An effective and efficient method of transfecting primary human chondrocytes in suspension.

An effective and efficient method of transfecting primary human chondrocytes in suspension.

Makki Mohammad Shahidul; Akhtar Nahid; Haqqi Tariq M

Analytical biochemistry

2017

2017-06

Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80-90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.

*Chondrocytes; *Osteoarthritis; *siRNA; *Transfection; Aggrecans/genetics; Articular/metabolism/pathology; Cartilage; Cells; Chondrocytes/cytology/*metabolism; Collagen Type II/genetics; Cultured; Humans; Messenger/*genetics; mRNA Cleavage and Polyadenylation Factors/antagonists & inhibitors/genetics; Nucleotidyltransferases/antagonists & inhibitors/genetics; Plasmids/*genetics; RNA; Small Interfering/*genetics; Transfection/*methods

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