{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T04:12:49Z","timestamp":1792210369257,"version":"4.4.0"},"reference-count":33,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,10,4]],"date-time":"2025-10-04T00:00:00Z","timestamp":1759536000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>Background: Cryopreservation of hematopoietic stem cells (HSCs) at \u221280 \u00b0C using uncontrolled-rate freezing is frequently employed in resource-constrained settings, yet concerns remain regarding long-term viability and clinical efficacy. Reliable post-thaw assessment is essential to ensure graft quality and engraftment success. Methods: This single-center, retrospective study evaluated 72 cryopreserved stem cell products from 25 patients stored at \u221280 \u00b0C for a median of 868 days. Viability was assessed using both acridine orange (AO) staining and 7-AAD (7-aminoactinomycin D) flow cytometry at three time points: collection (T0), pre-infusion (T1), and delayed post-thaw evaluation (T2). Associations between viability loss, storage duration, and clinical engraftment outcomes were analyzed. Results: Median post-thaw viability remained high (94.8%) despite a moderate time-dependent decline (~1.02% per 100 days; R2 = 0.283, p &lt; 0.001). Mean viability loss at T2 was 9.2% (AO) and 6.6% (flow cytometry). AO demonstrated greater sensitivity to delayed degradation, with a significant difference between methods (p &lt; 0.001). Engraftment kinetics were preserved in most patients, with neutrophil and platelet recovery primarily influenced by disease type rather than product integrity. Notably, storage duration and donor age were not significantly associated with engraftment outcomes or CD34+ cell dose. Conclusions: Long-term cryopreservation at \u221280 \u00b0C maintains HSC viability sufficient for durable engraftment, despite gradual decline. While transplant outcomes are primarily dictated by disease biology and remission status, AO staining provides enhanced sensitivity for detecting delayed cellular damage. Notably, our viability-loss model offers a practical framework for predicting product quality, potentially supporting graft selection and clinical decision-making in real-world, resource-constrained transplant settings.<\/jats:p>","DOI":"10.3390\/jcm14197032","type":"journal-article","created":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T13:33:41Z","timestamp":1759757621000},"page":"7032","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Viable and Functional: Long-Term \u221280 \u00b0C Cryopreservation Sustains CD34+ Integrity and Transplant Success"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9907-1498","authenticated-orcid":false,"given":"Ibrahim Ethem","family":"Pinar","sequence":"first","affiliation":[{"name":"Department of Hematology, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muge","family":"Sahin","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vildan","family":"Gursoy","sequence":"additional","affiliation":[{"name":"Department of Hematology, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5419-3221","authenticated-orcid":false,"given":"Tuba","family":"Ersal","sequence":"additional","affiliation":[{"name":"Department of Hematology, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7625-9148","authenticated-orcid":false,"given":"Ferah","family":"Budak","sequence":"additional","affiliation":[{"name":"Department of Immunology, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vildan","family":"Ozkocaman","sequence":"additional","affiliation":[{"name":"Department of Hematology, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9710-134X","authenticated-orcid":false,"given":"Fahir","family":"Ozkalemkas","sequence":"additional","affiliation":[{"name":"Department of Hematology, Bursa Uludag University Faculty of Medicine, 16059 Bursa, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"870","DOI":"10.21873\/invivo.13890","article-title":"Should we Cryopreserve Allogenic Hematopoietic Stem Cell Grafts?","volume":"39","author":"Bembnista","year":"2025","journal-title":"Vivo"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"777.e1","DOI":"10.1016\/j.jtct.2023.08.032","article-title":"Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at \u221280 \u00b0C: An Effective Option in Resource-Limited Settings","volume":"29","author":"Gokarn","year":"2023","journal-title":"Transplant. 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