Human cell-based assays for in vitro testing of drugs in preclinical and research studies, as well as in clinical practice, are gaining greater importance especially in view of personalized medicine, which is tailored to the individual needs and benefits of a patient. This chapter begins with an overview of contemporary cell-based assays, routinely used for a comparative in vitro potency testing of anti-TNF-α innovator biologics and their biosimilars. In sequel, based on the results of our original work, we will further discuss the establishment and use of 2D normal and osteoarthritic primary chondrocyte monolayer cultures and 3D microspheroidal articular cartilage tissues, prepared in hanging drops from osteoarthritic chondrocytes and chondrogenically differentiated mesenchymal stem cells. Both 2D and 3D cultures will be presented as models for assessing the neutralizing potency of the three well-known anti-TNF-α biological drugs: adalimumab, etanercept, and infliximab.
- in vitro cell-based assays
- anti-TNF-α biologics
- human articular chondrocytes
- mesenchymal stem cells
- 2D monolayer cultures
- 3D cell cultures
- gene expression
Following the discovery and characterization of tumor necrosis factor (TNF) in the mid-1980s, this pleiotropic proinflammatory cytokine continues to be the focus of numerous studies and represents an important therapeutic target [1, 2]. The venue of anti-TNF biological drugs has revolutionized treatment of autoimmune and inflammatory diseases like rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriasis, Crohn’s disease, ulcerative colitis, and others . Although expensive, biological drugs (biologics) at the moment represent the best-selling group of pharmaceuticals. Nowadays, following the expiry of originators patents, a plethora of less expensive biosimilar drugs (biosimilars) are available to patients. In order to confirm the biocomparability of original and biosimilar products and to prove their quality, safety, and efficacy, the use of reliable and standardized bioassays relevant in assessing their modes of action is of crucial importance.
In this chapter, after a short introductory review of TNF biology, anti-TNF biological drugs and their mechanisms of action, we will present a selection of
2. A short overview of TNF biology
TNF is produced in various cell types, mainly immune cells such as monocytes and macrophages, microglia, neutrophils, natural killer cells (NK), T lymphocytes, and also in neuronal cells, keratinocytes, and fibroblasts [2, 3]. The cytokine exists in two biologically active forms. The first being a transmembrane protein (tmTNF), which can be cleaved by the metalloproteinase TNF-α-converting enzyme (TACE) (also known as disintegrin and metalloproteinase domain-containing protein 17 (ADAM17)) into its second form, a homotrimeric soluble TNF (sTNF) .
There are two TNF-binding homotrimeric transmembrane receptors, namely the TNF receptor 1 (TNFR1 or CD120a) and the TNF receptor 2 (TNFR2 or CD120b) . While the TNFR1 is constitutively expressed on a vast majority of nucleated cells, the TNFR2 expression is inducible and tightly regulated, preferentially on endothelial, hematopoietic, neural, and immune cells [2, 4]. TNFR2 is also expressed on tumor cells where it is supposed to function as a tumor oncogene [5, 6].
Interestingly, tmTNF can induce signals in a bipolar way, as it acts as a ligand of both receptor types and as a receptor itself in cell-to-cell contacts [2, 4]. This means that tmTNF-α-expressing cells transmit signals to cells bearing TNFR1 and/or TNFR2. This phenomenon is called “outside-to-inside” or “reverse signaling,” the function of which has not been completely clarified yet [2, 4]. The receptor function of tmTNF has been demonstrated in human monocytes, macrophages, NK cells, and T lymphocytes .
While TNFR1 is activated by both tmTNF and sTNF, TNFR2 can only be triggered by tmTNF. Both types of membrane-bound receptors are prone to TACE cleavage, resulting in fragments termed soluble TNF receptors (sTNFR) . In turn, sTNFR may contribute to the regulation of cellular TNF responses by capturing and neutralizing circulating TNF (intrinsic TNF inhibitors). Additionally, due to increased receptor shedding, the number of functional signaling membrane TNFRs decreases. Consequently, this leads to a state of transient TNF desensitization .
