ee961b46adbe30f030e85ffe09f91166 283217.pdf ec5414e26082284fa8a3d72eb7eae425ed0c8f61 283217.pdf bb339c288101e2ad75ab8049d246ea669dd22b4555621e8453780e439787756a 283217.pdf Title: A Comparative Study Using Fluorescent Confocal Microscopy and Flow Cytometry to Evaluate Chondrocyte Viability in Human Osteochondral Allografts Subject: The preservation conditions of fresh osteochondral allografts for clinical applications are critical due their objective: to transplant mature hyaline cartilage containing viable chondrocytes, maintaining their metabolic activity and also preserving the structural and functional characteristics of the extracellular matrix. The aim of the present study was to compare fluorescence confocal microscopy and flow cytometry techniques to evaluate the viability of the chondrocytes present in the osteochondral tissue, in order to determine their effectiveness and thus ensure reproducibility and robustness of the analysis. To this end, osteochondral allografts from human cadaveric donors were preserved at 4 C for 3 weeks in a preservation medium supplemented with antibiotic and antifungal agents. Cell viability of chondrocytes was determined by monitoring the cartilage for 3 weeks of preservation by confocal fluorescence microscopy and flow cytometry, obtaining cell viabilities of 83.7 2.6% and 55.8 7.8% for week three, respectively. The confocal fluorescence microscopy approach is more advantageous and accurate, as it correlates better with actual cell viability values for monitoring osteochondral graft preservation, detecting only the cells that died a natural death associated with the preservation method. Keywords: osteoarthritis; osteochondral grafts; chondrocytes cell viability; graft preservation; confocal fluorescence microscopy; flow cytometry Author: Patricia López-Chicón, Tatiana Riba-Tietz, Oscar Fariñas, Pablo-Eduardo Gelber, Ricardo-Pedro Casaroli-Marano and Anna Vilarrodona Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Sep 29 13:46:27 2022 CEST ModDate: Thu Sep 29 13:53:51 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2830813 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- HHIVEO+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 CXSDFF+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 BXHDBT+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 VYVZGZ+CMSY10 Type 1 Builtin yes yes no 23 0 FPRZBG+VnURWPalladioL Type 1 Custom 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FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 177 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 193 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 199 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 205 0 FNTSBS+ArialMT CID TrueType Identity-H yes yes yes 211 0 FNTSBS+Arial-BoldMT CID TrueType Identity-H yes yes yes 217 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 228 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 234 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 240 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-11-04 02:29:19 CET RepresentationInformation: 283217.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-11-03 12:26:52 CET Size: 2830813 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 374 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Children: Item: Title: Ethical Considerations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Osteochondral Tissue Processing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Cell Viability by Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Cell Viability by Confocal Fluorescence Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Children: Item: Title: Cell Characterization by Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Cell Viability Calculation and Apoptosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Cell Viability by Confocal Fluorescence Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Info: Title: A Comparative Study Using Fluorescent Confocal Microscopy and Flow Cytometry to Evaluate Chondrocyte Viability in Human Osteochondral Allografts Author: Patricia López-Chicón, Tatiana Riba-Tietz, Oscar Fariñas, Pablo-Eduardo Gelber, Ricardo-Pedro Casaroli-Marano and Anna Vilarrodona Subject: The preservation conditions of fresh osteochondral allografts for clinical applications are critical due their objective: to transplant mature hyaline cartilage containing viable chondrocytes, maintaining their metabolic activity and also preserving the structural and functional characteristics of the extracellular matrix. The aim of the present study was to compare fluorescence confocal microscopy and flow cytometry techniques to evaluate the viability of the chondrocytes present in the osteochondral tissue, in order to determine their effectiveness and thus ensure reproducibility and robustness of the analysis. To this end, osteochondral allografts from human cadaveric donors were preserved at 4 C for 3 weeks in a preservation medium supplemented with antibiotic and antifungal agents. Cell viability of chondrocytes was determined by monitoring the cartilage for 3 weeks of preservation by confocal fluorescence microscopy and flow cytometry, obtaining cell viabilities of 83.7 2.6% and 55.8 7.8% for week three, respectively. The confocal fluorescence microscopy approach is more advantageous and accurate, as it correlates better with actual cell viability values for monitoring osteochondral graft preservation, detecting only the cells that died a natural death associated with the preservation method. 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