RESUMEN
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.
Asunto(s)
Resinas Acrílicas , Técnicas de Cultivo de Célula , Poliésteres , Temperatura , Resinas Acrílicas/química , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Matriz Extracelular , Adhesiones Focales , Humanos , Células Madre Mesenquimatosas , Poliésteres/química , Análisis Espectral , Propiedades de SuperficieRESUMEN
Alginate microbeads are extensively used in tissue engineering as microcarriers and cell encapsulation vessels. In this study, we used atomic force microscopy (AFM) based indentation using 20⯵m colloidal probes to assess the local reduced elastic modulus (Eâ¯*â¯) using a novel method to detect the contact point based on the principle of virtual work, to measure microbead mechanical stability under cell culture conditions for 2 weeks. The bead diameter and swelling were assessed in parallel. Alginate beads swelled up to 150% of their original diameter following addition of cell culture media. The diameter eventually stabilized from day 2 onwards. This behaviour was mirrored in Eâ¯*â¯where a significant decrease was observed at the start of the culture period before stabilization was observed at ~ 2.1â¯kPa. Furthermore, the mechanical properties of freeze dried alginate beads after re-swelling them in culture media were measured. These beads displayed vastly different structural and mechanical properties compared those that did not go through the freeze drying process, with around 125% swelling and a significantly higher Eâ¯*â¯at values over 3â¯kPa.
Asunto(s)
Alginatos/química , Técnicas de Cultivo de Célula , Ensayo de Materiales , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Microesferas , Módulo de ElasticidadRESUMEN
Mesenchymal stem cells (MSCs) are potential therapeutic candidates, owing to their ability to differentiate into several cell types. However, the gap between availability and demand of MSCs requires alternative expansion methods from 2D culture flasks. Microcarriers are a promising approach for MSC expansion due to their high surface area-to-volume ratio. However, current commercial microcarriers do not provide the highest cell yield due to low cell attachment efficiencies and difficulty in cell detachment. This study developed a hydrogel-based microcarrier from genipin cross-linked alginate-chitosan beads. Alginate beads were produced by electrospraying before being coated with chitosan and cross-linked in genipin. The degree of cross-linking was determined through fluorescence reading of the genipin-chitosan conjugates. MSCs cultured on these microcarriers had a 26% higher cell attachment and twice the proliferation rate compared to the commercial microcarrier Cytodex 1. Cells easily detached from the hydrogel beads and did not require extended incubation periods or intense agitation during cell harvest. There was no significant difference in gene expression between the two microcarriers for the positive MSC surface markers CD-90, CD-105, and CD-73 as well as showing either low or no signal for negative MSC surface markers CD-45 and CD-34. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 122-133, 2019.
Asunto(s)
Alginatos , Técnicas de Cultivo de Célula , Quitosano , Iridoides , Células Madre Mesenquimatosas/metabolismo , Microesferas , Alginatos/química , Alginatos/farmacología , Línea Celular Transformada , Quitosano/química , Quitosano/farmacología , Humanos , Iridoides/química , Iridoides/farmacología , Células Madre Mesenquimatosas/citologíaRESUMEN
OBJECTIVE: To demonstrate that uniform poly(vinyl butyral) (PVB) fibres can be safely electrospun onto a monolayer of human dermal fibroblasts using a portable device. RESULTS: PVB in solvent mixtures containing various amounts of ethanol and water was electrospun. Six percent (weight-to-volume ratio) PVB in a 9:1 ethanol:water ratio was the solution with the highest content in water that could be electrospun into consistent fibres with an average diameter of 0.9 µm (± 0.1 µm). Four and five percent PVB solutions created beaded fibres. A 8:2 ethanol:water solution lead to microbead formation while a 7:3 ethanol:water mix failed to fully dissolve. The selected solution was successfully electrospun onto a monolayer of human dermal fibroblasts and the process had no significant effect (p < 0.05) on cell viability compared to the control without fibres. CONCLUSIONS: PVB-ethanol-water solutions could be electrospun without damaging the exposed cell layer. However, further work is required to demonstrate the long-term effect of PVB as a wound healing material.