Cryopreservation Of Stem Cells Research Articles

Non-cryopreserved autologous peripheral blood stem cell transplantation for multiple myeloma and lymphoma in countries with limited resources: practice considerations from the Worldwide Network for Blood and Marrow Transplantation.

Autologous peripheral blood stem cell (PBSC) transplantation is a standard treatment of multiple myeloma (MM), Hodgkin lymphoma and various subtypes of non-Hodgkin lymphoma. Cryopreservation of hematopoietic stem cells is standard practice that allows time for delivery of conditioning regimen prior to cell infusion. The aim of this Worldwide Network for Blood & Marrow Transplantation (WBMT) work was to assess existing evidence on non-cryopreserved autologous transplants through a systematic review/meta-analysis, to study feasibility and safety of this approach. We searched PubMed, Web of Science and SCOPUS for studies that utilized non-cryopreserved autologous PBSC transplantation. Identified literature was reviewed for information on mobilization, apheresis, preservation and viability, conditioning regimen, engraftment, response, and survival. Results highlight collective experience from 19 transplant centers (1686 patients), that performed autologous transplants using non-cryopreserved PBSCs. The mean of infused CD34+ was 5.6 × 106/kg. Stem cell viability at transplantation was >90% in MM and >75% in lymphomas, after a storage time of 24-144 h at +4 °C. Mean time-to-neutrophil engraftment was 12 days and 15.3 days for platelets. Pooled proportion estimates of day 100 transplant-related mortality and graft failure were 1% and 0%, respectively. Non-cryopreservation of apheresed autologous PBSCs appears feasible and safe.

7911 High Dose Serial Radioactive Iodine Therapies Facilitated byAutologous Stem Cell Cryopreservation in a Patient with Exceptionally Avid Metastatic FTC

Abstract Disclosure: S. Paknikar: None. D. Toro Tobon: None. J. Durski: None. H. Alkhateeb: None. M.M. Ryder: None. Introduction: Radioactive iodine (RAI) therapy-induced myelosuppression is a dose-limiting toxicity for RAI avid metastatic differentiated thyroid cancers. We describe a patient with exceptionally RAI avid metastatic follicular thyroid cancer (FTC) who underwent autologous stem cell cryopreservation (ASCC) to enable high dose I-131 treatments. Case: A 60-year-old woman with T1bN0M0 FTC underwent two-stage thyroidectomy and three doses of I-131 between 2002-2007 (434 mCi cumulative activity) due to incomplete biochemical response. She presented to our institution in 2015 with rising thyroglobulin (Tg) levels and lung nodules that were intensely RAI avid. She enrolled in a clinical trial of selumetinib versus placebo (for 1 month) followed by 150 mci I-131. She had a profound response with an 80% reduction in Tg and 50% tumor shrinkage. The following year her Tg rose to 8381 ng/mL and she developed biopsy proven, well differentiated metastatic FTC in the hip and liver. Lenvatinib treatment resulted in partial tumor and Tg response but was halted due to grade 3/4 toxicities. Tumor genomic interrogation revealed NRAS, PIK3CA, and TSH-R activating mutations, providing a causal explanation of her intensely RAI avid and responsive disease. Repeat I-131 therapy was facilitated by GM-CSF stimulated ASCC (8.57 x10(6) CD34+ cells/kg for two cycles) as a tool to help mitigate future potential RAI-induced myelosuppression. The patient received 295 mci I-131 followed by a 98% decrease in Tg and 20-25% shrinkage of liver metastases. Transient pancytopenia ensued but did not require autologous stem cell transplantation (ASCT). Due to insurance issues, the patient was lost to follow-up for two years. She returned with cachexia from severe, untreated thyrotoxicosis due to widespread functional, thyroxine producing metastatic disease. Anti-thyroid drug therapy (ATD) restored euthyroid status with significant clinical improvement. A non-stimulated I-123 whole body scan showed diffuse RAI avid disease with 88% whole body radioiodine retention at 48h. She received 125 mci I-131 with initial tumor response but with progression of bone marrow, calvaria, and retinal metastases. Six months later, a final dose of 253 mCi I-131 (cumulative activity 1,370 mCi) resulted in mixed tumor responses. Unfortunately, resection of a RAI refractory calvaria lesion postoperatively resulted in serious complications. ASCT and salvage treatments could not be pursued due to her impaired functional status. The patient pursued hospice care and passed away 20 years after her diagnosis. Conclusion: We characterize a patient with NRAS, PIK3CA and TSH-R mutant metastatic, thyroxine-producing FTC whose recurrences over 20 years retained intense RAI avidity and response to I-131 therapy. In cases with potential benefit from multiple high dose RAI therapies, ASCT may be a tool to mitigate I-131-induced myelosuppression. Presentation: 6/1/2024

