Gene expression of cultured human chondrocytes as a model for assessing neutralization efficacy of soluble TNFα by TNFα antagonists

Barlič, Ariana; Žigon, Sara; Blejec, Andrej; Velikonja, Nevenka Kregar

doi:10.1016/j.biologicals.2015.03.001

Cited by 6 publications

(13 citation statements)

References 41 publications

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“…Based on our previous results, the levels of 8 genes that were found to be most differentially expressed in OA ( IL6, IL8, MCP1, MMP1, MMP13, VCAM1, ACAN , and SOX9 ) were assessed in microspheroids made of OACs or chondrogenically differentiated MSCs originating from 3 different donors (Figure a), following inflammation induction with TNF‐α, IL‐1β, or MCM h.s. .…”

Section: Resultsmentioning

confidence: 99%

“…In addition to their known catabolic effects, both cytokines inhibit proteoglycan and type II collagen synthesis . The previously determined in vitro effects of TNF‐α and IL‐1β on human MSCs and human and animal chondrocytes ,35,40,41, served as a basis for designing our 3D OACs/MSCs‐derived microspheroid tissue model for efficacy testing of anti‐TNF‐α biological drugs …”

Section: Discussionmentioning

confidence: 99%

“…OACs were enzymatically isolated and cryopreserved from cartilage samples as described previously . Briefly, samples were rinsed in sterile PBS, minced into small pieces and collected in DMEM Nutrient Mixture F‐12 (Gibco), supplemented with 50 μg/mL gentamicin, 2 μg/mL Fungizone ® , and 1 mg/mL collagenase type II (Gibco).…”

Section: Methodsmentioning

confidence: 99%

“…The results were recorded on StepOne Plus Real‐Time PCR System (Applied Biosystems, Singapore) and the amplification products were analyzed with the StepOne Soft program (Applied Biosystems, Singapore). In each sample, the level of particular gene expression was calculated as a ratio of the analyzed gene C q values versus the stably expressed glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) reference gene C q values and normalized to untreated control group, that is, ø or MCMø . For each group, three samples of cDNA were obtained from three individual microspheroids and for each of them qRT‐PCR analysis was performed in duplicate.…”

Section: Methodsmentioning

confidence: 99%

“…The specific cytokine‐neutralizing potencies of ADA, ETA, IFX, and ANA were determined by qRT‐PCR. Drug potencies were assessed from the expression levels of the eight most differently expressed genes involved in arthritis . The changes in gene expression levels measured in differently treated microspheroids were correlated with the concentrations of specific proteins found in microspheroid culture supernatants.…”

Section: Introductionmentioning

confidence: 99%

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Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

Žigon-Branc

Barlič

Knežević

et al. 2018

Biotechnology Progress

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Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). New in vitro testing systems are needed to evaluate efficacies of new anti-inflammatory biological drugs, ideally in a patient-specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti-inflammatory drugs, spheroids were exposed to TNF-α, IL-1β, or to supernatant containing secretome from activated macrophages (MCM). The anti-inflammatory efficacies of anti-TNF-α biologicals adalimumab, infliximab, and etanercept, and the anti-IL-1β agent anakinra were assessed in short-term microspheroid and long-term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF-α or IL-1β. The differences in potency of anti-TNF-α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short-term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti-TNF-α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045-1058, 2018.

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Section: Resultsmentioning

confidence: 99%