Maria E. Nash

Straightforward, One-Step Fabrication of Ultrathin Thermoresponsive Films from Commercially Available pNIPAm for Cell Culture and Recovery

The use of thermoresponsive surfaces as platforms for cell culture and cell regeneration has been explored over the last couple of decades. Poly-N-isopropylacrylamide (pNIPAm) is a well characterized thermoresponsive polymer which has an aqueous lower critical solution temperature (LCST) in a physiologically useful range, which allows it to reversibly attract (T < 32 °C) and repel water (T > 32 °C). It is this phenomenon that is exploited in temperature-controlled cell harvesting. pNIPAm coatings are generally poorly cell compatible and a number of complex or expensive techniques have been developed in order to overcome this issue. This study seeks to design a simple one-step system whereby commercially sourced pNIPAm is used to achieve similar results. Films were deposited using the operationally simple but rheologically complex spin coating technique. Reversible temperature modulated cell adhesion was achieved using a variety of different cell lines. This system offers a simplistic and cheaper alternative to methods used elsewhere.

Ultra-thin spin coated crosslinkable hydrogels for use in cell sheet recovery—synthesis, characterisation to application

The advantages of detaching adherent cells from thermoresponsive platforms over conventional cell detachment protocols has been well documented. This study focuses on the development of an alternative method to produce thermoresponsive surfaces for cell and cell sheet regeneration to already established techniques which are complex and expensive and may be inaccessible to many laboratories. A photcrosslinkable poly-N-isopropylacrylamide (pNIPAm) copolymer was synthesised and thin films of the copolymer were deposited using the operationally simple spin coating technique which were subsequently crosslinked upon exposure to ultraviolet (UV) irradiation. Characterisation of hydrogel properties and behaviour was achieved using UV spectroscopy, atomic force microscopy (AFM), advancing contact angle, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR) and white light interferometry analyses. Results indicate that 3T3 fibroblast cells adhere and proliferate to confluence on hydrogels of 30 nm was comparatively poor. Confluent cell sheets were harvested from the sub 30 nm ultra-thin hydrogels upon temperature reduction within 10 min. Spin coating allows for the facile control of film thickness via variation of the depositing polymer solution concentration and therefore the routine fabrication of ultra-thin hydrogels capable of hosting cells to confluence is easily achievable.

Cell and cell sheet recovery from pNIPAm coatings; motivation and history to present day approaches

The regeneration of cells and cell sheets mediated by thermoresponsive substrates represents an important and ever growing area in tissue engineering. This review seeks to track the development of this field from inception to the present day by highlighting the most significant breakthroughs as well as focusing on important physical and chemical characterization of substrates produced for this specific purpose. Furthermore, a critical evaluation encompassing the advantages and disadvantages of different techniques used for producing such surfaces will be included as well as suggestions for possible future directions.

Synthesis and characterization of a novel thermoresponsive copolymer series and their application in cell and cell sheet regeneration

A series of poly-(N-isopropyl acrylamide)-based copolymers were developed with a view to biomedical applications, specifically cell cultivation and recovery. Ethylpyrrolidone methacrylate (EPM), the monomer of poly-(ethylpyrrolidone methacrylate) (pEPM), which is itself thermoresponsive, was copolymerized with N-isopropylacrylamide in varying ratios to create this novel thermoresponsive copolymer series. Characterization indicated a moderate increase of copolymer lower critical solution temperature with increasing EPM content. Films of the copolymers successfully hosted cells to monolayer. Cells detached from the copolymers upon temperature reduction with cell to cell junctions maintained, avoiding the damage which can be caused using conventional detachment techniques. These results indicate that these copolymers are highly cell compatible and may be useful for a range of biomedical applications.

Pseudo-double network hydrogels with unique properties as supports for cell manipulation

Pseudo-double network hydrogels based on vinylpyrrolidone and anionic methacrylic units were prepared, for the first-time, via a simple one step radical polymerization procedure using thermal or photoinitiation. These networks showed improved mechanical properties, in the hydrated state, compared with their single network cousins and were capable of hosting cells to confluence. Rapid cell detachment can be induced through simple mechanical agitation and the cell sheets can be transplanted easily without the need for a cell superstrate. The results reported in this work suggest that these hydrogels could be used as support systems for cell manipulation and are candidates to compete with the conventionally used thermoresponsive cell platforms based on poly-N-isopropylacrylamide (pNIPAm).

Thermoresponsive Substrates Used for the Growth and Controlled Differentiation of Human Mesenchymal Stem Cells.

This communication outlines the advances made in the development of thermoresponsive substrates for human mesenchymal stem cell (hMSC) expansion and subsequent controlled specific and multilineage differentiation from a previous study performed by this group. Previously, the development of an inexpensive and technically accessible method for hMSC expansion and harvesting was reported, using the solvent casting deposition method and thermoresponsive poly(N-isopropylacrylamide). Here, the logical continuation of this work is reported with the multipassage expansion of hMSCs with phenotypic maintenance followed by induced adipogenic, osteogenic, and chondrogenic differentiation. Interestingly, 1 μm thick solvent cast films are not only capable of hosting an expanding population of phenotypically preserved hMSCs similar to tissue culture plastic controls, but also the cells detached via temperature control better maintain their ability to differentiate compared to conventionally trypsinized cells. This approach to hMSC expansion and differentiation can be highly attractive to stem cell researchers where clinical therapies have seen a collective deviation away from the employment of animal derived products such as proteolytic trypsin.

Nanometer-scale physically adsorbed thermoresponsive films for cell culture

ABSTRACT Physical adsorption was used to produce nanometer thick thermoresponsive films with a view to nonenzymatic cell detachment. Two polymers were investigated, poly-(N-isopropylacrylamide) and poly (N-isopropylacrylamide-co-N-tertbutylacrylamide). Substrates were prepared above and below the polymers’ LCST to investigate the effect of polymer conformation on the prepared substrates. Endothelial cells were seeded on the prepared films; cell proliferation was higher on the films produced below the polymers’ LCST than on those prepared above and cells detached from the surfaces upon temperature reduction. Physical adsorption of poly-(N-isopropylacrylamide)–based films is a viable approach to produce substrates compliant with cell growth and temperature modulated detachment. GRAPHICAL ABSTRACT

An investigation of cell growth and detachment from thermoresponsive physically crosslinked networks

The primary aim of this investigation was to determine the biocompatibility and cell culture potential of a newly designed class of thermoresponsive polymers. The attractiveness of these polymers lies in the fact that they swell rather than dissolve when the temperature is reduced below their respective lower critical solution temperature, due to the incorporation of octadecyl methacrylate (ODMA). The ODMA monomer acts as a physical crosslinker, preventing polymer dissolution upon temperature reduction. Two polymers were studied in this investigation poly(N isorpoylacrylamide (NIPAm)(99.25%)-co-ODMA(0.75%)) and poly(NIPAm(65%)-co-N-tert-butylacrylamide (NtBAm)(34.25%)-co-ODMA(0.75%)). Thin thermoresponsive films of the polymers were prepared via spin coating. 3T3 cells were then seeded on the prepared films and cell viability was assessed quantitatively through cell viability and activity assays and qualitatively by light microscopy. Cells were successfully seeded and grown on the poly(NIPAm-co-ODMA) and poly(NIPAm-co-NtBAm-co-ODMA) copolymer films after film modification with cell adhesion promoters (CAPs). Cell sheets successfully detached from the CAP coated poly(NIPAm-co-ODMA) platforms upon temperature reduction.