Controlled Ice Nucleation With a Sand-PDMS Film Device Enhances Cryopreservation of Mouse Preantral Ovarian Follicles.

Ovarian follicle cryopreservation is a promising strategy for fertility preservation; however, cryopreservation protocols have room for improvement to maximize post-thaw follicle viability and quality. Current slow-freezing protocols use either manual ice-seeding in combination with expensive programmable-rate freezers or other clinically incompatible ice initiators to control the ice-seeding temperature in the extracellular solution, a critical parameter that impacts post-cryopreservation cell/tissue quality. Previously, sand has been shown to be an excellent, biocompatible ice initiator, and its use in cryopreservation of human induced pluripotent stem cells enables high cell viability and quality after cryopreservation. This study applies sand as an ice initiator to cryopreserve multicellular microtissue, preantral ovarian follicles, using a simple slow-freezing protocol in the mouse model. Ovarian follicles cryopreserved using the sand partially embedded in polydimethylsiloxane (PDMS) film to seed ice in the extracellular solution exhibit healthy morphology, high viability, and the ability to grow similarly to fresh follicles in culture post-thaw. This sand-based cryopreservation strategy can facilitate convenient ovarian follicle cryopreservation using simple equipment, and this study further demonstrates the translatability of this strategy to not only single cells but also multicellular tissues.

Mesenchymal stem cell cryopreservation with cavitation-mediated trehalose treatment

Dimethylsulfoxide (DMSO) has conventionally been used for cell cryopreservation both in research and in clinical applications, but has long-term cytotoxic effects. Trehalose, a natural disaccharide, has been proposed as a non-toxic cryoprotectant. However, the lack of specific cell membrane transporter receptors inhibits transmembrane transport and severely limits its cryoprotective capability. This research presents a method to successfully deliver trehalose into mesenchymal stem cells (MSCs) using ultrasound in the presence of microbubbles. The optimised trehalose concentration was shown to be able to not only preserve membrane integrity and cell viability but also the multipotency of MSCs, which are essential for stem cell therapy. Confocal imaging revealed that rhodamine-labelled trehalose was transported into cells rather than simply attached to the membrane. Additionally, the membranes were successfully preserved in lyophilised cells. This study demonstrates that ultrasonication with microbubbles facilitated trehalose delivery, offering promising cryoprotective capability without the cytotoxicity associated with DMSO-based methods.

Effects of nanographene oxide on adipose-derived stem cell cryopreservation.

Cryoinjury mitigation is key in cell cryopreservation. Here, we aimed to assess the effectiveness of nanographene oxide (nano-GO) for improving cryoprotectant agents (CPAs) in human adipose stem cell (hADSC) cryopreservation. For in vitro experiments, nano-GO (5μg/mL) was added to the CPAs in the control, and passage (P) 2 hADSCs were collected and cryopreserved for around two weeks. We compared cytotoxicity, cell viability, immunophenotypes, proliferation, cell apoptosis, and tri-lineage differentiation. In vivo, studies used lipoaspirate to create non-enriched or hADSC-enriched fat tissues by combining it with PBS or hADSCs cryopreserved with the aforementioned CPAs. Each nude mouse received a 0.3mL subcutaneous injection of the graft. At 12weeks, the grafts were harvested. Histology, adipocyte-associated genes and protein, vascular density and angiogenic cytokines, macrophage infiltration, and inflammatory cytokines were analyzed. Nano-GO CPAcontributed to increased cell viability, improved cell recovery, and lowered levels of early apoptosis. Nano GO at concentrations of 0.01-100μg/mL caused no cytotoxicity to hADSCs. The absence of nano GOs in the intracellular compartments of the cells was confirmed by transmission electron microscopy. The fat grafts from the CPA-GO group showed more viable adipocytes and significantly increased angiogenesis compared to the PBS and CPA-C groups. Adding hADSCs from the CPA-GO group to the graftreduced macrophage infiltration and MCP-1 expression. Nano-GO plays an anti-apoptotic role in the cryopreservation of hADSCs, which could improve the survival of transplanted fat tissues, possibly via improved angiogenesis and lower inflammatory response in the transplanted adipose tissue.

