Gene expression profiles of 3D gene-edited bioprinted cells

Abstract

Cancer is the biggest cause of death in New Zealand each year and places a large economic strain on the health system. Previously, two-dimensional (2D) cell models have provided insight into the development of cancer but now, three-dimensional (3D) models provided more accurate information on how cell-cell interactions in the body affect the efficacy of different drug therapies. This research aims to investigate the gene expression profiles of 3D gene-edited bioprinted lung cancer cells to understand how mutations and the vital trace element Selenium (Se) affects gene expression. The human A549 lung cancer cell line was bioprinted into a set dome shape made of sodium alginate, gelatine, and cell media. The cells were then cultured for 21 days before a six-hour drug exposure with 2μM and 10μM Methylselenic Acid (MSA). RNA was also extracted from 18 bioprints and the spectrophotometer data suggested high quality with a total RNA concentration range of 9 – 671μg/μL, respectively. The RNA integrity was further examined using the RNA tape station. The solvent control (n=2) and the 2μM MSA treatment (n=3) from the second independent experiment resulted in intact ribosomal bands. The protein concentration was determined using a Bradford assay and ranged from 0.1 – 3.42mg/mL. The electrophoresed protein showed a molecular weight size range from 120kDa – 10kDa. The protein was then transferred onto a western blot membrane and detected by two primary antibodies; (1) HSP70 - correctly fold proteins that are under cellular stress and (2) GAPDH – a loading control reference antibody. The HSP70 protein expression was measured for the drug-treated bioprints for three bioprinting experiment. We hypothesised an upregulation in HSP70 expression based on previous studies. However, the data set could not be replicated to conclusively downregulation in response to 2 and 10μM MSA. Thus, further optimisation into drug accessibility, cell and protein loading concentration is required. Gene-editing using lipofectamine and CRISPR-Cas9 was attempted using A549 cells but no conclusive evidence that a mutation had been introduced into the Cyclin Dependent Kinase 4 (CDK4) gene was observed (n=5). Three research optimisations have been recommended for gene-editing and establishing a stable gene-edited cell line for future use in 3D bioprinting and gene expression analysis.