Evaluation of hematopoietic stem cell cryopreservation: A comparative study of liquid nitrogen versus storage at -80°C

Evaluation of hematopoietic stem cell cryopreservation: A comparative study of liquid nitrogen versus storage at -80°C | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of hematopoietic stem cell cryopreservation: A comparative study of liquid nitrogen versus storage at -80°C Zakaria This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7642582/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hematopoietic stem cell (HSC) cryopreservation is a critical component of cellular therapy that directly influences transplant outcomes. Although mechanical freezing at − 80°C is widely used in resource-limited settings, liquid nitrogen (LN₂) cryopreservation with controlled-rate freezing remains the gold standard for long-term storage. However, comparative data on the efficacy and operational feasibility of these methods remain scarce. This study aimed to compare the efficacy, cellular integrity, and viability of HSC grafts preserved via LN₂ cryopreservation with those preserved via mechanical freezing at − 80°C. Additionally, we sought to validate a controlled-rate freezing protocol and evaluate its clinical applicability within a cell therapy unit. We conducted four experimental trials: (1) validation of a controlled-rate freezing protocol, (2) assessment of postthaw recovery in low-cell-density samples, and (3–4) a direct comparison between LN₂ and − 80°C cryopreservation using paired aliquots. Postthaw CD34⁺ cell counts were analyzed via flow cytometry, and freezing curves were recorded to assess the consistency of the decrease in temperature. Both methods demonstrated comparable short-term CD34⁺ cell viability, with similar postthaw cell loss rates (~ 2 × 10⁶ cells/kg). However, LN₂ cryopreservation provided superior thermal control, as evidenced by strict adherence to the programmed freezing curve. Moreover, the LN₂ method reduced the reliance on scarce cryoprotectants such as Voluven®, enhancing reproducibility and long-term storage potential. No instances of bag leakage or contamination were observed with sterile packaging. While − 80°C cryopreservation remains a viable short-term option, LN₂ cryopreservation offers distinct advantages in terms of thermal stability, cryoprotectant flexibility, and long-term cell viability. Our validated LN₂ protocol meets clinical standards for safety, sustainability, and quality, confirming its role as the preferred method for high-quality HSC biobanking and transplantation. Further studies incorporating functional assays and extended follow-up are needed to assess long-term engraftment potential. Stem Cell & Developmental Cell Biology Hematopoietic stem cells cryopreservation liquid nitrogen –80°C storage CD34⁺ cells cell viability stem cell transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The cryopreservation of hematopoietic stem cells (HSCs) plays a vital role in modern transplantation medicine. These cells, which are essential for treating blood disorders and cancers, must maintain viability during storage [ 1 ]. While−80°C freezing has been the standard, liquid nitrogen (−196°C) offers potential advantages for long-term preservation [ 2 ]. This study compares both methods to determine which method better preserves cell viability and function. The importance of reliable cryopreservation cannot be overstated. Patients receiving stem cell transplants depend on these cells being healthy and functional after thawing [ 3 ]. Poor preservation can lead to transplant failure, putting lives at risk. Our research aims to provide clear data to help clinics choose the best storage method [ 4 ]. Recent advances in cryobiology have improved freezing techniques. However, few studies have directly compared liquid nitrogen and−80°C freezing for HSCs [ 5 ]. Our work fills this gap by systematically evaluating both methods under controlled conditions. We examine not only cell survival but also practical factors such as equipment needs and costs. This study aims to evaluate the feasibility, efficacy, and reliability of HSC cryopreservation in liquid nitrogen within our cell therapy unit. Three experimental assays were performed to validate the controlled-rate freezing protocol, verify the integrity of the storage bags and postthaw cell recovery, and compare this method to traditional freezing at−80°C in terms of its impact on graft richness. Methods & Materials To conduct this study on the cryopreservation of HSCs via both the−80°C and liquid nitrogen methods, all essential materials and logistical requirements were systematically organized, as presented in Tables 1 and 2 . Table 1 Equipment and consumables for cryopreservation with liquid nitrogen Equipment Consumables - Sterile connection device - +4°C refrigerator - Class 100 vertical laminar flow hood (PSM) - Precision scale - Tube sealer - Forceps - Refrigerated bag centrifuge - Plasma press - Scissors - Stainless steel strainer - Stainless steel trays - Cooling blocks - Cardboard cassette - Sterile drape - Manual tube stripper −18G needle −20% albumin stored at + 4°C −70% alcohol - Pediatric blood culture flask - Povidone-iodine - Sterile compresses - Cryotubes - DMSO (10 ml) - Labels - Surgical gloves - Sterile gloves - Blades for sterile connection −100 ml freezing bags −600 ml transfer bags - Sampling equipment - Syringes (60 ml, 10 ml, 5 ml, 1 ml) with needles −0.9% NaCl Table 2 Equipment and consumables for cryopreservation at−80°C Equipment Consumables - Sterile connection device - +4°C refrigerator - Class 100 vertical laminar flow hood (PSM) - Precision scale - Tube sealer - Forceps - Refrigerated bag centrifuge - Plasma press - Scissors - Stainless steel strainer - Stainless steel trays - Cooling blocks - Cardboard cassette - Sterile drape - Manual tube stripper −18G needle −20% albumin stored at + 4°C −70% alcohol - Pediatric aerobic blood culture flask - Povidone-iodine - Sterile compresses - Cryotubes - DMSO (10 ml) - Labels - Surgical gloves - Sterile gloves - Blades for sterile connection - Freezing bags (60 ml or 100 ml) −600 ml transfer bags - Sampling equipment - Syringes (60 ml, 10 ml, 5 ml, 1 ml) with needles - Voluven (hydroxyethyl starch) The experimental workflow followed a logical progression : 1. Equipment and protocol validation (Trial 1) 2. Low-concentration cell recovery assessment (Trial 2) 3. Comparative analysis of preservation methods (Trials 3–4) Trial 1: Freezing Protocol Validation and Temperature Gradient Evaluation This trial aimed to validate the controlled-rate freezing protocol using the Consarctic® device and assess cryobag integrity during liquid nitrogen storage. An HSC bag was processed through a standardized freezing cycle, beginning with gradual cooling