Conditioned media and DMSO enhance the cryopreservation of bovine adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells derived from adipose tissue (AD-MSCs) show great potential for repro ductive biotechnology in the livestock sector. However, enzyme-based isolation of MSCs is expensive and time-consuming, so it is still rarely done, especially for applications in the livestock sector in de veloping countries. So, MSCs must be cryopreserved with an efficient cryoprotective agent to be stored and reproduced in various laboratories after isolation. This study was aimed to optimize the cryopreser vation media for adipose-derived MSCs in cattle. This study evaluated the viability, proliferation, and morphology of AD-MSCs. The results of this study indicate that a combination of 10% DMSO, 45% DMEM, and 45% conditioned media significantly improves post-thaw viability, proliferation, and sur vival as compared to other mediums. Furthermore, AD-MSCs cryopreserved in this medium exhibit similar morphology as fresh cells. These findings suggest that the optimized cryopreservation medium can enhance the quality and safety of AD-MSCs for clinical applications in the livestock industry.

Histological Identification of Germline Stem Cells’ Recipients for Conservation of Balkan Trout Fish Species by Transplantation Technology

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful technique for long-term conservation of the genetic resources of threatened fish species. However, transplantation of GSCs has been limited by a failure to identify an appropriate recipient for surrogate production of donor-derived gametes. In the worst-case scenario, recipients may completely reject the transplanted GSCs due to an immunological reaction. This limitation can be overcome by specifying the gonadal status of both GSC donor and recipient species. This study used histological methods to identify possible GSC recipients for rescuing endangered trout fish species on the Balkan Peninsula. The study specified the gonadal status of brown trout, triploid rainbow trout, and tiger trout. The findings revealed that both triploid rainbow trout and tiger trout are sterile. The fish's testes contained only early-stage germ cells and did not develop past type-B spermatogonia, whereas the ovaries contained only oogonia. Both spermatogenesis and oogenesis were incomplete in these fish, and the gametes, i.e., functional spermatozoa and eggs, could not be formed. As a result, these fish make excellent recipients for GSC transplantation. Because most Balkan trout belong to the genus Salmo and closely related species, tiger trout would be a more feasible recipient of GSCs due to the small phylogenetic distance, as it is a hybrid of brown trout female and brook trout male.

Retrospective clinical study on cryopreservation-free integrated autologous hematopoietic stem cell transplantation model for newly diagnosed multiple myeloma

