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Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture.

ABSTRACT: Poly(N-isopropyl acrylamide) (PNIPAAm) is a well-known 'smart' material responding to external stimuli such as temperature. PNIPAAm was successfully conjugated to polycaprolactone (PCL) bead surfaces through amidation reaction. Functionalization steps were characterized and confirmed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and Energy Dispersion Spectroscopy. PNIPAAm-conjugated PCL allowed human dermal fibroblast cells (HDF) and mesenchymal stem cells (MSC) to adhere, spread, and grow successfully. By reducing the temperature to 30?°C, more than 70% of HDF were detached from PNIPAAm-conjugated PCL macrocarriers with 85% viability. The cell detachment ratio by trypsin treatment was slightly higher than that induced by reduced temperature, however, cell detachment from PNIPAAm-conjugated macrocarriers by lowering the temperature significantly reduced cell death and increased both cell viability and the recovery potential of the detached cells. HDF attachment and detachment were also observed by Live-Dead staining and phase contrast imaging. The expression of extracellular matrix proteins such as Laminin and Fibronectin was also affected by the trypsinization process but not by the reduced temperature process. Taken together, our results showed that thermo-responsive macrocarriers could be a promising alternative method for the non-invasive detachment of cells, in particular for tissue engineering, clinical applications and the use of bioreactors.

SUBMITTER: Nguyen LTB

PROVIDER: S-EPMC6401373 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture.

Nguyen Linh T B LTB Nguyen Linh T B LTB Odeleye Akinlolu O O AOO Chui Chih-Yao CY Baudequin Timothée T Cui Zhanfeng Z Ye Hua H

Scientific reports 20190305 1

Poly(N-isopropyl acrylamide) (PNIPAAm) is a well-known 'smart' material responding to external stimuli such as temperature. PNIPAAm was successfully conjugated to polycaprolactone (PCL) bead surfaces through amidation reaction. Functionalization steps were characterized and confirmed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and Energy Dispersion Spectroscopy. PNIPAAm-conjugated PCL allowed human dermal fibroblast cells (HDF) and m ...[more]

PMID: 30837639

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Project description:Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte-derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte-derived macrophages. In summary, we observed similar functionality and viability of primary monocyte-derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.

Project description:Thermoresponsive poly((N,N-dimethyl acrylamide)-co-(N-isopropyl acrylamide)) (P(DMA-co-NIPAM)) copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The monomer reactivity ratios were determined by the Kelen-Tüdős method to be rNIPAM = 0.83 and rDMA = 1.10. The thermoresponsive properties of these copo-lymers with varying molecular weights were characterized by visual turbidimetry and dynamic light scattering (DLS). The copolymers showed a lower critical solution temperature (LCST) in water with a dependence on the molar fraction of DMA in the copolymer. Chaotropic and kosmotropic salt anions of the Hofmeister series, known to affect the LCST of thermoresponsive polymers, were used as additives in the aqueous copolymer solutions and their influence on the LCST was demonstrated. Further on, in order to investigate the thermoresponsive behavior of P(DMA-co-NIPAM) in a confined state, P(DMA-co-NIPAM)-b-PS diblock copolymers were prepared via polymerization induced self-assembly (PISA) through surfactant-free RAFT mediated emulsion polymerization of styrene using P(DMA-co-NIPAM) as the macromolecular chain transfer agent (mCTA) of the polymerization. As confirmed by cryogenic transmission electron microscopy (cryoTEM), this approach yielded stabilized spherical micelles in aqueous dispersions where the PS block formed the hydrophobic core and the P(DMA-co-NIPAM) block formed the hydrophilic corona of the spherical micelle. The temperature-dependent behavior of the LCST-type diblock copolymers was further studied by examining the collapse of the P(DMA-co-NIPAM) minor block of the P(DMA-co-NIPAM)-b-PS diblock copolymers as a function of temperature in aqueous solution. The nanospheres were found to be thermosensitive by changing their hydrodynamic radii almost linearly as a function of temperature between 25 °C and 45 °C. The addition of kosmotropic salt anions, as a potentially useful tuning feature of micellar assemblies, was found to increase the hydrodynamic radius of the micelles and resulted in a faster collapse of the micelle corona upon heating.

Dataset Information

Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture.

Publications

Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture.

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