at−1°C/min to−4°C, followed by manual seeding and subsequent cooling phases (−1°C/min to−40°C, then−5°C/min to−125°C) before being transferred to vapor-phase liquid nitrogen storage (−196°C) in tank PS151. After 63 days of cryopreservation, the bag was thawed and evaluated, with no observed bag leakage or full equipment functionality. Trial 2: Low-Concentration HSC Recovery Assessment The freezing process was conducted via liquid nitrogen at−196°C with 10% DMSO as the cryoprotectant, following the validated protocol from the initial trial, to assess the freezing procedure, verify the effectiveness of the gradual freezing cycle, and test cell recovery after thawing in a low-cell-density bag. Postthaw, a sample was sent for flow cytometry analysis to evaluate cellular richness by quantifying CD34 + cells. Trials 3 and 4: Comparison of Preservation Methods This study aimed to evaluate the impact of liquid nitrogen cryopreservation on HSC graft quality by comparing two methods: liquid nitrogen freezing and freezing at−80°C. An initial HSC sample was equally divided into two aliquots. Aliquots A were cryopreserved in liquid nitrogen via the validated protocol, whereas aliquot B was frozen at−80°C following standard procedures. Following thawing, the samples were analyzed via flow cytometry to quantify CD34 + cell counts and assess potential cryopreservation-induced modifications to the cellular composition. This paired comparison design enabled direct evaluation of method-specific effects on cell preservation efficacy while controlling for donor-related variables. Results An HSC graft was collected at an initial concentration of 0.6 × 10⁶ CD34 + cells/kg with a postthaw concentration of 0.3 × 10⁶ CD34 + cells/kg. The temperature descent curve ( Fig. 2 ) illustrates adherence to the programmed controlled-rate freezing profile using the Consarctic® device, from room temperature to−125°C, before transfer into liquid nitrogen. In Trial 3, a cryocyte with an initial richness of 3.6 × 10⁶ cells/kg was thawed, resulting in a loss of 2.1 × 10⁶ cells/kg, likely due to the very high number of CNTs, measured at 213.12 × 10³. In Trial 4, after freezing at−80°C, the initial richness was 7.2 × 10⁶ cells/kg, and a loss of 2 × 10⁶ cells/kg was observed after thawing. The curve ( Fig. 4 ) shows a gradual and controlled temperature decrease to approximately−120°C, in accordance with the protocol established via the Consarctic device, before being transferred into liquid nitrogen for cryostorage. This curve confirms the proper execution of the freezing process, which is essential for preserving the integrity and richness of the graft. The bag stored at−80°C was not subjected to temperature recording but was frozen via standard procedures. Discussion This study presents a comparative assessment of two cryopreservation methods: mechanical freezing at−80°C and storage in liquid nitrogen for the preservation of HSCs. Our findings indicate that both techniques effectively maintain short-term cell viability and CD34⁺ marker preservation postthaw. However, critical differences emerge in long-term stability, cryoprotectant dependency, and clinical applicability. Short-term efficacy of mechanical freezing at − 80°C Analysis of postthaw outcomes from Trials 3 and 4 revealed no statistically significant difference in immediate cell loss between the two methods, suggesting that mechanical freezing at−80°C is sufficient for short-term HSC preservation. An equivalent degree of cell loss (−2 cells/kg) was observed following both liquid nitrogen and−80°C cryopreservation, indicating that both methods are comparably effective in maintaining HSC viability and the CD34⁺ cell concentration. These results align with those of Campos-Carli et al. (2024), who reported stable HSC viability at−80°C for up to six years when nitrogen storage remains unfeasible, with no significant correlation between storage duration, CD34⁺ cell concentration, and engraftment kinetics [ 6 ]. Despite these findings, mechanical freezing has notable limitations. Unlike controlled-rate freezing, it lacks precise thermal regulation, increasing the risk of intracellular ice crystal formation and subsequent cellular damage. Additionally, the clinical scalability of−80°C protocols is constrained by the limited global availability of Voluven® (hydroxyethyl starch, HES), a key extracellular cryoprotectant in many clinical-grade formulations; however, it may cause allergic reactions [ 8 ]. While adjunctive agents such as Albumine have demonstrated improved thermal stability and reduced recrystallization in experimental settings, their use remains nonstandardized for clinical HSC cryopreservation and has primarily been validated in pluripotent stem cell models to improve regenerative therapies [ 9 , 10 ]. Thus, while mechanical freezing may serve as a provisional alternative in resource-limited settings, its long-term reliability and reproducibility remain uncertain. Further investigations incorporating functional assays such as colony-forming unit (CFU) analysis and in vivo hematopoietic reconstitution models are necessary to comprehensively evaluate the regenerative potential of−80°C-preserved HSCs [ 11 ]. Liquid Nitrogen Cryopreservation: The Gold Standard In contrast, LN₂ cryopreservation, particularly when preceded by controlled-rate freezing, remains the gold standard for long-term HSC storage. The gradual temperature reduction minimizes intracellular ice formation, preserves membrane integrity, and mitigates cellular stress. As demonstrated by the curve ( Fig. 2 ), the stability and consistency of the freezing cycles, which are essential factors for ensuring postthaw cell viability, were confirmed. Furthermore, as shown by the freezing curve in Fig. 4 , our data support established cryobiological principles, confirming that controlled-rate freezing followed by ultralow-temperature (–196°C) storage is critical for maintaining HSC structural and functional integrity. Hornberger et al. (2019) emphasized that such conditions halt metabolic activity, preserving HSC clonogenicity and engraftment potential for over a decade [ 12 ]. Furthermore, their review underscores the importance of combining dimethyl sulfoxide (DMSO) with extracellular cryoprotectants (HESs) to reduce DMSO-associated cytotoxicity, which was implemented in our study to increase postthaw viability while sustaining long-term stem cell function [ 12 ]. From a biosafety perspective, we addressed potential microbial contamination risks in LN₂ storage by utilizing sterile, single-use protective packaging, ensuring cryobag integrity throughout storage and handling. Clinical and logistic considerations When cryopreservation methods are selected for clinical and biobanking applications, infrastructure, reagent availability, and protocol