Objective: To explore the efficacy and safety of cryopreservation-free integrated autologous hematopoietic stem cell transplantation (HSCT) model for patients with multiple myeloma. Methods: A total of 96 patients with newly diagnosed multiple myeloma (NDMM) between July 31, 2020, and December 31, 2022, were retrospectively analyzed, of which 41 patients in the observation group received integrated non-cryopreserved transplantation mode. After hematopoietic stem cells were mobilized and collected, melphalan was started immediately for pre-transplant conditioning, and non-cryopreserved grafts from the medical blood transfusion refrigerator were directly injected intravenously into the patient within 24-48 h after the melphalan conditioning. The control group consisted of 55 patients who received traditional transplantation mode. After hematopoietic stem cells were collected, stem cell cryopreservation was performed in liquid nitrogen, and then the transplant plans were started at the right time. All patients received mobilization of autologous hematopoietic stem cells using the G-CSF combined with the plerixafor. Results: ① A total of 34 patients (82.9% ) with VGPR plus CR in the observation group were significantly higher than 33 patients (60.0% ) in the control group (P=0.016). ②Compared with the control group, the incidence of grade 1 oral mucosal inflammation was higher in the observation group (P<0.001) ; however, the incidence of grades 2 and 3 oral mucosal inflammation was lower (P=0.004, P=0.048), and neither group experienced grade 4 or above oral mucosal inflammation. The incidence of grade 1 diarrhea was higher in the observation group (P=0.002), whereas the incidence of grade 3 diarrhea was lower (P=0.007). No statistically significant difference was observed in the incidence of grade 4 diarrhea (P=0.506), and neither group experienced grade 5 diarrhea. ③ The incidence of bacterial infection in the observation group was lower than that in the control group (34.1% vs 65.5%, P=0.002), whereas no statistically significant difference was observed in the incidence of fungal infection (29.3% vs 31.4%, P=0.863) and viral infection (4.88% vs 3.64%, P=0.831). ④No statistically significant difference was observed in the implantation time of granulocytes and platelets between the observation and control groups [10 (8-20) days vs 11 (8-17) days, P=0.501; 13 (10-21) days vs 15 (10-20) days, P=0.245]. ⑤ All patients did not receive lenalidomide treatment 100 days post-transplantation. At 30 days post-transplantation, the CTL, NK, and Th cell counts in the observation group were lower than those in the control group (P<0.001, P=0.002, P=0.049), and the NKT cell counts were higher than those in the control group (P=0.024). At 100 days post-transplantation, the CTL, NKT, and Th cell counts in the observation group were higher than those in the control group (P=0.025, P=0.011, P=0.007), and no statistically significant difference in NK cell counts was observed between the two groups (P=0.396). ⑥ The median follow-up was 18 (4-33) months. The overall 2-year survival rates of the observation and control groups post-transplantation were 91.5% and 78.2%, respectively (P=0.337). The recurrence-free survival rates were 85.3% and 77.6%, respectively (P=0.386), and the cumulative recurrence rates were 9.8% and 16.9%, respectively (P=0.373) . Conclusion: In NDMM, the cryopreservation-free integrated autologous HSCT model can achieve similar therapeutic effects as traditional transplantation models, with lower rates of severe mucosal inflammation and infection compared with traditional transplantation models.

Innovation in hematopoietic stem cell cryopreservation and cold chain management

Innovation in hematopoietic stem cell cryopreservation and cold chain management

Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies

Background aimsCryopreservation of hematopoietic stem cells (HSCs) is crucial for autologous transplantation, cord blood banking and other special circumstances. Dimethyl sulfoxide (DMSO) is used most commonly for cryopreserving HSC products but can cause infusional toxicities and affect cell viability and engraftment after transplant. A systematic review of controlled studies using lower concentrations of DMSO to cryopreserve HSC products in clinical transplant studies is needed to determine the effect of reducing DMSO concentrations on post-thaw cell viability, initial engraftment and adverse effects on patient health. MethodsAll studies identified in our systematic search (to July 11, 2023) examining the use of cryopreserved peripheral blood stem cells (PBSCs) for autologous stem cell transplantation (AHCT) were included. Meta-analysis was performed to determine how varying the concentration of DMSO during cryopreservation effects post-thaw cell viability, initial engraftment and adverse effects on patient health. ResultsA total of 1547 studies were identified in our systematic search, with seven published articles meeting eligibility for inclusion in meta-analysis. All patients underwent AHCT using (PBSCs) to treat hematologic malignancies. The viability of CD34+ cells post thaw was greater when cryopreserved with 5% DMSO compared with 10% DMSO, with lower rates of adverse side effects in patients. DMSO concentration had minimal impact on rates of initial engraftment. Significant heterogeneity in outcome reporting was observed and the potential for bias was identified in all studies. ConclusionsReducing the concentration of DMSO from 10% to 5% during cryopreservation of autologous PBSCs may improve cell viability and reduce DMSO-associated adverse effects in patients undergoing AHCT. Data from more studies with similar patients and standard outcome reporting are needed to increase confidence in our initial observations. Protocol registrationPROSPERO; registration number CRD42023476809 registered November 8, 2023.

Autologous hematopoietic stem cell transplant at a tertiary care centre in India: achieving comparable outcomes with adaptations.