3. Anti-TNF biological drugs and their mechanisms of action
Among currently available Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved originator and biosimilar anti-TNF drugs, there are three full-length monoclonal antibodies (mAbs); these are infliximab (IFX), a chimeric mouse/human mAb (Remicade® and its biosimilars: Remsima®, Inflectra®, Flixabi®, Ixifi®, Renflexis®, and Zessly®), adalimumab (ADA), a fully humanized mAb (Humira® and its biosimilars: Cyltezo®, Imraldi®, Amgevita®, Solymbic®, Hyrimoz®, Hulio®, Halimatoz®, and Heyifa®), and golimumab, another fully humanized mAb (Simponi®) (Figure 1) [2, 4]. The additional two anti-TNF biological drugs, which are not mAbs, are etanercept (ETA) (Enbrel® and its biosimilars: Erelzi® and Benepali®), a fusion protein consisting of two extracellular parts of the human TNFR2 and the Fc portion of human IgG1, and certolizumab pegol (Cimzia®) composed of a human Fab’ fragment, covalently attached to two cross-linked 20 kDa polyethylene glycol chains (Figure 1) [2, 4].
Although all anti-TNF biologics neutralize the same target (sTNF and tmTNF), they are not equally effective in treatment of certain inflammatory pathologies, for example, Crohn’s disease. This is due to differences in their characteristics (structure and binding affinities) and mechanisms of action (Figure 1) [2, 4]. Besides all of them being efficacious in neutralizing both forms of TNF, infliximab additionally induces “outside-to-inside” signaling via binding to tmTNF, thereby triggering apoptosis of tmTNF-expressing immune cells . Being full-length mAbs, adalimumab, golimumab, and infliximab can, after binding to cells expressing tmTNF via their effector Fc regions (IgG1), induce antibody-dependent cytotoxicity (ADCC) of NK cells and activate the classical complement pathway, resulting in a complement-dependent cytotoxicity (CDC) and apoptosis [2, 4]. While ADCC and CDC are also induced by etanercept, which contains a truncated form of IgG1 Fc domain (lacking a CH1 constant region), certolizumab pegol, due to its Fc domain missing structure, acts differently. In treating inflammatory bowel disease with anti-TNF mAbs, another mechanism of their action is based on the interaction between IgG1 Fc domains of therapeutic mAbs and macrophage Fcγ receptors (FcγR), resulting in increased numbers of regulatory M2 macrophages (CD206+). These cells in turn inhibit T cell proliferation [2, 7]. Additionally, in rheumatoid arthritis (RA), adalimumab enhances the expression of tmTNF on monocytes, thereby promoting the interaction between tmTNF and TNFR2 present on regulatory T cells (Tregs), which subsequently increase their immunosuppressive activities [2, 8]. Also in RA, infliximab promotes the generation of natural Tregs (CD4+CD25highFoxP3+), which inhibit a proinflammatory cytokine production and replenish a defective pool of these cells, typically found in this autoimmune disease [2, 9]. In RA patients, the adhesion molecules and chemokines are upregulated on their joint vasculature endothelium. Blockage of TNF-α with adalimumab, golimumab, infliximab, or etanercept deactivates inflamed vascular endothelium, thereby decreasing the numbers of inflammatory immune cells entering synovial joints and additionally improving the generation of new synovial blood vessels by increasing the circulating levels of vascular endothelial growth factor (VEGF) [10, 11].
In vitro cell-based bioassays for general potency assessment of anti-TNF biologics
Numerous well-established and standardized cell-based assays are available for assessing and comparing potencies of anti-TNF biologics and their biosimilars. Table 1 contains some basic information regarding the most frequently used routine TNF-α neutralization (A), ADCC (B), and CDC (C) tests. The majority of data on bioassays presented in Table 1 (see next page) were summarized from two publications describing the establishment of the first infliximab and etanercept World Health Organization (WHO) International Standards [12, 13]. These were performed within international collaborative studies, confirming their high degree of relevance and analytical laboratory utility.
Additionally, the capability of anti-TNF biological drugs to downregulate E-selectin adhesion molecules expressed on inflamed vascular endothelium can be determined on
Other bioassay readout approaches, like flow cytometry and measurement of induced endogenous gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR), are also being applied [16, 17, 18].
