A Novel 3D Co-Culture System for Isolation and Amplification of Primary Liquid Cancer Cells

Abstract 426

One of the important challenges in screening anti-cancer drugs is the lack of available “primary cultures systems” that is easy to use to screen new compounds or their combinations. The low yield of primary cancer cell cultures is mainly due to suboptimum environment in vitro and inefficient 2-dimensional cell culture conditions. To create an optimum in vitro environment, lymphoma cell lines were grown in 3-dimension model by using a scaffold and the stromal cells derived from neonatal foreskin was used as the feeder component. This 3-dimensional (3D) stromal co-culture generates an in-vitro model that may mimic the conditions/microenvironment of blood cancer cells interacting with stromal compartments. A specific 3D tissue culture scaffold 3D Insert-PS™ (300 μ m in fiber diameter and 400μ m in pore size) significantly enhances the cell proliferation and maintenance of liquid cancer cells in comparison to 2D stromal co-culture control. The combination of the neonatal stroma cells, a novel 3D scaffold, the constant gyration and a frequent nutrient stimulation allows the lymphoma cells to proliferate 10-fold faster than the cells grown in 2D under the same condition. Starting from the 2^nd day of 3D cell culture, these lymphoma cells grew to form layers of aggregated clusters and caused disappearance of single cells morphology and phenotype that is typical of cells growing in suspension. The cell aggregates are continuously produced from the 3D scaffold, subsequently dislodge from the scaffold and then remain viable at the bottom of the dish below the scaffold. When the cell clusters are harvested and cultured in 3D condition, the contamination of fibroblasts is over 1,000 fold less than the cell clusters that are generated from 2D environment. In addition, the clusters of cancer cells generated from 3D co-culture using 3D scaffolds contained the fibroblasts contamination that is less than 0.00001% of the total cell count, suggesting that this novel 3D environment can be implicated for the isolation of primary lymphoma/cancer cells from patient's blood or tissue specimen. To investigate this feasibility, <1% lymphoma cells were premixed with 100 fold excess of neonatal stroma cells, and the mixture was grown using our 3D scaffolds. In 7 days, the 3D culture system was able to amplify lymphoma cells over 100 fold or over 10,000 % of the starting cell number. This preliminary data indicate that this 3D scaffold and co-culturing environment can be customized to amplify primary cancer cells from blood or tumor tissues and subsequently used for personalized drug screening procedures.