standardization are critical factors [ 13 ]. Although−80°C systems offer logistical and cost advantages, their reliance on nonstandardized cryoprotectants (e.g., Voluven®) and the absence of long-term clinical validation for HSCs restrict their widespread adoption [ 14 ]. Conversely, LN₂-based systems provide a well-established, standardized platform for long-term HSC preservation [ 14 ]. The compatibility of these materials with controlled-rate freezing and their capacity for large-volume cryobanking reinforce their clinical superiority. Moreover, as demonstrated in this study, LN₂ protocols can be optimized to mitigate contamination risks, increasing their suitability for both research and therapeutic applications [ 15 ]. Future directions While our results confirm the short-term equivalence of−80°C and LN₂ cryopreservation in terms of viability metrics, functional assessments are essential for validating long-term regenerative capacity. Future research should prioritize functional characterization through clonogenic (CFU) assays and in vivo hematopoietic reconstitution studies, alongside comparative clinical analyses of engraftment efficiency in transplant recipients [ 16 , 17 ]. Additionally, economic and logistical evaluations of cryoprotectant utilization across preservation platforms should be conducted [ 18 , 19 ]. Further efforts must focus on optimizing−80°C protocols for clinical application, including the standardization of cryoprotectant formulations and extended validation of storage efficacy beyond several years. Study Limitations This study has several limitations. First, functional assessments (clonogenic potential, engraftment capacity) were not performed despite evaluating viability and CD34⁺ preservation. Second, the sample size may limit broader applicability. Third, the Voluven® dependence of the−80°C protocol affects reproducibility. Additionally, postthaw immunophenotypic changes were not assessed. Finally, longer-term storage effects, especially for−80°C preservation, require further study. Conclusion Experimental trials have established liquid nitrogen cryopreservation as a superior approach for long-term hematopoietic stem cell (HSC) preservation. Our validation of the controlled-rate freezing protocol of the Consarctic system confirmed both cryobag integrity and reliable postthaw cellular recovery. While−80°C cryopreservation results in comparable short-term viability, LN₂ provides critical advantages, including precise thermal regulation, elimination of volatile dependency, and indefinite storage capability without compromising cell function. Although−80°C remains viable for transient storage, its practical constraints position LN₂ as the optimal choice for clinical HSC banking. The implemented protocol has both immediate clinical utility and sustainable long-term preservation, meeting the rigorous demands of modern cellular therapies. Future refinements should focus on cryoprotectant optimization to increase recovery while preserving these demonstrated benefits. References Berz D, McCormack EM, Winer ES, Colvin GA, Quesenberry PJ (2007) Cryopreservation of hematopoietic stem cells. Am J Hematol 82(6):463–472. 10.1002/ajh.20707 Prisciandaro M, Santodirocco M (2024) Innovation in hematopoietic stem cell cryopreservation and cold chain management. Cell Gene Ther Insights 10(3):345–362. 10.18609/cgti.2024.050 Bennett B, Hanotaux J, Pasala AR, Hasan T, Hassan D, Shor R et al (2024) Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies. Cytotherapy 26(5):482–489. 10.1016/j.jcyt.2024.02.006 Mancias-Guerra C, Valdés-Burnes SL, Gonzalez-Llano O, Aguirre-Fernandez GC, Villarreal-Martinez L, Cantu-Rodriguez O et al (2013) Another method for thawing hematopoietic stem cells and its impact in the recovery of the transplanted hematological patient. Biol Blood Marrow Transpl 19(2 Suppl):S349–S350. 10.1016/j.bbmt.2012.11.543 Desoutter J, Ossart C, Lacassagne MN, Regnier A, Marolleau JP, Harrivel V et al (2019) Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: key role of granulocytes. Cytotherapy 21(6):612–618. 10.1016/j.jcyt.2019.04.004 Campos-Carli SM, D’Ávila A, de Oliveira CF, de Souza GF, Moraes-Vieira PMM, Silva-Costa AC (2024) Cryopreservation of hematopoietic stem cells at–80°C: Impact of storage time on cell viability and engraftment. Transfus Clin Biol 46(3):188–196. 10.1159/000528038 Gomes MRA, Ramos AS, Alves ACL, Zamperlini G, Granja PGG, Cruz DVB et al (2024) Validation of the use of hydroxyethyl starch (Voluven 6%) to remove dimethyl sulfoxide from hematopoietic stem cells. Hematol Transfus Cell Ther 46(Suppl 4):S991–S992 Lee JY, Hong SH (2019) Hematopoietic stem cells and their roles in tissue regeneration. Int J Stem Cells 13(1):1–12. 10.15283/ijsc19127 Kuten Pella O, Hornyák I, Horváthy D, Fodor E, Nehrer S, Lacza Z (2022) Albumin as a biomaterial and therapeutic agent in regenerative medicine. Int J Mol Sci 23(18):10557. 10.3390/ijms231810557 Wang J, Ye D, Li S, Lu X, Chi Y, Tang C, Zhou X, Chen M, Zheng Y, Ge J, Zou Q (2024) Generation of human induced pluripotent stem cells carrying albumin-sfGFP reporter. Stem Cell Res 81:103543. 10.1016/j.scr.2024.103543 Jahan S, Kaushal R, Pasha R, Pineault N (2021) Current and future perspectives for the cryopreservation of cord blood stem cells. Transfus Med Rev 35(2):95–102. 10.1016/j.tmrv.2021.01.003 Hornberger K, Borgmann S, Heinemann FM, Bär A, Wein F, Schmitz M (2019) Cryopreservation of hematopoietic stem cells: Advances and perspectives. Transfus Med Hemother 46(3):188–196. 10.1159/000498948 Yuan X, Yuan F, Wang Y, Cai J, Liao J, Liao L et al (2016) Efficient long-term cryopreservation of pluripotent stem cells at–80°C. Sci Rep 6:21054. 10.1038/srep21054 Gokarn A, Tembhare PR, Syed H, Sanyal I, Kumar R, Parab S et al (2023) Long-term cryopreservation of peripheral blood stem cell harvest using low concentration (4.35%) dimethyl sulfoxide with methyl cellulose and uncontrolled rate freezing at–80°C: An effective option in resource-limited settings. Transpl Cell Ther 29(12):777e1–777e8. 10.1016/j.jtct.2023.08.032 Lecchi L, Giovanelli S, Gagliardi B, Pezzali I, Ratti I, Marconi M (2016) An update on methods for cryopreservation and thawing of hemopoietic stem cells. Transfus Apher Sci 54(3):324–336. 10.1016/j.transci.2016.05.009 Fountain D, Ralston M, Higgins N, Gorlin JB, Uhl L, Wheeler C et al (1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 37(6):585–591. 10.1046/j.1537–2995.1997.37697335152.x Sarma NJ, Takeda A, Yaseen NR (2010) Colony forming cell (CFC) assay for human hematopoietic cells. J Vis Exp 462195. 10.3791/2195 Maqbool S, Nadeem M, Shahroz A, Naimat K, Khan I, Tahir H et al (2022) Engraftment syndrome following hematopoietic stem cell transplantation: a systematic approach toward diagnosis and management. Med Oncol 40(1):36. 