The reason why various human and murine cell lines are used in these assays is that such tests can be standardized and their results can be compared between laboratories. However, the use of different types of primary cells in such general tests is less appropriate due to their high interindividual differences and in certain cases also weak responsiveness to anti-TNF biologics. Therefore, the results obtained in this way can hardly be compared .
5. Two-dimensional (2D) and three-dimensional (3D) primary cell cultures for personalized
in vitro potency testing of anti-TNF biologics
Primary cells are indispensable for determining personal responses of patients to a given anti-TNF biologic, thereby generating important information for planning and performing optimal and cost-effective therapies. For this purpose, different cell types, especially those isolated from a patient’s disease-affected tissues or
In 2D cell cultures (monolayers), nutrients are evenly accessible to cells, but the communication between cells via secreted soluble molecules is restricted to their diffusion within the fluid, unless the medium is mixed or stirred regularly . On the other hand, in dense multicellular 3D cell constructs prepared and cultured
In the following subchapters, we will present our results after establishing 2D and 3D
5.1 Establishment of a 2D primary human chondrocyte-based cell model for
in vitro testing of anti-TNF-α biologicals
Cartilage, which covers joint surfaces, is one of the most affected tissues in RA and other inflammatory arthritic diseases. Its only living constituents are chondrocytes, which produce and maintain a cartilaginous matrix mainly consisting of collagen and proteoglycans .
For the establishment of our 2D model, two types of cells were used. Normal, healthy chondrocytes (NCs) were obtained from surplus cartilage biopsies of patients scheduled for an autologous chondrocyte implantation procedure or were acquired postmortem from donors with healthy cartilage, in accordance with National Medical Ethics Committee approvals. On the other hand, osteoarthritic chondrocytes (OACs) were obtained from cartilage samples of patients undergoing total knee replacement surgery, in accordance with National Medical Ethics Committee approval. Following chondrocyte isolation and cultivation, confluent cell cultures were incubated in serum-free conditions with 1 ng/mL of rhTNF-α (PeproTech, USA) ± 1 μg/mL of each of the two anti-TNF-α biologicals tested, infliximab (IFX; Remicade®, Centocor, Netherlands) and etanercept (ETA; Enbrel®, Wyeth Pharmaceuticals, UK). After 24 h of incubation, chondrocytes and cell culture media were sampled for gene and protein expression analyses, respectively. In experiments using OACs, only the most relevant genes were selected and analyzed. Names and symbols of screened genes are presented in Table 2. Data were analyzed by applying the 2−ΔΔCq formula (ABI PRISM® 7700 Sequence Detection System User Bulletin #2) with the nontreated chondrocyte samples used for normalization. Results are presented as relative quantities (RQ) or Log2 relative quantity values (Log2 RQ). For protein expression analysis, a custom antibody array (RayBiotech, USA) was designed to detect interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), monocyte chemoattractant protein-1 (MCP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), and vascular cell adhesion protein 1 (VCAM-1). All protein analysis data were normalized to nontreated controls.
The results of the first set of experiments obtained after stimulation of cultured NCs and OACs with rhTNF-α and after their preincubation with a combination of rhTNF-α and IFX or ETA are presented in Figure 2 (graphs A and B, respectively). Upon TNF-α stimulation of NCs, the highest gene upregulation was observed for
Because NCs are difficult to obtain, we performed the same IFX and ETA neutralization experiments with rhTNF-α-treated OACs, however, to a lesser extent. A selected group of the most responsive genes were tested using OAC biological samples from four donors (Figure 2B). We observed a similar response to NCs when OACs were treated by rhTNF-α alone (Figure 2, graph Ba) and after their preincubation with a combination of rhTNF-α and IFX or ETA (Figure 2, graphs Bb and Bd, respectively). In Figure 2, graphs Bc and Be show the responses of OACs after their exposure to each individual biological drug. The cartilage of OA patients represents a biological waste material, which can be obtained in joint replacement surgeries. Despite changes in gene expression observed during the
With the data obtained, we were able to establish a statistical model for the evaluation of IFX and ETA TNF-α neutralization efficacy. Expressions of the nine most representative genes were chosen for a graphical presentation of results. Geometrical means of RQ values were plotted on radial axes of radar graphs and connected by a polygon, forming a distinctive shape. A comparison of shapes obtained with IFX and ETA revealed differences in their inhibition of gene expressions. Value 0, depicted in the center of graphs, represents total gene inhibition. For easier comparisons of results, shaded areas of twofold changes were plotted as well. Arbitrary fold-change cutoffs >2 (0.5 for down- and 2 for upregulated genes) were considered biologically significant. In our experimental conditions, the twofold change rims only overlapped in case of
The presented statistical model is also suitable for a comparative neutralization efficacy determination of new bioactive molecules and biosimilars relative to well-established and approved biologics, according to effective criteria for the assessment of biosimilarity, nonsimilarity, and incomparability.
