Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes.

Marek Kawecki,Aleksander L Sieroń,Alicja Utrata-Wesołek,Diana Kitala,Wojciech Łabuś,Dawid Szweda,Mariusz Nowak,Agnieszka Klama-Baryła,Barbara Trzebicka,Małgorzata Kraut,Justyna Glik,Andrzej Dworak

doi:10.1007/s10856-016-5718-1

Abstract

In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell™ dishes (NUNC™) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P® and SUPRATHEL®, were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.

Highlights

The outer layer of the skin, the epidermis, is composed mostly of epithelial cells, pigment cells, cells responsible for immune reactions (Langerhans cells) and nervous system cells (Merkel’s cells), whereas fibroblasts are connective tissue cells that inhabit the dermis
Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCellTM dishes (NUNCTM) coated with thermoresponsive poly(Nisopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE))
Cell culture and detachment were performed on the thermoresponsive PNIPAM (UpCell) and P(TEGMA-EE) surfaces (Fig. 2)

Summary

Introduction

The outer layer of the skin, the epidermis, is composed mostly of epithelial cells (keratinocytes), pigment cells (melanocytes), cells responsible for immune reactions (Langerhans cells) and nervous system cells (Merkel’s cells), whereas fibroblasts are connective tissue cells that inhabit the dermis. Skin cells can proliferate ex vivo in cell culture under appropriate conditions. A homogeneous sheet of cells connected by extracellular matrix (ECM) can be obtained. The skin cells must be separated from the support [5]. There are two basic methods that are used for cell separation, mechanical and enzymatic separation. Mechanical separation is based on cell scraping with special scrapers. Cell separation can be performed with the use of proteases (e.g., dispase). This method is commonly used and is less invasive. Proteases cause the enzymatic degradation of the ECM, which leads to cell separation [6]. The layer of cells is disintegrated when full confluence has not been reached or

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