REGULAR CONTENT
Final ID
418
Type
Original Scientific Research-Oral or Pos
Authors
J Chen1, M Noji2, R Kiefer2, M Soulen1, S Hunt1, G Nadolski1, J Shi2, T Gade1
Institutions
1Hospital of the University of Pennsylvania, Philadelphia, PA, 2University of Pennsylvania School of Medicine, Philadelphia, PA
Purpose
Marked intertumoral genetic heterogeneity of hepatocellular carcinoma (HCC) poses a fundamental challenge to therapy development, as mutations found in existing immortalized tumor cell lines may not effectively recapitulate individual tumor profiles. The purpose of this study was to determine optimal methods for derivation and propagation of primary cell culture lines from HCC biopsy samples, as the foundation for a precision medicine approach to identifying targetable genetic vulnerabilities in HCC using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 approach.
Materials & Methods
Tumor samples were acquired by percutaneous core needle biopsy. A core was immediately processed for cell culture and another was frozen for DNA sequencing, to provide a baseline for evaluating genetic fidelity of propagated cell lines. Tissue cores were dissociated using combination of enzymatic and mechanical methods. HCC cells were enriched by lysis of red blood cells (RBC) and removal of endothelial cells. The robustness of cell propagation was evaluated using a 7-day growth curve assay. Variations in culture technique including the use of gelatin coated vs. uncoated plates, and hepatocyte-specific (HMM) vs. generic (RPMI-1640) cell culture media were evaluated. Cells were transduced with a lentivirus vector containing CRISPR/Cas-9 components and transduction efficiency was assessed.
Results
RBC lysis and endothelial cell removal were effective for enriching the HCC cell population. Gelatin coated plates supported 1/3 shorter cell adhesion times as compared to tissue culture plastic. Cells grown in generic media demonstrated a one-fold shorter doubling time compared to hepatocyte specific media. Cultured cells were able to be maintained for at least 6 passages. Lentiviral transduction of the CRISPR/Cas-9 vector was successful with transduction efficiency up to 95%.
Conclusions
Primary cell culture can be effectively established and propagated from HCC core needle biopsy samples and transduced with a CRISPR/Cas-9 vector construct providing the potential for functional genome editing to enable precision medicine in HCC.
Final ID
418
Type
Original Scientific Research-Oral or Pos
Authors
J Chen1, M Noji2, R Kiefer2, M Soulen1, S Hunt1, G Nadolski1, J Shi2, T Gade1
Institutions
1Hospital of the University of Pennsylvania, Philadelphia, PA, 2University of Pennsylvania School of Medicine, Philadelphia, PA
Purpose
Marked intertumoral genetic heterogeneity of hepatocellular carcinoma (HCC) poses a fundamental challenge to therapy development, as mutations found in existing immortalized tumor cell lines may not effectively recapitulate individual tumor profiles. The purpose of this study was to determine optimal methods for derivation and propagation of primary cell culture lines from HCC biopsy samples, as the foundation for a precision medicine approach to identifying targetable genetic vulnerabilities in HCC using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 approach.
Materials & Methods
Tumor samples were acquired by percutaneous core needle biopsy. A core was immediately processed for cell culture and another was frozen for DNA sequencing, to provide a baseline for evaluating genetic fidelity of propagated cell lines. Tissue cores were dissociated using combination of enzymatic and mechanical methods. HCC cells were enriched by lysis of red blood cells (RBC) and removal of endothelial cells. The robustness of cell propagation was evaluated using a 7-day growth curve assay. Variations in culture technique including the use of gelatin coated vs. uncoated plates, and hepatocyte-specific (HMM) vs. generic (RPMI-1640) cell culture media were evaluated. Cells were transduced with a lentivirus vector containing CRISPR/Cas-9 components and transduction efficiency was assessed.
Results
RBC lysis and endothelial cell removal were effective for enriching the HCC cell population. Gelatin coated plates supported 1/3 shorter cell adhesion times as compared to tissue culture plastic. Cells grown in generic media demonstrated a one-fold shorter doubling time compared to hepatocyte specific media. Cultured cells were able to be maintained for at least 6 passages. Lentiviral transduction of the CRISPR/Cas-9 vector was successful with transduction efficiency up to 95%.
Conclusions
Primary cell culture can be effectively established and propagated from HCC core needle biopsy samples and transduced with a CRISPR/Cas-9 vector construct providing the potential for functional genome editing to enable precision medicine in HCC.