Celsius for 30 minutes to allow the collagen gel to polymerize.

Next, cut P1000 tips with a heated scalpel to create wide-bore tips. Then, use the wide orifice tips to place a selected cystic structure with a small amount of medium on the lid of a 10-centimeter Petri dish. Carefully excise any byproducts of the structure using a sterile scalpel, while stabilizing it with sterile forceps on the opposite side.

Excise approximately one millimeter from both ends of a cystic skin organoid adjacent to the first incision using a scalpel. Using sterile forceps, gently unfold the upper layer of skin. Cut the tissue into two to four pieces, depending on size.

Then, using sterile forceps, move each piece onto a collagen-coated insert with the epidermis facing up. Add 600 microliters of OMM to each well of a standard 12-well plate. Transfer the insert back to the 12-well plate containing OMM.

Then place the plate into a 37 degrees Celsius incubator with 5%carbon dioxide, A single, dense cellular aggregate incorporating the majority of the cells was formed by day zero through careful handling and centrifugation. Proper surface epiderm induction led to a thin, clear epithelium forming on the aggregate's outer layer by day three. By day 12, a thin layer of mesenchymal cells had accumulated at one pole of the cyst.

Hair placodes and pegs became visible between days 50 and 80, following epithelial and mesenchymal co-induction. Organoids lacking visible placodes displayed extremely thin epithelium and were excluded from planar transition. Only cysts at least five millimeters in diameter with polarized byproducts were selected for planar skin organoid generation.

Hair follicles elongated progressively throughout the 27 days of air-liquid interface culture. Sebaceous glands became visible around day 14 in the planar configuration. Pigmentation gradually increased in the organoids over time.

Hematoxylin and eosin staining revealed a stratified epithelium and a basement membrane separating it from the dermis. Immunofluorescence staining confirmed human skin-like architecture in planar skin organoids.