RESUMO
Natural polymeric hydrogels represent an optimal framework for 3D culture development. This study demonstrates a freeze-thaw-based ionic crosslinking technique for fabricating alginate/carboxymethylcellulose scaffold for culturing human hepatocellular carcinoma, Huh-7 cells to generate 3D spheroids. Consolidating morphological and biomechanical characterization of Alg/CMC scaffolds shows the formation of uniform hydrogels with significant crosslinking (ATR-FTIR), multiscale pores (FE-SEM), swelling/water absorbance, softer texture, viscoelasticity (rheology), spreading nature (contact angle), and degradation rate optimal for 3D culture establishment. The influence of cell seeding density and time with spheroid formation reveals a maximal size of 250-300 µm on day 7. Calcein AM and Propidium iodide staining confirm that a culmination of viable and dead cells generates spheroidal heterogeneity. RT-qPCR in 3D culture against RPL-13 and 2D culture controls indicate an upregulation of E-cadherin, N-cadherin, fibronectin, and integrin α9/ß6. Further, western blotting and immunofluorescence confirm the collective display of cellular interactions in 3D spheroids. Thus, the expression profile signifies the role of key genes during the assembly and formation of 3D spheroids in 1%Alg/1%CMC scaffolds with a profound epithelial characteristic. In the future, this study will bring a 3D spheroid model in a platter for elucidating epithelial to mesenchymal transition of cells during in vitro disease modeling.
Assuntos
Alginatos , Neoplasias Hepáticas , Humanos , Esferoides Celulares , Transição Epitelial-Mesenquimal , Carboximetilcelulose Sódica , Hidrogéis , Engenharia Tecidual/métodosRESUMO
This article describes the changes in the microstructure, cooling curve characteristics and mechanical properties of cast Nickel Aluminium Bronze alloy (NAB) alloy that were produced by the addition of various amounts of Tin (Sn). The solidification parameters were recorded using a computer-aided cooling curve analysis setup, and optical and scanning electron microscopes were utilised to study the evolution of the microstructure. The chemical composition of different phases generated in the NAB alloy with and without Tin was investigated using an X-ray diffraction technique. With the addition of tin, the alloy's microstructure changed from columnar to equiaxed grain structures, and the ideal microstructure was produced at a Tin concentration of roughly 1.0 weight percent. The formation of the high temperature α and the grain boundary Sn rich phases across the alloy microstructure as a result of further addition has a considerable impact on the alloy's increased hardness (upto 69%) and tensile strength (upto 28.4%) compared to untreated NAB alloy. Influence of Sn on microstructure transformation is confirmed by the decline in alloy nucleation temperatures, the reduction in undercooling intensity, and the decrease in cooling rate during solidification. The addition of Tin to the NAB alloy caused morphological changes in the kappa (K) phases, which are also reported in the this article. In addition to this, the research makes an attempt to describe the mechanism underlying the formation of equiaxed grains and phase transformations in Sn-treated NAB alloys.