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.

journal_name

Cartilage

journal_title

Cartilage

authors

Parreno J,Delve E,Andrejevic K,Paez-Parent S,Wu PH,Kandel R

doi

10.1177/1947603515609399

subject

Has Abstract

pub_date

2016-01-01 00:00:00

pages

82-91

issue

1

eissn

1947-6035

issn

1947-6043

pii

10.1177_1947603515609399

journal_volume

7

pub_type

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