Gene Transfer to Synovial Fibroblast: Methods and Evaluation in the SCID Mouse Model

Meinecke, Ingmar; Rutkauskaite, Edita; Cinski, Antje; Müller‐Ladner, Ulf; Pap, Thomas

doi:10.1385/1-59745-401-x:393

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…Although intra-articular injection of fibroblasts into the knee joint is feasible in mice [ 27 ] this invasive procedure may destroy regular structures in the knee joint. In contrast, subcutaneous implantation is easier to perform, is non-invasive, and offers the advantage that several test items may be applied in one mouse [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP)

et al. 2015

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BackgroundThe clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair.MethodsThe testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC).ResultsNo human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin.ConclusionsIn summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0517-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP)

et al. 2015

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“…The Matrigel Transwell assay (9, 10) and the matrix‐associated transepithelial resistance invasion assay (11) have shown the invasive potential of RA as compared with OA synovial fibroblasts. Further, the RA SCID mouse model is a potent system for examining the invasiveness of synovial fibroblasts in vivo (12). In this model, synovial fibroblasts are coimplanted with cartilage under the renal capsule in SCID mice, which lack an intact immune system, thereby preventing the rejection of the transplant.…”

mentioning

confidence: 99%

“…In this model, synovial fibroblasts are coimplanted with cartilage under the renal capsule in SCID mice, which lack an intact immune system, thereby preventing the rejection of the transplant. Previous studies have shown that only RA, and not OA, synovial fibroblasts migrate, attach to, and destroy the adjacent cartilage (12).…”

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confidence: 99%

The synovial lining micromass system: Toward rheumatoid arthritis in a dish?

Perlman¹,

Pope²

2010

Arthritis & Rheumatism

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“…Quantification of the invasive potential of RASFs, as well as the assessment of treatment strategies with respect to matrix destruction, has been a major challenge. Until now, invasion has been measured by methods such as the Boyden chamber assay or in vivo in the SCID mouse model (5, 6). However, all of these assays have several disadvantages, the most striking of which is the imprecise nature of the readout data that serve to quantify the invasive potential of RASFs.…”

mentioning

confidence: 99%

Establishment of a matrix‐associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts

Wunrau¹,

Schnaeker²,

Freyth³

et al. 2009

Arthritis & Rheumatism

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Objective. Synovial fibroblasts (SFs) contribute to several aspects of the pathogenesis of rheumatoid arthritis (RA) and have been implicated most prominently in the progressive destruction of articular cartilage. Targeting the invasive phenotype of RASFs has therefore gained increasing attention, but the precise measurement of their invasive capacity and the evaluation of potential treatment effects constitute a challenge that needs to be addressed. This study used a novel in vitro invasion assay based on the breakdown of transepithelial electrical resistance to determine the course of fibroblast invasion into extracellular matrix.Methods. A matrix-associated transepithelial resistance invasion (MATRIN) assay was used to assess SFs from patients with RA in comparison with SFs from patients with osteoarthritis (OA). The SFs were grown on a commercially available collagen mix that was placed onto the upper side of a Transwell polycarbonate membrane. In addition, freshly isolated cartilage extracts were studied to assess the conditions in vivo. Under this membrane, a monolayer of MDCK-C7 cells was seeded to create a high electrical resistance.Results. Invasion of fibroblasts into the matrix affected the integrity of the MDCK-C7 monolayer and led to a measurable decrease and subsequent breakdown of electrical resistance. Unlike in the assay with OASFs, which did not achieve a breakdown of resistance up to 72 hours, RASFs exhibited a pronounced invasiveness in this assay, with a 50% breakdown after 42 hours. Treatment of fibroblasts with either a matrix metalloproteinase inhibitor or antibodies against ␤1 integrin significantly reduced the invasiveness of RASFs.Conclusion. The MATRIN assay is a valuable and sensitive biologic assay system that can be used to determine precisely the invasive potential of RASFs in vitro, and thus would be suitable for screening antiinvasion compounds.

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Gene Transfer to Synovial Fibroblast: Methods and Evaluation in the SCID Mouse Model

Cited by 3 publications

References 12 publications

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP)

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP)

The synovial lining micromass system: Toward rheumatoid arthritis in a dish?

Establishment of a matrix‐associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts

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