Autologous stem cell transplantation (ASCT) is the standard treatment for many high-risk solid tumors. Patients undergoing ASCT should be managed in a dedicated hematopoietic stem cell transplantation (HSCT) unit with isolation rooms, high-efficiency particulate air (HEPA) filters, and positive pressure. We report the outcomes of the first 20 pediatric patients who underwent ASCT in isolation rooms with no HEPA filters or positive pressure. Moreover, the isolation rooms were not part of a dedicated HSCT unit. Data from 20 patients were analyzed. All patients included in the study underwent ASCT after harvest and cryopreservation of the hematopoietic stem cells (HSC). Furthermore, all patients also underwent myeloablative conditioning. The most common indications for ASCT included high-risk neuroblastoma (HR-NB) (n=9) and refractory/relapsed Hodgkin's lymphoma (HL) (n=6). The median CD-34 positive HSC administered was 4.5 (0.8-21.9) million per kg. The median time to neutrophil and platelet engraftment was 16.5 (10-35) and 19 (10-87) days, respectively. Additionally, only one transplant-related mortality was observed and the mean time to discharge from the hospital was 27.6+8.3 days. The overall survival for all our patients was 75% at a median follow-up of 33.2 months (15 out of 20 patients survived), and the disease-free survival was 60% (median follow-up, 28.4 months). The overall survival for the patients with HL was 85.7% at a median of 45.3 months and for the HR-NB was 66.7% at a median of 34.9 months. This study provides evidence that ASCT can be safely performed in isolation rooms without HEPA filters and positive pressure if expertise and supportive care are available. In settings with limited resources, such a model could help establish low-cost HSCT units.

Cryopreservation of adipose tissue-derived mesenchymal stem cells: An alternative to serial fat grafting in breast reconstruction

Cryopreservation of adipose tissue-derived mesenchymal stem cells: An alternative to serial fat grafting in breast reconstruction

Development of Electrodes for Cryopreservation of Stem Cells by Electroporation-Assisted Loading with Sucrose

Development of Electrodes for Cryopreservation of Stem Cells by Electroporation-Assisted Loading with Sucrose

Optimization of Deep Eutectic Solvents Enables Green and Efficient Cryopreservation.

Cryopreservation presents significant opportunities for biomedical applications including cell therapy, tissue engineering, and assisted reproduction. Dimethyl sulfoxide (DMSO), the most commonly used cryoprotectant (CPA), can be added to cells to prevent cryogenic damage. However, the toxicity of cryoprotectants restrains its further development in many areas with safety concerns such as clinical treatment. Therefore, the development of low-toxicity cryoprotectants is essential for medical research. This work reports deep eutectic solvents (DES) as naturally biocompatible osmoprotectants for green and efficient cryopreservation of human umbilical cord mesenchymal stem cells (HuMSC), which may be an ideal alternative to DMSO. The six types of DESs were explored for thermal properties, toxicity, and permeability in cells. Raman spectroscopy and viscosity studies showed that DES exhibited an improved hydrogen-bonding system as the temperature decreased. By optimizing the freezing process (cooling rate, incubation time, and loading procedure) of DES, the viability of mouse embryonic fibroblast cells (NIH-3T3) after thawing was significantly improved. The HuMSC were successfully preserved with no significant difference (p > 0.05) in cell viability (94.65%) after thawing compared with DMSO, which preserved the cell differentiation function and improved the cell proliferation rate. The mechanism of DES in cryopreservation was investigated, and it was found that DES could bind water molecules and effectively inhibit the growth of ice crystals during ice recrystallization, reducing mechanical damage to cells. This study highlights the excellent performance of DES as a low-toxicity CPA for stem cell preservation, which may be a significant advance for future clinical cell therapy.

