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Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System.

ABSTRACT: Angiogenesis is critical for local tumor growth. This study aimed to develop a three-dimensional two-layer co-culture system to investigate effects of cancer cells on the growth of endothelial cells (ECs). Poly(?-caprolactone) (PCL) nanofibrous membranes were generated via electrospinning of PCL in chloroform (C-PCL-M) and chloroform and dimethylformamide (C/DMF-PCL-M). We assembled a two-layer co-culture system using C-PCL-M and C/DMF-PCL-M for EC growth in the upper layer with co-cultured cancer cells in the lower layer. In the absence of vascular endothelial growth factor (VEGF), growth of bEND.3 ECs decreased on C/DMF-PCL-M but not on C-PCL-M with time. Growth of bEND.3 cells on C/DMF-PCL-M was enhanced through co-culturing of CT26 cancer cells and enhanced growth of bEND.3 cells was abrogated with anti-VEGF antibodies and sorafenib. However, EA.hy926 ECs displayed steady growth and proliferation on C/DMF-PCL-M, and their growth was not further increased through co-culturing of cancer cells. Moreover, chemical hypoxia in CT26 cancer cells upon treatment with CoCl2 enhanced the growth of co-cultured bEND.3 cells in the two-layer system. Thus, EC growth on the nanofibrous scaffold is dependent on the types of ECs and composition of nanofibers and this co-culture system can be used to analyze EC growth induced by cancer cells.

SUBMITTER: Oh YS

PROVIDER: S-EPMC7312426 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System.

Oh Ye-Seul YS Choi Min-Ho MH Shin Jung-In JI Maza Perry Ayn Mayson A PAMA Kwak Jong-Young JY

International journal of molecular sciences 20200610 11

Angiogenesis is critical for local tumor growth. This study aimed to develop a three-dimensional two-layer co-culture system to investigate effects of cancer cells on the growth of endothelial cells (ECs). Poly(ε-caprolactone) (PCL) nanofibrous membranes were generated via electrospinning of PCL in chloroform (C-PCL-M) and chloroform and dimethylformamide (C/DMF-PCL-M). We assembled a two-layer co-culture system using C-PCL-M and C/DMF-PCL-M for EC growth in the upper layer with co-cultured canc ...[more]

PMID: 32531897

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Project description:Background:The main issue in cutaneous regeneration is to develop engineered scaffolds based on natural extracellular matrix to promote dynamics of skin progenitor cells and accelerate differentiation into mature keratinocytes. Methods:In this study, nanofibrous scaffolds composed of a blend poly (?-caprolactone) (PCL), silk fibroin (SF), soluble eggshell membrane (SESM), and Aloe vera (AV) gel were developed by electrospinning method and human basal cells were used to examine differentiation capacity toward keratinocyte-like cells. For this propose, cells were allocated to four distinct groups; control, PCL/SF, PCL/SF/SESM, and PCL/SF/SESM/AV. In all groups, cells were incubated with differentiation medium. Morphology, composition, hydrophilicity and mechanical features of PCL/SF, PCL/SF/SESM and PCL/SF/SESM/AV nanofibers were studied by scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FT-IR), water contact angle and tensile tests. To examine the orientation of basal cells to mature keratinocytes, we performed immunofluorescence analysis by monitoring cytokeratin-19. The expression of genes such as involucrin, keratin-14 and -5 was monitored by real-time PCR assay. Results:PCL/SF, PCL/SF/SESM, and PCL/SF/SESM/AV had suitable physic chemical indices and biological activities to be applied as biomimetic scaffolds for the restoration cutaneous tissue. Compared to control, we found an increased basal cell proliferation at 7 and 14?days after plating on scaffolds and reach maximum levels in group PCL/SF/SESM/AV on day 14 (p?<?0.05). Electron microscopy showed cell flattening, morphological adaptation. An integrated cell-to-cell connection was generated after cell seeding on scaffolds in all groups. Immunofluorescence imaging showed the ability of basal cells to synthesize cytokeratin-19 in PCL/SF, PCL/SF/SESM, and positive control cells after exposure to differentiation medium. However, these values were less in PCL/SF/SESM/AV compared to other groups. Real-time PCR analysis showed the potency of all scaffolds to induce the transcription of involucrin, keratin-14 and -5, especially involucrin in PCL/SF/SESM/AV group compared to the negative control. Conclusion:Modulation of scaffolds with natural biopolymers could enable us to synthesize structures appropriate for cutaneous regeneration.

Dataset Information

Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System.

Publications

Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System.

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