Transfection Information

Transfecting HuH-7 cells

HuH-7 cells are an immortalized hepatocellular carcinoma cell line originally resected from a male, 57 year old liver tumor.  The HuH-7 cell line is an epithelial-like adherent cell line containing multiple mutations including a point mutation in p53.  HuH-7 is receptive to transfection with certain viruses.  As an example, HuH-7 cells have been used to elucidate how HCV, the hepatitis C virus, interacts with hepatocytes in vivo.  HCV is a flavivirus which causes acute hepatitis, chronic hepatitis and cancer in the host organism.  The HCV virus has affected approximately 170 million people worldwide, a number which greatly incentivizes to control the infection and downstream effects of the HCV virus.

HuH-7 Cell Transfection Protocol

An optimized HuH-7 transfection kit is available from Altogen Biosystems – HuH-7 Transfection Reagent.  The kit is a biodegradable polymer reagent optimized to deliver siRNA, miRNA or DNA plasmid intracellularly with low toxicity.  The 24-well plate protocol to transfect HuH-7 cells is here:

  1. Prepare HuH-7 cell suspension:
    • Trypsinize cells with 0.05% Trypsin for 3-5 minutes at 37°C
    • Dilute the cell suspension in complete growth medium to a concentration of 5 x 104 cells/mL
  2. Transfection complexes are prepared by mixing 40 µL of serum-free medium, 4.5 µL of transfection reagent, and one of the following: 750 ng DNA (or mRNA), or 30 nM – 50 nM of siRNA (or microRNA)
  3. The transfection complexes are then incubated at RT for 15 – 30 minutes
  4. Optional: 2 µL of Complex Condenser can be added to reduce the size of the transfection complex; this increases transfection efficiency but may increase cell toxicity
  5. Pipette 20,000 – 30,000 cells per well in 0.5 ml of complete growth medium (from step #1) into cell culture plate
  6. Add the prepared transfection complexes (from step 3 or 4)
  7. Incubate the cells at 37°C in a CO2 incubator
  8. Assay the cells for phenotype or target gene expression changes 48 – 72 hours after transfection

HuH-7 Transfection Services

Transfection services using HuH-7 cells (ELISA, IC-50, assay development, cell cycle, proliferation, apoptosis, HuH-7 CDX xenograft mouse models) are offered by Altogen Labs (www.altogenlabs.com).

Publications

  • The HuH-7 cell line derived xenograft mouse model was utilized in this study to test pitavastatin as an anti-tumor drug.  Previously, pitavastatin had only been approved as a therapeutic for lowering blood cholesterol.  Experimental results of the study exhibited that pitavastatin inhibited tumor growth and colony formation and may be developed further as a drug for liver cancer treatment.  LINK: https://www.dovepress.com/pitavastatin-suppressed-liver-cancer-cells-in-vitro-and-in-vivo-peer-reviewed-fulltext-article-OTT
  • HuH-7 cells grown in RWV suspension are incubated in a horizontally rotating cylindrical culture vessel that allows cells to grow in 3D aggregates, without the turbulence associated with conventional, stirred bioreactors.  Cells cultured in RWV are grown much like cells of a fetus in a mother’s uterus.  The HuH-7 cells experience an environment with aspects resembling microgravity and “suspended animation”, where the cells are in constant free-fall.  The fluid dynamics of the RWV cell culture allows cells to develop as they would grow in vivo.  See image below for aggregate bead formation.  LINK: https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-6-103

 

Figure: RMV Aggregate Images: Phase contrast image of HuH-7 aggregates (top image).  Bright field image of H&E stained sections of HuH-7 aggregates.

 

 

 

*”Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection.”  Sainz B, TenCate V and Uprichard SL. Virology Journal. V6:103.  15July2009.

 

 

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