Cryopreservation of stem cells by electroporation-assisted loading with sucrose

Cryopreservation of stem cells by electroporation-assisted loading with sucrose

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

Trends in hematopoietic stem cell utilization and cost of cryopreservation

Abstract Autologous stem cell transplantation (ASCT) is the standard of care treatment for multiple myeloma. Standard ASCT procedures collect hematopoietic stem cells (HPSC) in excess for multiple transplants due to reports of high mobilization failure and unsuccessful ASCT collections following myeloablative treatments. Cost analysis studies estimate that one year of cryopreservation and one day of HPSC collection would each cost $5000 per patient. Limited data is available on the usage of the second transplant and the storage costs of non-transplanted products. This study evaluates the utilization of cryopreserved HPSC products and calculates the cost of cryopreservation. A retrospective chart review study of a cohort of 240 deceased MM patients, who received ASCT between January 2010 to December 2015. Demographic and clinical data were obtained from the laboratory and electronic medical record. The cohort was categorized into two groups, with 130 patients in Group 1 (≤60 years) and 110 patients in Group 2 (&amp;gt;60 years). Cumulative incidence of a second transplant was computed from the life table using the Kaplan Meier method. The cost of cryopreservation was calculated based on the time HPSC products remained in the freezers before disposal. PBSC was mobilized using Granulocyte Colony Stimulating Factor (GCSF), or GCSF and Mozobil. The average age of patients at first collection is 61 (IQR: 54-66) years and the average age of death is 64 (IQR: 58 -70) years. The average life span of patients after receiving the first transplant is 3 (IQR: 2-5) years. The average number of collection days is 2 (IQR: 1-3). 65 patients had 1-day collections, 105 had 2-day collections and 70 had ≥3-day collections. 175 patients (73%) had &amp;gt;1 day collection. Only 22 patients (9%) received a second transplant and 18 of them (82%) were in Group 1 (≤60). The cumulative incidence of a second transplant by 5 years is 23% for Group 1 and 4% for Group 2. Two-transplant collections required 492 days of apheresis with an overall cost of $2,500,000; however, one-transplant collections would cost $1,400,000 for 279 apheresis days. The median cost of cryopreservation for each patient who did not receive the second transplant is $15,000 (IQR: $8000 - $25,000). Therefore, the estimated cost for all patients in the study is $3,600,000 until the disposal of HPSC products. The incidence of a second transplant is low across all patients and significantly lower in patients greater than 60 years of age. Cryopreserved stem cell products remain under-utilized, leading to substantial financial burden. The duration of HPSC collection and cryopreservation must be re-evaluated for future treatment plans. Institutional policies should be revised based on the utilization trend to lower the operational cost of storage.

In Vivo Potency of Hematopoietic Stem Cells after Long-Term Storage at -80C in DMSO+HES Cryprotectant Mixture: Comparing Engraftment Kinetics and Efficacy Data in First and Second Autologous Stem Cell Transplantation in Myeloma