5.2 Establishment of a 3D human osteoarthritic model for
in vitro efficacy testing of anti-TNF-α biologicals, using primary human osteoarthritic chondrocytes and mesenchymal stem cells
As discussed in the introduction, 2D and 3D cell culture conditions have different impacts on cell phenotype and biological behavior, which were also confirmed for primary chondrocytes and chondrogenically differentiated MSCs [30, 31, 32, 33, 34, 35]. In the last decade, cell-based research shifted toward 3D tissue/organ models, providing more physiologically realistic biochemical and biomechanical microenvironments. However, besides their biological relevance, in order to meet the expectations of the pharmaceutical industry, drug screening assays should be high-throughput, widely applicable, and low cost. With this in mind, we established a new
As already stated, OACs represent an attractive source of cells for cell-based models as besides being rather easily accessible and free of ethical concerns, they are also genetically stable during their long-term
Among the numerous commercially available 3D cell culture systems, we have chosen Perfecta 3D® scaffolds (3D Biomatrix Inc., USA) to create tissues in hanging drops. Generation of scaffold-free spheroids of micrometric dimensions (microspheroids) by gravity-enforced self-assembly in hanging drops allows cell aggregation and tissue formation in a natural manner, without interference from the scaffold material [19, 32]. This technique has important advantages, especially the drop size control and consequent uniformity of formed microspheroids. Moreover, it is compatible with automated liquid handling systems, a prerequisite for high-throughput screening in drug discovery. The microspheroid formation in hanging drops mimics the condensation process of MSCs, which is one of the earliest phases of
Isolated OACs were first expanded in 2D monolayer cultures and then, from passage 2 and on, 10,000 cells were transferred into each hanging drop. In this way, the loss of chondrogenic phenotype of OACs in 2D was restored in 3D conditions, as already reported [30, 43]. Similarly as in our previously described 2D primary chondrocyte model, the TNF-α neutralizing efficiencies of ADA (Humira®, Abbott Laboratories, USA), ETA (Enbrel®, Immunex Corp., USA), IFX (Remicade®, Janssen Biotech, USA), and the anti-IL-1β drug anakinra (ANA; Kineret®, Swedish Orphan Biovitrum AB, Sweden) were assessed with both cell types by determining the extent of downregulation of six selected genes (
According to our criteria, Log2 RQ ≥1 and ≤ −1, TNF-α significantly upregulated the expression of
When microspheroids were incubated with MCM, a superior anti-IL-1β neutralization capacity of ANA compared to the three tested anti-TNF-α biologics was observed. This difference was probably due to the fact that MCM contained a much higher concentration of IL-1β (0.45 ng/mL) than TNF-α (0.05 ng/mL). Nevertheless, these concentrations of both cytokines are much higher than those measured in synovial fluids of OA and RA patients (0.028 ng/mL TNF-α and 0.1 ng/mL IL-1β) . Although MCM proved to be an excellent