10.1007/s12032-022-01894–7 Turney TL, Reese-Koç J, de Lima M, Otegbeye F (2019) Optimizing cryopreservation of hematopoietic stem cells collected for autologous stem cell transplantation in patients with multiple myeloma. Cytotherapy 21(5 Suppl):S39. 10.1016/j.jcyt.2019.03.375 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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07:01:05","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70946,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7642582/v1/a7ee2c1dbb58e7333d3d7850.html"},{"id":91817943,"identity":"7adf15dc-157b-4283-a5fe-7e4528221c7b","added_by":"auto","created_at":"2025-09-22 07:01:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of CD34⁺ cell richness before and after freezing in a low-concentration bag (×10⁶ cells/kg), Trial 2.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7642582/v1/9485e49960d6001db8686d46.png"},{"id":91817918,"identity":"61ed905d-2123-43b5-bb63-9d40d536e705","added_by":"auto","created_at":"2025-09-22 07:00:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":202497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCurves of the temperature decrease recorded during the freezing of the HSC bag – Trial 2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7642582/v1/0ce14d891cbcd108d9acec1a.png"},{"id":91817909,"identity":"2fab54e7-44da-4174-87ab-3661353447b0","added_by":"auto","created_at":"2025-09-22 07:00:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative impact of cryopreservation methods (liquid nitrogen vs. -80°C) on HSC viability after 13 days of storage (short-term cryopreservation)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7642582/v1/0a8b2656356b9a2ba4fdd74d.png"},{"id":91817962,"identity":"5d862324-e62a-454c-bc2d-35ed20e49b37","added_by":"auto","created_at":"2025-09-22 07:01:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemperature profile of HSC cryopreservation (liquid nitrogen method) - Trial 3\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7642582/v1/24648171f37a9b021a738399.png"},{"id":91818834,"identity":"07e1e0e7-e7ca-4983-ae21-12dc70b152c9","added_by":"auto","created_at":"2025-09-22 07:05:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":997540,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7642582/v1/1f0f81ac-96fe-4ebe-acc7-951dae749653.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluation of hematopoietic stem cell cryopreservation: A comparative study of liquid nitrogen versus storage at -80°C\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe cryopreservation of hematopoietic stem cells (HSCs) plays a vital role in modern transplantation medicine. These cells, which are essential for treating blood disorders and cancers, must maintain viability during storage\u003c/span\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWhile\u0026minus;80\u0026deg;C freezing has been the standard, liquid nitrogen (\u0026minus;196\u0026deg;C) offers potential advantages for long-term preservation\u003c/span\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThis study compares both methods to determine which method better preserves cell viability and function.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe importance of reliable cryopreservation cannot be overstated. Patients receiving stem cell transplants depend on these cells being healthy and functional after thawing\u003c/span\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ePoor preservation can lead to transplant failure, putting lives at risk. Our research aims to provide clear data to help clinics choose the best storage method\u003c/span\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eRecent advances in cryobiology have improved freezing techniques. However, few studies have directly compared liquid nitrogen and\u0026minus;80\u0026deg;C freezing for HSCs\u003c/span\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eOur work fills this gap by systematically evaluating both methods under controlled conditions. We examine not only cell survival but also practical factors such as equipment needs and costs. This study aims to evaluate the feasibility, efficacy, and reliability of HSC cryopreservation in liquid nitrogen within our cell therapy unit. Three experimental assays were performed to validate the controlled-rate freezing protocol, verify the integrity of the storage bags and postthaw cell recovery, and compare this method to traditional freezing at\u0026minus;80\u0026deg;C in terms of its impact on graft richness.\u003c/span\u003e\u003c/p\u003e"},{"header":"Methods \u0026 Materials","content":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eTo conduct this study on the cryopreservation of HSCs via both the\u0026minus;80\u0026deg;C and liquid nitrogen methods, all essential materials and logistical requirements were systematically organized, as presented in\u003c/span\u003e Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEquipment and consumables for cryopreservation with liquid nitrogen\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEquipment\u003c/span\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eConsumables\u003c/span\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile connection device\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- +4\u0026deg;C refrigerator\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Class 100 vertical laminar flow hood (PSM)\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Precision scale\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Tube sealer\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Forceps\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Refrigerated bag centrifuge\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Plasma press\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Scissors\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Stainless steel strainer\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Stainless steel trays\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Cooling blocks\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Cardboard cassette\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile drape\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Manual tube stripper\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;18G needle\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;20% albumin stored at +\u0026thinsp;4\u0026deg;C\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;70% alcohol\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Pediatric blood culture flask\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Povidone-iodine\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile compresses\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Cryotubes\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- DMSO (10 ml)\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Labels\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Surgical gloves\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile gloves\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Blades for sterile connection\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;100 ml freezing bags\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;600 ml transfer bags\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sampling equipment\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Syringes (60 ml, 10 ml, 5 ml, 1 ml) with needles\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;0.9% NaCl\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEquipment and consumables for cryopreservation at\u0026minus;80\u0026deg;C\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEquipment\u003c/span\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eConsumables\u003c/span\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile connection device\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- +4\u0026deg;C refrigerator\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Class 100 vertical laminar flow hood (PSM)\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Precision scale\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Tube sealer\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Forceps\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Refrigerated bag centrifuge\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Plasma press\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Scissors\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Stainless steel strainer\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Stainless steel trays\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Cooling blocks\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Cardboard cassette\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile drape\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Manual tube stripper\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;18G needle\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;20% albumin stored at +\u0026thinsp;4\u0026deg;C\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;70% alcohol\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Pediatric aerobic blood culture flask\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Povidone-iodine\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile compresses\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Cryotubes\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- DMSO (10 ml)\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Labels\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Surgical gloves\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sterile gloves\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Blades for sterile connection\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Freezing bags (60 ml or 100 ml)\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026minus;600 ml transfer bags\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Sampling equipment\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Syringes (60 ml, 10 ml, 5 ml, 1 ml) with needles\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e- Voluven (hydroxyethyl starch)\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe experimental workflow followed a logical progression\u003c/span\u003e:\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEquipment and protocol validation (Trial 1)\u003c/span\u003e\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eLow-concentration cell recovery assessment (Trial 2)\u003c/span\u003e\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section4\"\u003e\u003ch2\u003e3. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eComparative analysis of preservation methods (Trials 3\u0026ndash;4)\u003c/span\u003e\u003c/h2\u003e\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eTrial 1: Freezing Protocol Validation and Temperature Gradient Evaluation\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThis trial aimed to validate the controlled-rate freezing protocol using the Consarctic\u0026reg; device and assess cryobag integrity during liquid nitrogen storage. An HSC bag was processed through a standardized freezing cycle, beginning with gradual cooling at\u0026minus;1\u0026deg;C/min to\u0026minus;4\u0026deg;C, followed by manual seeding and subsequent cooling phases (\u0026minus;1\u0026deg;C/min to\u0026minus;40\u0026deg;C, then\u0026minus;5\u0026deg;C/min to\u0026minus;125\u0026deg;C) before being transferred to vapor-phase liquid nitrogen storage (\u0026minus;196\u0026deg;C) in tank PS151. After 63 days of cryopreservation, the bag was thawed and evaluated, with no observed bag leakage or full equipment functionality.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eTrial 2: Low-Concentration HSC Recovery Assessment\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe freezing process was conducted via liquid nitrogen at\u0026minus;196\u0026deg;C with 10% DMSO as the cryoprotectant, following the validated protocol from the initial trial, to assess the freezing procedure, verify the effectiveness of the gradual freezing cycle, and test cell recovery after thawing in a low-cell-density bag. Postthaw, a sample was sent for flow cytometry analysis to evaluate cellular richness by quantifying CD34\u0026thinsp;+\u0026thinsp;cells.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eTrials 3 and 4: Comparison of Preservation Methods\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThis study aimed to evaluate the impact of liquid nitrogen cryopreservation on HSC graft quality by comparing two methods: liquid nitrogen freezing and freezing at\u0026minus;80\u0026deg;C. An initial HSC sample was equally divided into two aliquots. Aliquots A were cryopreserved in liquid nitrogen via the validated protocol, whereas aliquot B was frozen at\u0026minus;80\u0026deg;C following standard procedures. Following thawing, the samples were analyzed via flow cytometry to quantify CD34\u0026thinsp;+\u0026thinsp;cell counts and assess potential cryopreservation-induced modifications to the cellular composition. This paired comparison design enabled direct evaluation of method-specific effects on cell preservation efficacy while controlling for donor-related variables.\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAn HSC graft was collected at an initial concentration of 0.6 × 10⁶ CD34 + cells/kg with a postthaw concentration of 0.3 × 10⁶ CD34 + cells/kg.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe temperature descent curve (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e) illustrates adherence to the programmed controlled-rate freezing profile using the Consarctic® device, from room temperature to−125°C, before transfer into liquid nitrogen.