Background: The use of a cryoprotectant combining both intracellular and extracellular protectants (DMSO+HES) permits large volume hematopoietic stem cell (HSC) cryopreservation in mechanical freezers at -80C without the development of clumping after thawing (Stiff et al, Blood 1983. PMID: 6187518) and was recently utilized for allograft cryopreservation during the COVID-19 pandemic. However, there are competing methods for cryopreservation. Many centers are utilizing vapor phase liquid nitrogen (VPLN) storage protocols with DMSO alone instead. While HSC stored with DMSO alone in VPLN have shown to remain viable after long-term storage (Makhani et al, TCT 2022. PMID:3524877), this has not yet been proven for DMSO+HES stored HSCs at -80C. Here we report on second autologous hematopoietic stem cell transplants (AHSCT2) for relapsed multiple myeloma (RRMM) performed &amp;gt; 2 years after cell storage began at - 80C to determine if there were differences between engraftment and outcomes between initial and long-term storage in DMSO+HES at -80C. Methods: We searched our own institution's records from 1990-2023 to find patients who received two separate AHSCT for MM after only one cell collection prior to first AHSCT (AHSCT1). Prior allogeneic transplants patients were excluded. The primary outcome was neutrophil and platelet engraftment times. Secondary outcomes were progression free survival (PFS) and overall survival (OS). We reviewed patient characteristics, disease state prior to both transplants, engraftment kinetics for both, and survival outcomes. Outcomes were analyzed by Kaplan Meier (KM) and longrank test where applicable, and median PFS (mPFS) and median OS (mOS) were determined by reverse KM. Differences in engraftment were compared using the Kruskal-Wallis test. Results: 35 patients met inclusion criteria, of whom 57% of patients were male. Median age at AHSCT1 was 56.3 years and at AHSCT2 was 60.1 years. Median follow up was 8 years from second transplant and 12.3 years from stem cell collection. Median time from stem cell collection to AHSCT1 was 11 days (range 3-39 days) and to AHSCT2 was 4.6 years (range 2.2-15.8 years). Prior to AHSCT1, 65% of patients were in PR, 20% in VGPR, and 14% in CR. Prior to AHSCT2, 44% were in PR, 38% in VGPR, and 18% in CR. 80% and 83% of patients were conditioned with melphalan alone in AHSCT1 and 2 respectively. Median CD34+ doses were 5.1x10^6/kg and 4.2x10^6/kg (p=0.028) for AHSCT1 and 2 respectively. There was no statistical difference between the engraftment kinetics of the first or second transplant with a median ANC engraftment time of 11 days for both groups (p=0.88), and median platelet engraftment of 17.0 and 17.5 days (p=0.55) for AHSCT1 and AHSCT2 respectively. Between categories of cells stored between 2-5 years and &amp;gt;5 years, there was no statistical difference in ANC (p=0.85) or platelet engraftment (p=0.29). mPFS from AHSCT1 for the overall study was 3.7 years and for AHSCT2 was 3.6 years. The mOS was 4.8 years from AHSCT2. There was a statistical difference in 2-year PFS between ASHCT1 and 2 at 86% and 62% (p=0.0001) respectively. The mPFS differed by disease state prior to AHSCT2 dramatically; those in CR had mPFS of 7.8 years, those in VGPR 4.3 years, and those in PR 1.3 years. Discussion: Our results suggest that HSC stored long-term at -80°C using the DMSO + HES combination does not adversely affect product quality even for cells stored up to 15.8 years. Furthermore, our method is simpler, more cost-effective and poses no risk to the working environment when compared to current freezing method where VPLN storage is mainly used. This is particularly important to expand access to cryopreserved HSC for centers and countries where resources are more limited. These data also add evidence to the efficacy of AHSCT2 as we saw a 2-year PFS of 63% for these patients, which compares favorably to 50% 1-year PFS in AHSCT2 noted in the large CIBMTR retrospective by Dhakal (Leukemia 2021, PMID 32747684). In addition, some patients benefit greatly, best seen in those achieving a CR prior to AHSCT2 who here had a mPFS of 7.8 years. This is an important selection criteria we now evaluate when considering AHSCT2 in RRMM. AHSCT2 is cost effective and compared to recently approved CAR-T and bispecifics is likely a more economical approach in select patients relapsing after long remission following AHSCT1 who achieve deep responses to salvage therapy.

Cryopreserved Versus Non-Cryopreserved Peripheral Blood Haematopoietic Stem Cells for Autologous Stem Cell Transplantation in Multiple Myeloma: A Comparative Analysis from the Chronic Malignancies Working Party of EBMT and WBMT