In our 3D microspheroidal rhTNF-α-induced inflammation model, the neutralization capacity of ADA was superior over that of ETA and the even weaker IFX (Figure 4b). Similar results were obtained with both microspheroids, regardless of whether they were made of OACs or chondrogenically differentiated MSCs. The observed differences in neutralizing efficiencies of ADA, ETA, and IFX can be attributed to differences in their molecular structures and sTNF-α-binding affinities . The superior anti-TNF-α efficacy of ADA over ETA and IFX has already been reported together with data, showing that the sTNF-α-binding affinity of ADA is higher for ADA (Kd = 7.05 × 10−11) than ETA (Kd = 2.35 × 10−11) and IFX (Kd = 1.17 × 10−10) [46, 47, 48]. However, according to our criterion, a particular biologic would be statistically more efficient than the compared one if it would cause a ≥2-fold decrease in a selected gene expression. This was not the case in any of our 3D microspheroidal model experiments. Consequently, we assumed that the observed differences in TNF-α neutralizing potency of ADA, ETA, and IFX were comparable (Figure 4b). Interestingly, although we showed in our 2D OACs model that ETA was significantly more efficient than IFX, the same kind of experiments carried out in a 3D microspheroidal model did not confirm this finding [26, 27]. We assume that compared to the 2D model, the diffusion of tested biologics in our 3D microspheroidal model was much slower and limited. Undoubtedly, the 3D model better resembles
We found that OACs and chondrogenically differentiated MSCs are suitable sources for hanging drop chondral 3D microspheroid cultures formation, which are useful for the assessment of neutralization potencies of anti-inflammatory biologics . Although the use of these two types of microspheroids resulted in different gene expression profiles following their incubation with tested combinations of rhTNF-α, and each of the three tested anti-TNF-α biological drugs (Figure 4b), these differences were rather small. Therefore, we concluded that MSCs can be used as an alternative and probably even more accessible cell source for
Cell-based assays are complex analytical tools, susceptible to multiple variables that are virtually impossible to control. Therefore, they have to be precise, reliable, and well standardized so that the results are reproducible and can be compared among different laboratories. When used for drug potency testing, such assays usually rely on the use of reference standards. Recently, the WHO has prepared two international standards for the two anti-TNF-α biologics, etanercept and infliximab. These have been tested by several laboratories within an international collaborative study using a number of different cell-based assays [12, 13]. In this chapter, we have presented an overview of the most routinely used tests for potency testing of anti-TNF-α biologics, which measure
Nowadays, with an expanding personal medicine approach, laboratory assay-guided pharmacotherapeutical strategies are becoming more and more important. In order to obtain relevant data on drug potencies for a particular patient, these kinds of tests should be based on the patient’s own, that is, autologous primary cells, as these can significantly reduce costs and enable safer and more effective therapies. Therefore, we dedicated a part of this chapter to our experience in establishing
The authors wish to express their gratitude to Prof. Andrej Blejec, Dr. Miomir Knežević, Asst. Prof. Nevenka Kregar Velikonja, and Prof. Gordana Vunjak-Novakovic for their contribution to our common, published original research work, partly presented in this chapter. We are also grateful to Rosmarijn E. Vandenbroucke for approving the use of a picture presented in Figure 1 and to Andrej Branc for proofreading the manuscript.
Conflict of interest
The authors declared that no competing interests exist.
