title: Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.
abstract: Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting.
authors.affiliation: Lunenfeld-Tanenbaum Research Insitute, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
authors.forename: Justin
authors.initials: J
authors.lastname: Parreno
authors.affiliation: Lunenfeld-Tanenbaum Research Insitute, Toronto, Ontario, Canada; Institute of Biomaterials and Bioengineering, University of Toronto, Ontario, Canada.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Delve
authors.affiliation: Lunenfeld-Tanenbaum Research Insitute, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
authors.forename: Katarina
authors.initials: K
authors.lastname: Andrejevic
authors.affiliation: Lunenfeld-Tanenbaum Research Insitute, Toronto, Ontario, Canada.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Paez-Parent
authors.affiliation: Lunenfeld-Tanenbaum Research Insitute, Toronto, Ontario, Canada.
authors.forename: Po-Han
authors.initials: PH
authors.lastname: Wu
authors.affiliation: Lunenfeld-Tanenbaum Research Insitute, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada; Institute of Biomaterials and Bioengineering, University of Toronto, Ontario, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Rita
authors.initials: R
authors.lastname: Kandel
keywords: cartilage; chondrocytes; gene therapy; nucleofection; transfection