Human adipose-derived stromal cells as a feeder layer to improve keratinocyte expansion for clinical applications

Marta Cecilia Tosca, Theodora Chlapanidas, Marta Galuzzi, Barbara Antonioli, Sara Perteghella, Barbara Vigani, Melissa Mantelli, Daniela Ingo, Maria Antonietta Avanzini, Daniele Vigo, Massimo Faustini, Maria Luisa Torre, Mario Marazzi

Research output: Contribution to journalArticlepeer-review


The aim of this work is to propose a keratinocytes (KC) culture method for clinical practice with irradiated adipose-derived mesenchymal stromal cells (ASCs) as human feeder layer, avoiding murine immortalized fibroblasts, commonly request for producing skin substitutes. ASCs were isolated, expanded, irradiated, and co-cultured with autologous or allogeneic KC. All experiments were performed using murine fibroblasts as control. Cell counts, flow cytometric analysis and ELISA were carried out, in order to define cell yield, viability and cytokine secretion. Results indicate that the optimal X-ray dose for ASCs is 120 Gy and the optimal seeding density is 625 cells/cm2; moreover, flow cytometric analysis shows that the percentage of feeder layer cells reaches values lower than 1%, within 8 days of co-culture. KC reach confluence in 6.9 days on ASCs substrate and, after confluence, the number of live cells increases again in a multilayered structure. Moreover, results show higher levels of interleukin (IL)-1a in co-culture with ASCs compared with 3T3, while no differences were observed for IL-6 and IL-8. Therefore, human ASCs enable to obtain effectively in vitro expanded KC and represent a viable alternative to murine fibroblasts for the production of clinical use skin substitutes.

Original languageEnglish
Pages (from-to)249-258
Number of pages10
JournalTissue Engineering and Regenerative Medicine
Issue number4
Publication statusPublished - Aug 18 2015


  • Adipose stromal cells
  • Feeder cell layers
  • Keratinocytes

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Biomedical Engineering


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