Abstract 5018: Bioprinted (3D) co-cultured spheroids with NSCLC PDX cells and cancer associated fibroblasts (CAFs) using alginate/gelatin hydrogel
Authors: Mondal Arindam , Gebeyehu Aragaw , Subramanian Ramakrishnan , Rishi Arun , Singh Mandip 
Published: August 2018

Abstract
3D bioprinting which is a significant improvement over 2D models offers layer by layer positioning of various cells which can provide superior cell-matrix interactions in the tumor microenvironment in vitro. The objective of the present study was to develop a co-culture model using Non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) cells and lung cancer associated fibroblasts (CAFs) in sodium alginate/ gelatin hydrogel using a 3D bioprinter.
Various concentrations of sodium alginate/gelatin hydrogels (w/w) were prepared and rheological properties were evaluated. PDX cells (EGFR T790M, obtained from Dr. Rishi's Laboratory, Wayne State University) and CAFs (AA0022, obtained from Dr. Noyes Laboratory, Moffit Cancer Center) were mixed with the hydrogel and printed usingINKREDIBLE bioprinter (Cellink, Sweden). A two layered cube was designed in Slic3r software, converted to g-code file and printed through a 22G needle. Printed scaffolds were cross linked by Crosslinking Agent (Cellink, Sweden) for 5 minutes, washed with HBSS and maintained with media at 37°C and 5% CO2. Spheroid formation was observed using an inverted microscope (Olympus IX70). Live/Dead and NucBlue/ActinGreen staining was performed to determine cell viability and spheroid formation. Immuno-fluorescence staining was performed for E-cadherin, α-SMA and vimentin to study cellular crosstalk within the spheroid.
Printable hydrogels were prepared by adding 3.25% sodium alginate to 4% gelatin which was found to be the most optimal mixture. Decrease in storage modulus (G') from 995.29± 30.69 Pa to 12.16± 8.83 Pa and loss modulus (G”) from 314.10± 17.54 Pa to 22.22± 12.69 Pa was observed from 18°C to 37°C when using a temperature sweep test. At lowest shear rate (1s-1), the viscosity of the hydrogel was 2.75± 0.37 Pa.s and at 100s-1 the viscosity was 0.50± 0.12 Pa.s. Decrease in hydrogel viscosity with increasing shear rate confirmed shear thinning. The height, width and length of the printed scaffolds were found as 1.45± 0.06 mm, 9.56± 0.15 mm and10.67± 0.13 mm respectively. Formation of spheroids was observed after 4 days and after 15 days, 59.88± 14.81% small (0-400µm2), 15.43± 4.86% medium (401-1000 µm2) and 24.67± 10.14% large size spheroids (1001 µm2-above) were observed which indicated proliferation of individual cells within the scaffolds. LIVE/ DEAD assay showed approximately 82% cells were viable in the printed scaffold. Up-regulation of vimentin and α-SMA and concomitant loss of E-cadherin was observed in the printed co-cultures indicating cross talk between the two cells.
This study demonstrates the preparation and characterization of NSCLC lung PDX and CAF co-culture spheroids in 3D bio-printed sodium alginate/ gelatin scaffold. This model can be further used for high throughput drug screening platform.
Citation Format: Arindam Mondal, Aragaw Gebeyehu, Ramakrishnan Subramanian, Arun Rishi, Mandip Singh. Bioprinted (3D) co-cultured spheroids with NSCLC PDX cells and cancer associated fibroblasts (CAFs) using alginate/gelatin hydrogel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5018.