Appendices and nomenclature
two-dimensional three-dimensional adalimumab anakinra etanercept infliximab interleukin 1β macrophage conditioned medium mesenchymal stem cells normal human articular chondrocytes osteoarthritic human articular chondrocytes relative quantity of gene expression recombinant human tumor necrosis factor α soluble form of tumor necrosis factor α transmembrane form of tumor necrosis factor α
macrophage conditioned medium
mesenchymal stem cells
normal human articular chondrocytes
osteoarthritic human articular chondrocytes
relative quantity of gene expression
recombinant human tumor necrosis factor α
soluble form of tumor necrosis factor α
transmembrane form of tumor necrosis factor α
Aggarwal BB, Gupta SC, Kim JH. Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey. Blood. 2012; 119:651-665. DOI: 10.1182/blood-2011-04-325225
Steeland S, Libert C, Vandenbroucke RE. A new venue of TNF targeting. International Journal of Molecular Sciences. 2018; 19:1442. DOI: 10.3390/ijms19051442
Yan L, Hu R, Tu S, Cheng W-J, Zheng Q, Wang J-W, et al. Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC. International Journal of Clinical and Experimental Medicine. 2015; 8:20065-20071
Mitoma H, Horiuchi T, Tsukamoto H, Ueda N. Molecular mechanisms of action of anti-TNF-α agents—Comparison among therapeutic TNF-α antagonists. Cytokine. 2018; 101:56-63. DOI: 10.1016/j.cyto.2016.08.014
Chen X, Oppenheim JJ. Targeting TNFR2, an immune checkpoint stimulator and oncoprotein, is a promising treatment for cancer. Science Signaling. 2017; 10:eaal2328. DOI: 10.1126/scisignal.aal2328
Torrey H, Butterworth J, Mera T, Okubo Y, Wang L, Baum D, et al. Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumor-associated Tregs. Science Signaling. 2017; 10:eaaf8608. DOI: 10.1126/scisignal.aaf8608
Vos ACW, Wildenberg ME, Duijvestein M, Verhaar AP, van den Brink GR, Hommes DW. Anti-tumor necrosis factor-α antibodies induce regulatory macrophages in an Fc region-dependent manner. Gastroenterology. 2011; 140:221-230.e3. DOI: 10.1053/j.gastro.2010.10.008
Nguyen DX, Ehrenstein MR. Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis. The Journal of Experimental Medicine. 2016; 213:1241-1253. DOI: 10.1084/jem.20151255
Nadkarni S, Mauri C, Ehrenstein MR. Anti-TNF-α therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-β. The Journal of Experimental Medicine. 2007; 204:33-39. DOI: 10.1084/jem.20061531
Shealy DJ, Cai A, Staquet K, Baker A, Lacy ER, Johns L, et al. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α. MAbs. 2010; 2:428-439
Paleolog E. Target effector role of vascular endothelium in the inflammatory response: Insights from the clinical trial of anti-TNF alpha antibody in rheumatoid arthritis. Molecular Pathology. 1997; 50:225-233
Wadhwa M, Bird C, Dilger P, Rigsby P, Jia H, Gross MEB. Establishment of the first WHO International Standard for etanercept, a TNF receptor II Fc fusion protein: Report of an international collaborative study. Journal of Immunological Methods. 2017; 447:14-22. DOI: 10.1016/j.jim.2017.03.007
Metcalfe C, Dougall T, Bird C, Rigsby P, Behr-Gross M-E, Wadhwa M, et al. The first World Health Organization International Standard for infliximab products: A step towards maintaining harmonized biological activity. MAbs. 2019; 11:13-25. DOI: 10.1080/19420862.2018.1532766
Hofmann H-P, Kronthaler U, Fritsch C, Grau R, Müller SO, Mayer R, et al. Characterization and non-clinical assessment of the proposed etanercept biosimilar GP2015 with originator etanercept (Enbrel(®)). Expert Opinion on Biological Therapy. 2016; 16:1185-1195. DOI: 10.1080/14712598.2016.1217329
Lallemand C, Kavrochorianou N, Steenholdt C, Bendtzen K, Ainsworth MA, Meritet J-F, et al. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists. Journal of Immunological Methods. 2011; 373:229-239. DOI: 10.1016/j.jim.2011.08.022
