Adipocytes Constitutively Release Factors That Accelerate Keratinocyte Proliferation In Vitro

Keratinocytes grown directly on adipose tissue have greater proliferation rates than keratinocytes grown alone. It is unknown if factors released by adipose tissue into culture media could increase keratinocyte proliferation without requiring incorporation of adipose tissue into skin graft models, or serve as a substitute for the fibroblast feeder layer. Human keratinocytes were grown with and without NIH 3T3 fibroblast feeder layer in the following conditions (12 cultures per group) adipose tissue coculture (AT), cultures supplemented with medium from whole adipose tissue referred to as adipose-conditioned medium (ACM), and control. Proliferation was measured with a colorimetric proliferation assay and digital calculations of percent confluence over time. Culture morphology was assessed by light microscopy. ACM cultures without 3T3s, AT cultures with and without 3T3s, and 3T3 control cultures demonstrated a similarly significant keratinocyte proliferation increase over non-3T3 control (P < 0.05) corresponding with a 2-fold increase in percent confluence by day 7. ACM cultures with 3T3s proliferated significantly faster than all other treatment groups ( P < 0.05) resulting in complete confluence by day 5. ACM cultures with and without 3T3s produced a thick keratinized layer by day 7 whereas all other cultures including AT cultures did not. Engineered tissue replacement can be accelerated and simplified by ACM without requiring the addition of adipose tissue or a fibroblast feeder layer to keratinocyte culture systems. ACM supplementation provides an additive proliferation benefit when combined with a feeder layer producing mature grafts in approximately half the time as keratinocytes alone by accelerating proliferation and increasing keratinization.