Abstract
A gelatin@non-woven fabric (gelatin@NWF) hybrid scaffold with tailored micropore structures was fabricated by lyophilizing, using gelatin to support cells and the NWF matrix as a frame to enforce the mechanical stability of gelatin. By freezing the gelatin and NWF hybrid in liquid nitrogen and subsequently lyophilizing and crosslinking the process, the gelatin@NWF scaffold was prepared to support cell growth and promote cell aggregation and spheroids’ formation. The results indicated that by tuning the lyophilizing temperature, the micropore size on the gelatin could be tailored. Consequently, tumor spheroids can be formed on gelatin@NWF scaffolds with honeycomb-like pores around 10 µm. The cell spheroids formed on the tailored gelatin@NWF scaffold were characterized in cancer stem cell (CSC)-associated gene expression, chemotherapy drug sensitivity, and motility. It was found that the expression of the CSC-associated biomarkers SOX2, OCT4, and ALDH1A1 in gene and protein levels in DU 145 cell spheres formed on gelatin@NWF scaffolds were significantly higher than in those cells grown as monolayers. Moreover, cells isolated from spheroids grown on gelatin@NWF scaffold showed higher drug resistance and motility. Tumor spheroids can be formed on a long-term storage scaffold, highlighting the potential of gelatin@NWF as a ready-to-use scaffold for tumor cell sphere generation and culturing.
Highlights
Two-dimensional (2D) cell culture, a well-established methodology, facilitates the understanding of tumor biology and accelerates drug discovery and development research
ALDH1A1 in cells grown qPCR characterized relative mRNA expression of OCT4, SOX2, and ALDH1A1 in cells grown on on gelatin@non-woven fabric (NWF) scaffold for 5 and 7 days, * denotes p < 0.05, ** denotes p < 0.01; (b) Western blot gelatin@NWF scaffold for 5 and 7 days, * denotes p < 0.05, ** denotes p < 0.01; (b) Western blot analysis to compare the Sox2 (35 kDa), Oct4 (45 kDa), and ALDH1A1 (55 kDa) level compared to analysis to compare the Sox2 (35 kDa), Oct4 (45 kDa), and ALDH1A1 (55 kDa) level compared to Glyceraldehyde3-phosphate dehydrogenase (GAPDH) (36 kDa) in cells grown on tissue culture plate (TCP) and gelatin@NWF scaffold for 5 and 7 days
The results suggest cells grown as a sphere on gelatin@NWF scaffold have a more substantial tolerance to chemotherapy drugs (Figure 6)
Summary
Two-dimensional (2D) cell culture, a well-established methodology, facilitates the understanding of tumor biology and accelerates drug discovery and development research. The rapid, uncontrolled growth phenotype of 2D cell culture was challenged by lacking an in vivo-like microenvironment, cell–cell contacting and nutrient distribution, etc. Increasing debates argued that those limitations, in mimicking the physiological and pathological conditions, might partially account for the high discrepancy rate between in vitro and in vivo tests [1–5]. Growing interests and attention have been attracted by three-dimensional (3D) cell culture among clinicians, researchers, and the pharmaceutical industry in the part of its potential in reassembling complexity and heterogeneity of microenvironment to mimic in vivo conditions [6,7]. Compared to 2D tumor cell culture, tumor spheroids are unique in recapitulating tissue architectures, mimicking the nutrition, metabolic, and proliferative gradients of in vivo tumors, and demonstrating clinically relevant chemoresistance [8–11].
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