Mitoma H, Horiuchi T, Tsukamoto H, Tamimoto Y, Kimoto Y, Uchino A, et al. Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor α-expressing cells: Comparison among infliximab, etanercept, and adalimumab. Arthritis and Rheumatism. 2008; 58:1248-1257. DOI: 10.1002/art.23447
Ueda N, Tsukamoto H, Mitoma H, Ayano M, Tanaka A, Ohta S, et al. The cytotoxic effects of certolizumab pegol and golimumab mediated by transmembrane tumor necrosis factor α. Inflammatory Bowel Diseases. 2013; 19:1224-1231. DOI: 10.1097/MIB.0b013e318280b169
Moore M, Ferguson J, Burns C. Applications of cell-based bioassays measuring the induced expression of endogenous genes. Bioanalysis. 2014; 6:1563-1574. DOI: 10.4155/bio.14.98
Rimann M, Graf-Hausner U. Synthetic 3D multicellular systems for drug development. Current Opinion in Biotechnology. 2012; 23:803-809. DOI: 10.1016/j.copbio.2012.01.011
Baker BM, Chen CS. Deconstructing the third dimension—How 3D culture microenvironments alter cellular cues. Journal of Cell Science. 2012; 125:3015-3024. DOI: 10.1242/jcs.079509
Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay and Drug Development Technologies. 2014; 12:207-218. DOI: 10.1089/adt.2014.573
Duval K, Grover H, Han L-H, Mou Y, Pegoraro AF, Fredberg J, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology. 2017; 32:266-277. DOI: 10.1152/physiol.00036.2016
Alépée N. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. ALTEX. 2014; 31(4):441-477. DOI: 10.14573/altex1406111
Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: Structure, composition, and function. Sports Health: Multidisciplinary Approach. 2009; 1:461-468. DOI: 10.1177/1941738109350438
Otero M, Favero M, Dragomir C, Hachem KE, Hashimoto K, Plumb DA, et al. Human chondrocyte cultures as models of cartilage-specific gene regulation. In: Mitry RR, Hughes RD, editors. Human Cell Culture Protocols. Vol. 806. Totowa, NJ: Humana Press; 2012. pp. 301-336
Žigon-Branc S, Barlič A, Knežević M, Jeras M, Vunjak-Novakovic G. 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. Biotechnology Progress. 2018; 34:1045-1058. DOI: 10.1002/btpr.2629
Žigon-Branc S, Jeras M, Blejec A, Barlič A. Applicability of human osteoarthritic chondrocytes for in vitro efficacy testing of anti-TNFα drugs. Biologicals. 2017; 45:96-101. DOI: 10.1016/j.biologicals.2016.09.013
Barlič A, Žigon S, Blejec A, Kregar Velikonja N. Gene expression of cultured human chondrocytes as a model for assessing neutralization efficacy of soluble TNFα by TNFα antagonists. Biologicals. 2015; 43:171-180. DOI: 10.1016/j.biologicals.2015.03.001
Burns CJ, Silva MMCG, Gray E, Robinson CJ. Quantitative RT-PCR as an alternative to late-stage bioassays for vascular endothelial growth factor. Journal of Pharmaceutical and Biomedical Analysis. 2008; 47:460-468. DOI: 10.1016/j.jpba.2008.02.011
Caron MMJ, Emans PJ, Coolsen MME, Voss L, Surtel DAM, Cremers A, et al. Redifferentiation of dedifferentiated human articular chondrocytes: Comparison of 2D and 3D cultures. Osteoarthritis and Cartilage. 2012; 20:1170-1178. DOI: 10.1016/j.joca.2012.06.016
Mata-Miranda MM, Martinez-Martinez CM, Noriega-Gonzalez JE, Paredes-Gonzalez LE, Vázquez-Zapién GJ. Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes. Histochemistry and Cell Biology. 2016; 146(2):183-189. DOI: 10.1007/s00418-016-1437-4
Lehmann M, Martin F, Mannigel K, Kaltschmidt K, Sack U, Anderer U. Three-dimensional scaffold-free fusion culture: The way to enhance chondrogenesis of in vitro propagated human articular chondrocytes. European Journal of Histochemistry. 2013; 57:31. DOI: 10.4081/ejh.2013.e31
Bhumiratana S, Vunjak-Novakovic G. Engineering physiologically stiff and stratified human cartilage by fusing condensed mesenchymal stem cells. Methods. 2015; 84:109-114. DOI: 10.1016/j.ymeth.2015.03.016
Baraniak PR, McDevitt TC. Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell and Tissue Research. 2012; 347:701-711. DOI: 10.1007/s00441-011-1215-5
