Surface Characterization of the Extracellular Matrix Remaining after Cell Detachment from a Thermoresponsive Polymer

Abstract

The temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) directly affects the attachment and detachment of cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS), while at room temperature, cells cultured on pNIPAM spontaneously detach as a confluent sheet. In comparison, cells grown on TCPS remain attached indefinitely after the temperature drop, requiring enzymatic or mechanical removal. In this work, we present an examination of the response of bovine aortic endothelial cells (BAECs) and extracellular matrix (ECM) proteins to plasma polymerized NIPAM (ppNIPAM) surfaces using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and immunostaining. Immunoassay results reveal that, although fibronectin, laminin, and collagen closely associate with the cell sheet, some collagen may be associated with the surface, as well. Our XPS results indicate that ppNIPAM surfaces after cell liftoff differ from their blank counterparts, the primary distinction being the presence of amide and alcohol species on ppNIPAM surfaces used for cell culture, possibly owing to the presence of a proteinaceous film. Finally, a comparison between ppNIPAM-treated surfaces used for cell culture versus control surfaces by principal component analysis of the ToF-SIMS data confirms that the surfaces differ; the presence of molecular ion fragments from amino acids (e.g., alanine, glycine, and proline) is the chief reason for this difference. Therefore, from our surface characterization of ppNIPAM-coated TCPS after cell liftoff, we conclude that although low-temperature liftoff of the BAEC monolayer is accompanied by the majority of the components of the ECM, some of the ECM proteins still remain at the surface.

Plasma-Generated Poly(allyl alcohol) Antifouling Coatings for Cellular Attachment

“…These assignments are summarized in Table 3. Bacterial adhesion to surfaces, and subsequent biofilm growth, is facilitated by biospecific (protein-protein, carbohydrate-protein) and non-specific (hydrophobic or electrostatic) surface interactions of pili and flagella [29,39].…”

“…and isopropylacrylamide [29], as well as co-polymers of allyl alcohol [30], acrylic acid [Error! Bookmark not defined.]…”

Surface Chemical and Mechanical Properties of Plasma-Polymerized N-Isopropylacrylamide

“…15,16 When immobilized onto a flat substrate, the LCST behavior of pNIPAM leads to a temperature controlled wettability change on the surface and has been used for control of protein adsorption 17,18 and cell adhesion. [19][20][21][22] The recent work by Okano and colleagues to recover cell sheets from pNIPAM-grafted surfaces 23,24 brings exciting applications of the grafted polymer to 2-D and 3-D tissue engineering.…”

PNIPAM grafted surfaces through ATRP and RAFT polymerization: Chemistry and bioadhesion

“…Various cells adhere to heated PNIPAM surfaces and are released during cooling [122]. Typically, cell detachment is achieved at T = 20 • C [118].…”