Introduction: Globally, most transplant centres cryopreserve peripheral blood haematopoietic stem cells (PBSC) for use in a subsequent autologous haematopoietic cell transplant (AHCT), utilizing Dimethyl sulfoxide (DMSO) as a cryoprotectant. In contrast, non-vryopreserved PBSC are used in autologous transplants for multiple myeloma (MM) in some countries with more limited resources. We therefor compared standard MM AHCT outcomes between a large transplant centre in Algeria and EBMT registry data. Patients and Methods: This was a retrospective, international, multi-centre, matched-pair analysis between a tertiary transplant center in Algeria and EBMT-affiliated centres in 23 countries. A total of 437 consecutive adult patients undergoing a first MM AHCT in Oran, Algeria, between 2009 and 2020 using non-cryopreserved PBSC. All patients had given informed consent for approved research. PBSC mobilization was performed using G-CSF alone (10µg/kg) for 5 days. Stem cell collection was performed on either the Cobe Spectra or Optia using standard protocols. PBSCs were stored in a 4°C refrigerator immediatly post-collection. PBSC viability was calculated by standard Trypan Blue and flow cytometric techniques. The conditioning regimen was either Melphalan 140 mg/m 2 or 200 mg/m 2. PBSCs were infused 24 hours following Melphalan administration. Patients from Oran were matched to a maximum of 4 patients from an overall cohort of 64,030 patients from 420 EBMT-affiliated centres receiving cryopreserved stem cells over the same time-period. Pair matching was based on the following criteria: (1) patient gender (M,F), (2) MM sub-classification (IgG, IgA, light-chain only), (3) disease status at AHCT (CR, VGPR, PR, relapse/progression), (4) Melphalan conditioning dosage (140 or 200), (5) interval from diagnosis to AHCT, (6) date of AHCT, and (7) age at AHCT. Exact matching was used for the first four variables; propensity score matching was used for the remaining three. Primary endpoints were neutrophil and platelet engraftment as defined by standard EBMT criteria. Secondary endpoints included non-relapse mortality (NRM). The cumulative incidence of neutrophil and platelet engraftment was estimated using the crude cumulative incidence estimator with death was as competing event. The same method was used to estimate NRM as a competing event. Groups were compared using Gray's test. Results: In Oran, 437 MM patients were autografted between 2009 and 2020. Induction regimens (available in 436/437) were heterogeneous (VAD (11.2%), VD (13.3%), CTD (1.4%), VTD (43.1%), VRD (1.4%), VCD (28.0%), TD (0.5%), PAD (0.5%) or others (0.7%)) reflective of local practice. Most patients had one line of induction therapy (n=333 (76.4%); 98 patients (22.5%) had a second line of therapy prior to mobilization. The mean number of collection procedures was two (range (r), 1-3). The average PBSC viability was 95% (r, 93.8-98.5%). Complete matching variable data was available in 405 Oran patients (93%) and 19/405 (4.7%) could not be matched to EBMT patients. These were mostly younger patients (16/19&amp;lt;50 years at AHCT) who received low dose melphalan (17/19 received 140 mg/m 2 melphalan). Of the 386 patients, 357 patients had the required matches of 4 patients from the EBMT database and were used in this analysis. For the patients from Oran, the median dose of PBSCs collected was 3.2x10 6 CD34+ cells/kg (interquartile range (IQR, 2.4-4.5); for EBMT patients (available in 12%), it was 3.7x10 6CD34+ cells/kg (IQR, 2.8-4.9) (p=0.22). Regarding engraftment kinetics, the median times to neutrophil engraftment for the non-cryopreserved and EBMT cryopreserved cohorts were 12 (IQR, 11 to 14) days, and 12 (IQR, 11 to 13) days, respectively (p=0.11). Moreover, no significant difference was evident in the median time to platelet engraftment&amp;gt;20x10 9/L: non-cryopreserved 12 (r, 11 to 14) days and EBMT cryopreserved 12 (r, 11 to 14) (p=0.39) (Figure 1). Non-relapse mortality at day 100 (NRM) rate was 1%(0-2%) in Oran and 1% (1-1%) in EBMT (p=0.4). Conclusion: This large co-operative study between Oran and EBMT found that neutrophil and platelet engraftment rates were similar in the cryopreserved and non-cryopreserved matched cohorts. Moreover, there was no difference in NRM. The use of non-cryopreserved PBSC for MM AHCT in countries with limited resources is more straighforward and less costly.

Successful Cryopreservation of Spermatogonia Stem Cells of Neotropical Catfish (Rhamdia quelen) and Enriched Germ Cell Transplantation into Common Carp (Cyprinus carpio) Testes

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) offer new possibilities in the conservation of valuable genetic resources. Therefore, the present study developed a cryopreservation method for whole testicular tissue and for spermatogonial stem cells of jundia catfish (Rhamdia quelen) and developed an enriched germ cell transplantation of jundia catfish into depleted common carp (Cyprinus carpio) testes. Our findings from whole testes indicate that the cryoprotectants MeOH (1.3 M), DMSO (1.4 M), and EG (1.4 M) resulted in high cell viability rates of 67%, 62%, and 51.5%, respectively. Notably, in the case of enriched post-thaw SSCs, DMSO exhibited the highest cell viability at 27%, followed by EG at 16% and MeOH at 7%. Additionally, we observed the successful colonization and proliferation of jundia germ cells within the recipient gonads of common carp following transplantation. Notably, Sertoli cells were identified in the recipient gonads, providing support to the stained donor germ cells and indicated the formation of cysts. Our data suggest that cryopreserving entire testicular tissue presents a viable alternative to cryopreserving isolated testicular cells, and the spermatogonial cells isolated from testes of jundia retained transplantability characteristics. Nonetheless, more investigations are required to reach the goal of functional gamete and to assess the effectiveness of transplantation using these cryopreserved tissues. Taken together, proper cryopreservation methodology and transplantation technology could aid the preservation practice of fish genetic resources.