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

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

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.

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Authors and Affiliations

  1. Struttura Semplice Tissue Therapy, Niguarda Ca’ Granda Hospital, Milan, Italy

    Marta Cecilia Tosca, Marta Galuzzi, Barbara Antonioli & Mario Marazzi

  2. Department of Drug Sciences, University of Pavia, Pavia, Italy

    Theodora Chlapanidas, Marta Galuzzi, Sara Perteghella, Barbara Vigani & Maria Luisa Torre

  3. Department of Pediatric Hematology/Oncology, Immunology and Transplantation Laboratory, Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy

    Melissa Mantelli & Maria Antonietta Avanzini

  4. Department of Surgery, Experimental Surgery, Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy

    Daniela Ingo

  5. Department of Veterinary Science and Public Health, University of Milan, Milan, Italy

    Daniele Vigo & Massimo Faustini

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  1. Marta Cecilia Tosca
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  2. Theodora Chlapanidas
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Correspondence to Theodora Chlapanidas.

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Tosca, M.C., Chlapanidas, T., Galuzzi, M. et al. Human adipose-derived stromal cells as a feeder layer to improve keratinocyte expansion for clinical applications. Tissue Eng Regen Med 12, 249–258 (2015). https://doi.org/10.1007/s13770-015-0007-5

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  • DOI: https://doi.org/10.1007/s13770-015-0007-5

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