An effective and efficient method of transfecting primary human chondrocytes in suspension.
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.
Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, United States.
Mohammad Shahidul
MS
Makki
Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, United States.
Nahid
N
Akhtar
Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, United States. Electronic address: thaqqi@neomed.edu.
Tariq M
TM
Haqqi
D053674:Aggrecans / Q000235:genetics; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D006801:Humans; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011062:Polynucleotide Adenylyltransferase; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000037:antagonists & inhibitors / Q000235:genetics
C508319:ACAN protein, human; D053674:Aggrecans; C499177:COL2A1 protein, human; D024043:Collagen Type II; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D039221:mRNA Cleavage and Polyadenylation Factors; D009713:Nucleotidyltransferases; C586325:TUT1 protein, human; D011062:Polynucleotide Adenylyltransferase; C550174:TENT2 protein, human
Chondrocytes; Osteoarthritis; Transfection; siRNA