Sart S, Tsai A-C, Li Y, Ma T. Three-dimensional aggregates of mesenchymal stem cells: Cellular mechanisms, biological properties, and applications. Tissue Engineering. Part B, Reviews. 2014; 20:365-380. DOI: 10.1089/ten.teb.2013.0537
Laganà M, Arrigoni C, Lopa S, Sansone V, Zagra L, Moretti M, et al. Characterization of articular chondrocytes isolated from 211 osteoarthritic patients. Cell and Tissue Banking. 2014; 15:59-66. DOI: 10.1007/s10561-013-9371-3
Neri S, Mariani E, Cattini L, Facchini A. Long-term in vitro expansion of osteoarthritic human articular chondrocytes do not alter genetic stability: A microsatellite instability analysis. Journal of Cellular Physiology. 2011; 226:2579-2585. DOI: 10.1002/jcp.22603
Tallheden T, Bengtsson C, Brantsing C, Sjogren-Jansson E, Carlsson L, Peterson L, et al. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis Research & Therapy. 2005; 7:R560-R568
Kafienah W, Mistry S, Dickinson SC, Sims TJ, Learmonth I, Hollander AP. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis and Rheumatism. 2007; 56:177-187. DOI: 10.1002/art.22285
Jagielski M, Wolf J, Marzahn U, Völker A, Lemke M, Meier C, et al. The influence of IL-10 and TNFα on chondrogenesis of human mesenchymal stromal cells in three-dimensional cultures. International Journal of Molecular Sciences. 2014; 15:15821-15844. DOI: 10.3390/ijms150915821
Dudics V, Kunstár A, Kovács J, Lakatos T, Géher P, Gömör B, et al. Chondrogenic potential of mesenchymal stem cells from patients with rheumatoid arthritis and osteoarthritis: Measurements in a microculture system. Cells, Tissues, Organs. 2009; 189:307-316. DOI: 10.1159/000140679
Agar G, Blumenstein S, Bar-Ziv Y, Kardosh R, Schrift-Tzadok M, Gal-Levy R, et al. The chondrogenic potential of mesenchymal cells and chondrocytes from osteoarthritic subjects: A comparative analysis. Cartilage. 2011; 2:40-49. DOI: 10.1177/1947603510380899
Schulze-Tanzil G, de Souza P, Villegas Castrejon H, John T, Merker H-J, Scheid A, et al. Redifferentiation of dedifferentiated human chondrocytes in high-density cultures. Cell and Tissue Research. 2002; 308:371-379. DOI: 10.1007/s00441-002-0562-7
Westacott CI, Whicher JT, Barnes IC, Thompson D, Swan AJ, Dieppe PA. Synovial fluid concentration of five different cytokines in rheumatic diseases. Annals of the Rheumatic Diseases. 1990; 49:676-681
Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necrosis factor antagonist mechanisms of action: A comprehensive review. Pharmacology & Therapeutics. 2008; 117:244-279. DOI: 10.1016/j.pharmthera.2007.10.001
Hu S, Liang S, Guo H, Zhang D, Li H, Wang X, et al. Comparison of the inhibition mechanisms of adalimumab and infliximab in treating tumor necrosis factor α-associated diseases from a molecular view. The Journal of Biological Chemistry. 2013; 288:27059-27067. DOI: 10.1074/jbc.M113.491530
Scallon B, Cai A, Solowski N, Rosenberg A, Song X-Y, Shealy D, et al. Binding and functional comparisons of two types of tumor necrosis factor antagonists. The Journal of Pharmacology and Experimental Therapeutics. 2002; 301:418-426
Granneman RG, Zhang YM, Noertersheuser PA, Velagapudi RB, Awni WM, Locke CS, et al. Pharmacokinetic/pharmacodynamic (PK/PD) relationships of adalimumab (HUMIRA™, Abbott) in rheumatoid arthritis (RA) patients during phase II/III clinical trials. In: Arthritis and Rheumatism. Vol. 48. Div John Wiley & Sons Inc, 605 Third Ave, New York, NY 10158–0012 USA: Wiley-Liss; 2003. pp. S140-S141
Hyrich KL, Lunt M, Watson KD, Symmons DPM, Silman AJ. British Society for Rheumatology Biologics Register. Outcomes after switching from one anti-tumor necrosis factor alpha agent to a second anti-tumor necrosis factor alpha agent in patients with rheumatoid arthritis: Results from a large UK national cohort study. Arthritis and Rheumatism. 2007; 56:13-20. DOI: 10.1002/art.22331