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eIn Trial 3, a cryocyte with an initial richness of 3.6 × 10⁶ cells/kg was thawed, resulting in a loss of 2.1 × 10⁶ cells/kg, likely due to the very high number of CNTs, measured at 213.12 × 10³. In Trial 4, after freezing at−80°C, the initial richness was 7.2 × 10⁶ cells/kg, and a loss of 2 × 10⁶ cells/kg was observed after thawing.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe curve (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e) shows a gradual and controlled temperature decrease to approximately−120°C, in accordance with the protocol established via the Consarctic device, before being transferred into liquid nitrogen for cryostorage. This curve confirms the proper execution of the freezing process, which is essential for preserving the integrity and richness of the graft. The bag stored at−80°C was not subjected to temperature recording but was frozen via standard procedures.\u003c/span\u003e\u003c/p\u003e\n\n\n\n\n\n"},{"header":"Discussion","content":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThis study presents a comparative assessment of two cryopreservation methods: mechanical freezing at−80°C and storage in liquid nitrogen for the preservation of HSCs. Our findings indicate that both techniques effectively maintain short-term cell viability and CD34⁺ marker preservation postthaw. However, critical differences emerge in long-term stability, cryoprotectant dependency, and clinical applicability.\u003c/span\u003e\u003c/p\u003e\u003ch3\u003eShort-term efficacy of mechanical freezing at − 80°C\u003c/h3\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAnalysis of postthaw outcomes from Trials 3 and 4 revealed no statistically significant difference in immediate cell loss between the two methods, suggesting that mechanical freezing at−80°C is sufficient for short-term HSC preservation. An equivalent degree of cell loss\u003c/span\u003e \u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003e(−2 cells/kg)\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewas observed following both liquid nitrogen and−80°C cryopreservation, indicating that both methods are comparably effective in maintaining HSC viability and the CD34⁺ cell concentration. These results align with those of Campos-Carli et al. (2024), who reported stable HSC viability at−80°C for up to six years when nitrogen storage remains unfeasible, with no significant correlation between storage duration, CD34⁺ cell concentration, and engraftment kinetics\u003c/span\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDespite these findings, mechanical freezing has notable limitations. Unlike controlled-rate freezing, it lacks precise thermal regulation, increasing the risk of intracellular ice crystal formation and subsequent cellular damage. Additionally, the clinical scalability of−80°C protocols is constrained by the limited global availability of Voluven® (hydroxyethyl starch, HES), a key extracellular cryoprotectant in many clinical-grade formulations; however, it may cause allergic reactions\u003c/span\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWhile adjunctive agents such as Albumine have demonstrated improved thermal stability and reduced recrystallization in experimental settings, their use remains nonstandardized for clinical HSC cryopreservation and has primarily been validated in pluripotent stem cell models to improve regenerative therapies\u003c/span\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThus, while mechanical freezing may serve as a provisional alternative in resource-limited settings, its long-term reliability and reproducibility remain uncertain. Further investigations incorporating functional assays such as colony-forming unit (CFU) analysis and in vivo hematopoietic reconstitution models are necessary to comprehensively evaluate the regenerative potential of−80°C-preserved HSCs\u003c/span\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eLiquid Nitrogen Cryopreservation: The Gold Standard\u003c/h2\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eIn contrast, LN₂ cryopreservation, particularly when preceded by controlled-rate freezing, remains the gold standard for long-term HSC storage. The gradual temperature reduction minimizes intracellular ice formation, preserves membrane integrity, and mitigates cellular stress. As demonstrated by the curve (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), the stability and consistency of the freezing cycles, which are essential factors for ensuring postthaw cell viability, were confirmed. Furthermore, as shown by the freezing curve in\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eour data support established cryobiological principles, confirming that controlled-rate freezing followed by ultralow-temperature (–196°C) storage is critical for maintaining HSC structural and functional integrity. Hornberger et al. (2019) emphasized that such conditions halt metabolic activity, preserving HSC clonogenicity and engraftment potential for over a decade\u003c/span\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eFurthermore, their review underscores the importance of combining dimethyl sulfoxide (DMSO) with extracellular cryoprotectants (HESs) to reduce DMSO-associated cytotoxicity, which was implemented in our study to increase postthaw viability while sustaining long-term stem cell function\u003c/span\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eFrom a biosafety perspective, we addressed potential microbial contamination risks in LN₂ storage by utilizing sterile, single-use protective packaging, ensuring cryobag integrity throughout storage and handling.