Functional Tissue Analysis Reveals Successful Cryopreservation of Human Osteoarthritic Synovium

<div><p>Osteoarthritis (OA) is a degenerative joint disease affecting cartilage and is the most common form of arthritis worldwide. One third of OA patients have severe synovitis and less than 10% have no evidence of synovitis. Moreover, synovitis is predictive for more severe disease progression. This offers a target for therapy but more research on the pathophysiological processes in the synovial tissue of these patients is needed. Functional studies performed with synovial tissue will be more approachable when this material, that becomes available by joint replacement surgery, can be stored for later use. We set out to determine the consequences of slow-freezing of human OA synovial tissue. Therefore, we validated a method that can be applied in every routine laboratory and performed a comparative study of five cryoprotective agent (CPA) solutions. To determine possible deleterious cryopreservation-thaw effects on viability, the synovial tissue architecture, metabolic activity, RNA quality, expression of cryopreservation associated stress genes, and expression of OA characteristic disease genes was studied. Furthermore, the biological activity of the cryopreserved tissue was determined by measuring cytokine secretion induced by the TLR ligands lipopolysaccharides and Pam3Cys. Compared to non frozen synovium, no difference in cell and tissue morphology could be identified in the conditions using the CS10, standard and CryoSFM CPA solution for cryopreservation. However, we observed significantly lower preservation of tissue morphology with the Biofreeze and CS2 media. The other viability assays showed trends in the same direction but were not sensitive enough to detect significant differences between conditions. In all assays tested a clearly lower viability was detected in the condition in which synovium was frozen without CPA solution. This detailed analysis showed that OA synovial tissue explants can be cryopreserved while maintaining the morphology, viability and phenotypical response after thawing, offering enhanced opportunities for human <i>in vitro</i> studies.</p></div

Histological analysis of cryopreserved synovial explants.

<p>(A) Histological preservation was determined by assessing global histological parameters using an arbitrary score. Mean score is displayed. A significantly lower score was determined for the CS2, Biofreeze and without CPA condition. Blue dots: patient 1, black dots: patient 2, grey dots: patient 3. For statistical analysis, one-way ANOVA was performed, comparing the non frozen control to the cryopreserved conditions. *** p<0.001. Displayed is the mean. (B) Non frozen highly cellular synovium from patient 1. (C) Synovium of low cellularity cryopreserved with CryoSFM of patient 2 displaying an intact and smooth intimal lining layer (arrow), intact cells and blood vessels (*). (D) Synovium from patient 3 cryopreserved with CryoSFM, displaying mainly intimal lining hyperplasia (arrow), the lining is smooth and intact, cells and blood vessels (*) are intact. (E) Synovium of patient 2 cryopreserved using the Biofreeze medium, showing a disrupted intimal lining layer (arrow), a high number of disrupted cells and disrupted blood vessels (*). (F) Synovium of patient 1 frozen without protecting CPA solution showing almost complete disruption of cells.</p

Cryopreserved synovial tissue is responsive to inflammatory stimuli.

<p>The secretion of proinflammatory cytokines IL1β (A), TNFα (B), IL6 (C) and IL8 (D) was determined before and after 24 hour stimulation with P3C/LPS. Displayed is the mean +/- SD. Statistical analysis was performed by Students t-test, comparing the unstimulated control to the stimulated conditions. ** p<0.01; *** p<0.001.</p

No effect of cryopreservation on RNA integrity in synovial explants.

<p>The RIN was determined in triplo of OA synovial tissue from four patients, 24 hours after thawing. Numbers indicate the number of biopsies of which the RIN could be successfully determined. Displayed is the mean +/- the SD. Statistical analysis was performed by one-way ANOVA, comparing the non-frozen control to the cryopreserved conditions.</p

No effect of cryopreservation on disease characteristic gene expression.

<p>Gene expression of <i>TIMP3</i> (A), <i>CCL18</i> (B) and <i>MMP9</i> (C) was determined after thawing and 24 hours culture of synovial biopsies of four patients. Per patient 3 biopsies were included in the analysis. Patient 6: green dots, patient 7: black dots, patient 8: dark blue dots, patient 4: light blue dots. Displayed is the mean +/- SD. Statistical analysis was performed by one-way ANOVA, comparing the non-frozen control to the cryopreserved conditions.</p

Analysis of cryopreservation-induced changes in disease- characteristic gene expression compared to non-frozen tissue in individual OA patients.

<p>Analysis of cryopreservation-induced changes in disease- characteristic gene expression compared to non-frozen tissue in individual OA patients.</p

No increase in stress gene expression after cryopreservation.

<p>Gene expression of (A) <i>HSPA1a</i>, <i>(B) HSP27</i>, <i>(C) CASP3</i>, <i>(D) BAX</i>, <i>(E) CD95</i> and <i>(F) MCL1</i> was determined by qPCR analysis in four patients, 24 hours after thawing. Patient 6: green dots, patient 7: black dots, patient 8: dark blue dots, patient 4: light blue dots. Displayed is the mean. Statistical analysis was performed by one-way ANOVA, comparing the non-frozen control to the cryopreserved conditions. ** p<0.01; *** p<0.001.</p

No change in metabolic activity in synovial explants after thawing.

<p>ATP and XTT levels were determined in triplo in OA synovial tissue of 3 patients. Patient 4: blue dots, patient 5: black dots, patient 1: grey dots. (A) Shown are the relative light units (RLU) (B) Shown is the percentage increase in absorbance over a 4 hour period, depicted as the mean +/- the SD. Statistical analysis was performed by one-way ANOVA, comparing the non-frozen control to the cryopreserved conditions. *** p<0.001.</p

Highly reduced Th17 differentiation in the absence of both IL-6 and IL-21 signaling.

<p>WT and IL-21R^-/- naïve T cells were stimulated for four days with a differentiation cocktail either with or without IL-6 as described in <i>Methods</i>. Representative flow cytometry dot plots reflecting the IL-17+ fraction of the CD4+ population. Gates were set at a maximum of 0.3% IL-17-positive cells in the ‘fluorescence minus one’ control per condition, without addition of IL-17A antibodies (A). Summary of the relative proportion of IL-17+ cells within the CD4+ population (B). Culture supernatant levels of IL-17 (C), IL-21 (D), and IL-22 (E). 6–10 mice/group; *p<0.05, **p<0.01, ***p<0.001 versus WT + IL-6; ^###p<0.001 versus WT—IL-6; ^p<0.05, ^^^p<0.001 versus IL-21R^-/- + IL-6; A—Kruskal-Wallis, B-D—One-way ANOVA.</p

IL-6 and IL-21 play an important role during in vivo Th17 differentiation and antibody production.

<p>AIA was induced in WT, IL-6^-/-, IL-21R^-/-, and IL-6^-/- x IL-21R^-/- mice. Draining lymph node CD4+IL-17+ cell fraction two days after arthritis induction as measured using flow cytometry (A; n = 10/group). Serum IgG1, IgG2b, and total IgG levels as measured by ELISA (B; n = 5/group). *p<0.05, **p<0.01, ***p<0.001 versus WT; ^##p<0.01 versus IL-6^-/-; ^^^p<0.001 versus IL-21R^-/-; A—One-way ANOVA, B—Kruskal-Wallis.</p