Adhesion of Epithelial Cells to PNIPAm Treated Surfaces for Temperature-Controlled Cell-Sheet Harvesting.

Hyejeong Kim,Marco Tarantola,Hannes Witt,Tabea A Oswald

doi:10.1021/acsami.0c09166

Abstract

Stimuli responsive polymer coatings are a common motive for designing surfaces for cell biological applications. In the present study, we have characterized temperature dependent adhesive properties of poly(N-isopropylacrylamide) (PNIPAm) microgel coated surfaces (PMS) using various atomic force microscopy based approaches. We imaged and quantified the material properties of PMS upon a temperature switch using quantitative AFM imaging but also employed single-cell force spectroscopy (SCFS) before and after decreasing the temperature to assess the forces and work of initial adhesion between cells and PMS. We performed a detailed analysis of steps in the force–distance curves. Finally, we applied colloid probe atomic force microscopy (CP-AFM) to analyze the adhesive properties of two major components of the extracellular matrix to PMS under temperature control, namely collagen I and fibronectin. In combination with confocal imaging, we could show that these two ECM components differ in their detachment properties from PNIPAm microgel films upon cell harvesting, and thus gained a deeper understanding of cell-sheet maturation and harvesting process and the involved partial ECM dissolution.

Highlights

The generation of cell sheets is a milestone in a broad range of biomedical fields, from cell sorting, analysis, confinement, crowding, and differentiation to regenerative tissue engineering of organs, e.g. heart, cornea, skin, or bones.[1−4] Cells are cultured in customized 2D/3D scaffold to suit their needs and transferred to specific wounds or disease sites.[5−7] Often cell adhesion on standard cell culture surfaces is so strong that cell detachment requires a chemical or physical treatment with potentially harmful enzymes or mechanical force.[8]
Since the mechanical properties of the cell culture surface have a huge influence on the initial cell adhesion and cell-sheet detachment from the surface, we quantitatively characterized the mechanical properties of the PNIPAm microgel coated surfaces (PMS) surface upon temperature variation
Contact angle measurements, which relate to the wettability of the surface, show that the PMS at 45 °C yields a higher degree of hydrophobicity, as indicated by the increased static water contact angle (SCA), while the wettability of the gold surface does not change significantly with temperature (Figure 1F, S4)

Summary

Introduction

The generation of cell sheets is a milestone in a broad range of biomedical fields, from cell sorting, analysis, confinement, crowding, and differentiation to regenerative tissue engineering of organs, e.g. heart, cornea, skin, or bones.[1−4] Cells are cultured in customized 2D/3D scaffold to suit their needs and transferred to specific wounds or disease sites.[5−7] Often cell adhesion on standard cell culture surfaces is so strong that cell detachment requires a chemical or physical treatment with potentially harmful enzymes or mechanical force.[8] A more gentle approach is controllable cell adhesion on designed substrates in the more unique way of cell-sheet engineering. It has been successfully applied to produce many types of cell sheets, for example endothelial cells, cardiomyocytes, or keratinocytes.[9,20−23]

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Results

Discussion

Conclusion