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A10.08 Detailed analysis of the effect of cryopreservation on the viability and cytokine release of human synovial tissue
  1. M de Vries,
  2. MGA Broeren,
  3. MB Bennink,
  4. PLEM van Lent,
  5. PM van der Kraan,
  6. MI Koenders,
  7. RM Thurlings,
  8. FAJ van de Loo
  1. Experimental Rheumatology, Department of Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands

Abstract

Background and objectives Human synovial tissue derived from joint surgery of osteoarthritis (OA) and rheumatoid arthritis (RA) patients is valuable material for studying fundamental research questions. It would be ideal to store and collect all this valuable tissue for later use. In this study cryopreservation solutions are validated for their capability to preserve the tissue as freshly obtained material.

Materials and methods Human synovial OA tissue was derived (n = 4), with informed consent, from joint surgery and frozen as 3 mm biopsies. Cryosections were made to prove synovial origin. Biopsies were cryopreserved by slow freezing using several cryoprotectant (CPA) solutions (Cryostor CS2, Cryostor CS10, CryoSFM, Biofreeze and standard freezing medium containing 10% DMSO and 10% FCS). For slow freezing an isopropanol container was used assuring a temperature decrease of 1°C/min. Frozen tissues were stored for 7 days in liquid nitrogen. Thawing was performed at 37°C, followed by quick removal of the CPA solution.

Results To asses general viability after thawing the tissue was cultured overnight. Both the ATP and XTT assays showed viability rates up to 90% of non frozen tissue. However, decreased viability (60%) was observed with higher concentrations of DMSO (CS10 and standard media, XTT) and the complete absence of DMSO (Biofreeze, ATP). Next, the RNA integrity number (RIN) was determined 24 h after thawing to ensure the RNA is suitable for gene array experiments. A high RIN (˜8) was found in all conditions. Stress genes (CASP3, HSPA1A, HSP27, MCL1, BAX, CD95, p53) described to be up/downregulated after cryopreservation did not show deviations from the non frozen tissue in the four donors tested. However, studying OA characteristic gene expression (TIMP3, CCL18, MMP9) showed that the Biofreeze medium had most changes (up/down) compared to non frozen tissue. Furthermore, we showed that the tissue was able to respond to inflammatory stimuli (Pam3Cys/LPS) as cytokine secretion (IL1b, TNFa, IL6, IL8) was highly upregulated.

Conclusion Evaluation by two viability assays, stress – and disease characteristic gene expression, and cytokine secretion showed that during a short-time culture of human synovial tissue the disease characteristic phenotype was unchanged after cryopreservation.

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