\u003c/span\u003e\u003c/p\u003e\u003ch3\u003eClinical and logistic considerations\u003c/h3\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWhen cryopreservation methods are selected for clinical and biobanking applications, infrastructure, reagent availability, and protocol standardization are critical factors\u003c/span\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAlthough−80°C systems offer logistical and cost advantages, their reliance on nonstandardized cryoprotectants (e.g., Voluven®) and the absence of long-term clinical validation for HSCs restrict their widespread adoption\u003c/span\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eConversely, LN₂-based systems provide a well-established, standardized platform for long-term HSC preservation\u003c/span\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe compatibility of these materials with controlled-rate freezing and their capacity for large-volume cryobanking reinforce their clinical superiority. Moreover, as demonstrated in this study, LN₂ protocols can be optimized to mitigate contamination risks, increasing their suitability for both research and therapeutic applications\u003c/span\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003ch3\u003eFuture directions\u003c/h3\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWhile our results confirm the short-term equivalence of−80°C and LN₂ cryopreservation in terms of viability metrics, functional assessments are essential for validating long-term regenerative capacity. Future research should prioritize functional characterization through clonogenic (CFU) assays and in vivo hematopoietic reconstitution studies, alongside comparative clinical analyses of engraftment efficiency in transplant recipients\u003c/span\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAdditionally, economic and logistical evaluations of cryoprotectant utilization across preservation platforms should be conducted\u003c/span\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eFurther efforts must focus on optimizing−80°C protocols for clinical application, including the standardization of cryoprotectant formulations and extended validation of storage efficacy beyond several years.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eStudy Limitations\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThis study has several limitations. First, functional assessments (clonogenic potential, engraftment capacity) were not performed despite evaluating viability and CD34⁺ preservation. Second, the sample size may limit broader applicability. Third, the Voluven® dependence of the−80°C protocol affects reproducibility. Additionally, postthaw immunophenotypic changes were not assessed. Finally, longer-term storage effects, especially for−80°C preservation, require further study.\u003c/span\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eExperimental trials have established liquid nitrogen cryopreservation as a superior approach for long-term hematopoietic stem cell (HSC) preservation. Our validation of the controlled-rate freezing protocol of the Consarctic system confirmed both cryobag integrity and reliable postthaw cellular recovery. While−80°C cryopreservation results in comparable short-term viability, LN₂ provides critical advantages, including precise thermal regulation, elimination of volatile dependency, and indefinite storage capability without compromising cell function. Although−80°C remains viable for transient storage, its practical constraints position LN₂ as the optimal choice for clinical HSC banking. The implemented protocol has both immediate clinical utility and sustainable long-term preservation, meeting the rigorous demands of modern cellular therapies. Future refinements should focus on cryoprotectant optimization to increase recovery while preserving these demonstrated benefits.\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBerz D, McCormack EM, Winer ES, Colvin GA, Quesenberry PJ (2007) Cryopreservation of hematopoietic stem cells. Am J Hematol 82(6):463\u0026ndash;472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ajh.20707\u003c/span\u003e\u003cspan address=\"10.1002/ajh.20707\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrisciandaro M, Santodirocco M (2024) Innovation in hematopoietic stem cell cryopreservation and cold chain management. Cell Gene Ther Insights 10(3):345\u0026ndash;362. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18609/cgti.2024.050\u003c/span\u003e\u003cspan address=\"10.18609/cgti.2024.050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBennett B, Hanotaux J, Pasala AR, Hasan T, Hassan D, Shor R et al (2024) Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies. Cytotherapy 26(5):482\u0026ndash;489. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcyt.2024.02.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jcyt.2024.02.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMancias-Guerra C, Vald\u0026eacute;s-Burnes SL, Gonzalez-Llano O, Aguirre-Fernandez GC, Villarreal-Martinez L, Cantu-Rodriguez O et al (2013) Another method for thawing hematopoietic stem cells and its impact in the recovery of the transplanted hematological patient. Biol Blood Marrow Transpl 19(2 Suppl):S349\u0026ndash;S350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbmt.2012.11.543\u003c/span\u003e\u003cspan address=\"10.1016/j.bbmt.2012.11.543\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesoutter J, Ossart C, Lacassagne MN, Regnier A, Marolleau JP, Harrivel V et al (2019) Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: key role of granulocytes. Cytotherapy 21(6):612\u0026ndash;618. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcyt.2019.04.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jcyt.2019.04.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampos-Carli SM, D\u0026rsquo;\u0026Aacute;vila A, de Oliveira CF, de Souza GF, Moraes-Vieira PMM, Silva-Costa AC (2024) Cryopreservation of hematopoietic stem cells at\u0026ndash;80\u0026deg;C: Impact of storage time on cell viability and engraftment. Transfus Clin Biol 46(3):188\u0026ndash;196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1159/000528038\u003c/span\u003e\u003cspan address=\"10.1159/000528038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomes MRA, Ramos AS, Alves ACL, Zamperlini G, Granja PGG, Cruz DVB et al (2024) Validation of the use of hydroxyethyl starch (Voluven 6%) to remove dimethyl sulfoxide from hematopoietic stem cells. Hematol Transfus Cell Ther 46(Suppl 4):S991\u0026ndash;S992\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee JY, Hong SH (2019) Hematopoietic stem cells and their roles in tissue regeneration. Int J Stem Cells 13(1):1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15283/ijsc19127\u003c/span\u003e\u003cspan address=\"10.15283/ijsc19127\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuten Pella O, Horny\u0026aacute;k I, Horv\u0026aacute;thy D, Fodor E, Nehrer S, Lacza Z (2022) Albumin as a biomaterial and therapeutic agent in regenerative medicine. Int J Mol Sci 23(18):10557. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms231810557\u003c/span\u003e\u003cspan address=\"10.3390/ijms231810557\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Ye D, Li S, Lu X, Chi Y, Tang C, Zhou X, Chen M, Zheng Y, Ge J, Zou Q (2024) Generation of human induced pluripotent stem cells carrying albumin-sfGFP reporter. Stem Cell Res 81:103543. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.scr.2024.103543\u003c/span\u003e\u003cspan address=\"10.1016/j.scr.2024.103543\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJahan S, Kaushal R, Pasha R, Pineault N (2021) Current and future perspectives for the cryopreservation of cord blood stem cells. Transfus Med Rev 35(2):95\u0026ndash;102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tmrv.2021.01.003\u003c/span\u003e\u003cspan address=\"10.1016/j.tmrv.2021.01.