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

ABSTRACT: 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.

SUBMITTER: Makki MS

PROVIDER: S-EPMC5490990 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Makki Mohammad Shahidul MS Akhtar Nahid N Haqqi Tariq M TM

Analytical biochemistry 20170314

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 ...[more]

PMID: 28315317

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Project description:There is growing evidence in support of the involvement of inflammatory response in the pathogenesis of osteoarthritis (OA). Harpagoside, one of the bioactive components of Harpagophytum procumbens (Hp), has been shown to possess anti-inflammatory properties. Here we used an in vitro model of inflammation in OA to investigate the potential of harpagoside to suppress the production of inflammatory cytokines/chemokines such as IL-6 and matrix degrading proteases. We further investigated the likely targets of harpagoside in primary human OA chondrocytes. OA chondrocytes were pre-treated with harpagoside before stimulation with IL-1β. mRNA expression profile of 92 cytokines/chemokines was determined using TaqMan Human Chemokine PCR Array. Expression levels of selected mRNAs were confirmed using TaqMan assays. Protein levels of IL-6 and MMP-13 were assayed by ELISA and immunoblotting. Total protein levels and phosphorylation of signaling proteins were determined by immunoblotting. Cellular localization of IL-6 and c-Fos was performed by immunofluorescence and confocal microscopy. DNA binding activity of c-FOS/AP-1 was determined by ELISA. Harpagoside significantly altered the global chemokine expression profile in IL-1β-stimulated OA chondrocytes. Expression of IL-6 was highly induced by IL-1β, which was significantly inhibited by pre-treatment of OA chondrocytes with harpagoside. Harpagoside did not inhibit the IL-1β-induced activation of NF-κB and C/EBPβ transcription factors but suppressed the IL-1β-triggered induction, phosphorylation, and DNA binding activity of c-FOS, one of the main components of AP-1 transcription factors. Further, harpagoside significantly inhibited the expression of MMP-13 in OA chondrocytes under pathological conditions. siRNA-mediated knockdown of IL-6 resulted in suppressed expression and secretion of MMP-13 directly linking the role of IL-6 with MMP-13 expression. Taken together, the present study suggests that harpagoside exerts a significant anti-inflammatory effect by inhibiting the inflammatory stimuli mediated by suppressing c-FOS/AP-1 activity in OA chondrocytes under pathological conditions. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:311-320, 2017.

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

Publications

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

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