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHornberger K, Borgmann S, Heinemann FM, B\u0026auml;r A, Wein F, Schmitz M (2019) Cryopreservation of hematopoietic stem cells: Advances and perspectives. Transfus Med Hemother 46(3):188\u0026ndash;196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1159/000498948\u003c/span\u003e\u003cspan address=\"10.1159/000498948\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan X, Yuan F, Wang Y, Cai J, Liao J, Liao L et al (2016) Efficient long-term cryopreservation of pluripotent stem cells at\u0026ndash;80\u0026deg;C. Sci Rep 6:21054. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep21054\u003c/span\u003e\u003cspan address=\"10.1038/srep21054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGokarn A, Tembhare PR, Syed H, Sanyal I, Kumar R, Parab S et al (2023) Long-term cryopreservation of peripheral blood stem cell harvest using low concentration (4.35%) dimethyl sulfoxide with methyl cellulose and uncontrolled rate freezing at\u0026ndash;80\u0026deg;C: An effective option in resource-limited settings. Transpl Cell Ther 29(12):777e1\u0026ndash;777e8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jtct.2023.08.032\u003c/span\u003e\u003cspan address=\"10.1016/j.jtct.2023.08.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLecchi L, Giovanelli S, Gagliardi B, Pezzali I, Ratti I, Marconi M (2016) An update on methods for cryopreservation and thawing of hemopoietic stem cells. Transfus Apher Sci 54(3):324\u0026ndash;336. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.transci.2016.05.009\u003c/span\u003e\u003cspan address=\"10.1016/j.transci.2016.05.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFountain D, Ralston M, Higgins N, Gorlin JB, Uhl L, Wheeler C et al (1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 37(6):585\u0026ndash;591. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1537\u0026ndash;2995.1997.37697335152.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1537\u0026ndash;2995.1997.37697335152.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarma NJ, Takeda A, Yaseen NR (2010) Colony forming cell (CFC) assay for human hematopoietic cells. J Vis Exp 462195. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/2195\u003c/span\u003e\u003cspan address=\"10.3791/2195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaqbool S, Nadeem M, Shahroz A, Naimat K, Khan I, Tahir H et al (2022) Engraftment syndrome following hematopoietic stem cell transplantation: a systematic approach toward diagnosis and management. Med Oncol 40(1):36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12032-022-01894\u0026ndash;7\u003c/span\u003e\u003cspan address=\"10.1007/s12032-022-01894\u0026ndash;7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurney TL, Reese-Ko\u0026ccedil; J, de Lima M, Otegbeye F (2019) Optimizing cryopreservation of hematopoietic stem cells collected for autologous stem cell transplantation in patients with multiple myeloma. Cytotherapy 21(5 Suppl):S39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcyt.2019.03.375\u003c/span\u003e\u003cspan address=\"10.1016/j.jcyt.2019.03.375\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hematopoietic stem cells, cryopreservation, liquid nitrogen, –80°C storage, CD34⁺ cells, cell viability, stem cell transplantation","lastPublishedDoi":"10.21203/rs.3.rs-7642582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7642582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHematopoietic stem cell (HSC) cryopreservation is a critical component of cellular therapy that directly influences transplant outcomes. Although mechanical freezing at \u0026minus;\u0026thinsp;80\u0026deg;C is widely used in resource-limited settings, liquid nitrogen (LN₂) cryopreservation with controlled-rate freezing remains the gold standard for long-term storage. However, comparative data on the efficacy and operational feasibility of these methods remain scarce.\u003c/p\u003e\u003cp\u003eThis study aimed to compare the efficacy, cellular integrity, and viability of HSC grafts preserved via LN₂ cryopreservation with those preserved via mechanical freezing at \u0026minus;\u0026thinsp;80\u0026deg;C. Additionally, we sought to validate a controlled-rate freezing protocol and evaluate its clinical applicability within a cell therapy unit.\u003c/p\u003e\u003cp\u003eWe conducted four experimental trials: (1) validation of a controlled-rate freezing protocol, (2) assessment of postthaw recovery in low-cell-density samples, and (3\u0026ndash;4) a direct comparison between LN₂ and \u0026minus;\u0026thinsp;80\u0026deg;C cryopreservation using paired aliquots. Postthaw CD34⁺ cell counts were analyzed via flow cytometry, and freezing curves were recorded to assess the consistency of the decrease in temperature.\u003c/p\u003e\u003cp\u003eBoth methods demonstrated comparable short-term CD34⁺ cell viability, with similar postthaw cell loss rates (~\u0026thinsp;2 \u0026times; 10⁶ cells/kg). However, LN₂ cryopreservation provided superior thermal control, as evidenced by strict adherence to the programmed freezing curve. Moreover, the LN₂ method reduced the reliance on scarce cryoprotectants such as Voluven\u0026reg;, enhancing reproducibility and long-term storage potential. No instances of bag leakage or contamination were observed with sterile packaging.\u003c/p\u003e\u003cp\u003eWhile \u0026minus;\u0026thinsp;80\u0026deg;C cryopreservation remains a viable short-term option, LN₂ cryopreservation offers distinct advantages in terms of thermal stability, cryoprotectant flexibility, and long-term cell viability. Our validated LN₂ protocol meets clinical standards for safety, sustainability, and quality, confirming its role as the preferred method for high-quality HSC biobanking and transplantation. Further studies incorporating functional assays and extended follow-up are needed to assess long-term engraftment potential.\u003c/p\u003e","manuscriptTitle":"Evaluation of hematopoietic stem cell cryopreservation: A comparative study of liquid nitrogen versus storage at -80°C","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 05:13:01","doi":"10.21203/rs.3.rs-7642582/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6596be0c-8aa0-442f-b0d4-2901a901ef38","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54897619,"name":"Stem Cell \u0026 Developmental Cell Biology"}],"tags":[],"updatedAt":"2025-09-19T05:13:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-19 05:13:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7642582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7642582","identity":"rs-7642582","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}