Cryopreservation of mononuclear cells before extracorporeal photochemotherapy does not impair their anti-proliferative capabilities

Background aimsThe clinical benefits of extracorporeal photochemotherapy (ECP) are well recognized, but its clinical use is limited by logistical difficulties, especially because of the need to perform repeated aphereses. The cryopreservation of mononuclear cells could allow maintenance of the ECP schedule while reducing the number of aphereses. The aim of this work was to assess whether previous cryopreservation impairs the immunomodulatory function of ECP-treated peripheral blood mononuclear cells (PBMC).MethodsFresh or previously cryopreserved PBMC were exposed to ECP and added on day 0 into a mixed leukocyte reaction. Proliferation of alloreactive lymphocytes was measured by carboxyfluorescein succinimidyl ester (CFSE) dye dilution. Apoptosis was quantified by annexin–7AAD staining.ResultsECP-induced apoptosis was slightly increased in cryopreserved cells but the kinetics of apoptosis were similar to fresh cells. Lymphocytes stimulated in the presence of ECP-treated PBMC displayed a significant decrease in proliferation. The suppression was enforced when ECP-treated cells had been activated previously by allogeneic stimulation. Cryopreservation before ECP exposure did not impact apoptosis triggering or anti-proliferative properties of ECP-treated cells.ConclusionsCryopreservation before ECP does not impair the immunomodulatory effects of treated cells. These data warrant investigation of the clinical use of cryopreserved PBMC for ECP.     

Cryopreservation of Chlamydomonas reinhardtii: A cause of low viability at high cell density

Cryopreservation is a practical method for stabilizing the genetic content of living algae over long periods of time. Yet, Chlamydomonas reinhardtii, the algal species most often utilized in studies requiring genetically defined strains, is difficult to cryopreserve with a consistently high post-thaw viability. Work described here demonstrates that C. reinhardtii retains high viability only when cryopreserved at a low cell density. Low viability at high cell density was caused by the release of an injurious substance into the culture medium. Rapid freezing and thawing under non-cryoprotective conditions released large amounts of the injurious substance. Heat denaturation of cells prevented the release of the injurious substance, but heating did not inactivate it after it was released. Even when concentrated, the injurious substance was non-toxic to cells under normal culture conditions. Reduced viability of cells cryopreserved in the presence of the injurious substance could not be attributed to changes in the tonicity of the medium. A mutant strain of C. reinhardtii (cw10) with a greatly diminished cell wall did not release a substance that reduced the post-thaw viability of wild-type or cw10 cryopreserved cells. Cryopreservation of cw10 cells was achieved with approximately the same post-thaw viability irrespective to the cell concentration at the time of freezing. Acid treatment of the injurious substance was able to partially diminish its injurious effect on cells during cryopreservation. We propose that diminished viability of C. reinhardtii cells cryopreserved at high cell densities is caused by the enzymatic release of a cell-wall component.     

Evaluation of a clinical-grade,cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34⁺ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP).MethodscGMP VPA-mediated expansion was initiated with CD34⁺ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts.ResultsThe authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products.ConclusionsThe authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34⁺ cells for clinical utilization.     

Cryopreserving human peripheral blood progenitor cells

High-dose chemotherapy followed by autologous peripheral blood progenitor cell (PBPC) transplantation is used in the treatment of chemosensitive malignancies. Cryopreservation of PBPC in 10% dimethyl sulfoxide (DMSO) has been the standard procedure in most institutions. Infusion of PBPC cryopreserved with DMSO can be associated with toxic reactions such as vomiting, cardiac dysfunction, anaphylaxia and acute renal failure. The grade of toxicity experienced by patients is related to the amount of DMSO present in the PBPC. Cryopreservation with lower DMSO concentrations would be expected to reduce the toxicity. In recent studies done with PBPC cells cryopreserved with 5%, 4% and 2% DMSO, using 10% DMSO as a reference control, CD34+ cells were investigated for preservation of viability, apoptosis, and necrosis. Also preservation of mature colony-forming (CFU) cells, specifically mature myeloid, erythroid progenitors, CFU-megakaryocytes and long-term culture-initiating cells (LTC-ICs) were investigated, using 5% and 10% DMSO as cryoprotectant. All samples were frozen in a rate-controlled programmed freezer and stored in the vapor phase of liquid nitrogen until used. Conclusion: 5% DMSO is the optimal concentration for cryopreserving human PBPC in vitro. Consequently, some hospitals have started using 5% DMSO as cryoprotectant for the autologous PBPC as a standard procedure.     

Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood

Bone marrow transplantation (BMT) is a therapeutic procedure that involves transplantation of hematopoietic stem cells (HSC). To date, there are three sources of HSC for clinical use: bone marrow; mobilized peripheral blood; and umbilical cord blood (UCB). Depending on the stem cell source or type of transplantation, these cells are cryopreserved. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO) 10% (v/v), but infusion of Me₂SO-cryopreserved cells is frequently associated with serious side effects in patients. In this study, we assessed the use of trehalose and sucrose for cryopreservation of UCB cells in combination with reduced amounts of Me₂SO. The post-thawed cells were counted and tested for viability with Trypan blue, the proportion of HSC was determined by flow cytometry, and the proportion of hematopoeitic progenitor cells was measured by a colony-forming unit (CFU) assay. A solution of 30 mmol/L trehalose with 2.5% Me₂SO (v/v) or 60 mmol/L sucrose with 5% Me₂SO (v/v) produced results similar to those for 10% (v/v) Me₂SO in terms of the clonogenic potential of progenitor cells, cell viability, and numbers of CD45⁺/34⁺ cells in post-thawed cord blood cryopreserved for a minimum of 2 weeks. Thus, cord blood, as other HSC, can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides. The use of Me₂SO at low concentrations in the cryopreservation solution may improve the safety of hematopoietic cell transplantation by reducing the side effects on the patient.     

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay

PurposeThe prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification.MethodsFreshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density.ResultsA nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used.ConclusionsFreshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.     

Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation

Introduction

Although there is considerable variability in methodology among umbilical cord blood banks, their common goal is to achieve optimal product quality for transplantation. Cryopreservation is a critical issue for a long-term maintenance of cord blood viability and colony-forming capacities.

Materials and methods

We designed a prospective study to compare controlled (CRF) vs. non-controlled freezing (URF) of volume-reduced cord blood units. In addition, the influence of hydroxy ethyl starch (HES) on cryopreservation was also assayed. To assess the efficiency of protocols used, cell recoveries were measured and the presence of hematopoietic colony-forming units was quantified.

Results

In the study phase, we observed similar CB haematopoietc recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume reduced CB units in the URF group. When we analysed the data of routine processed CB units in samples from satellite cryovials, we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner.

Conclusions

URF of CB units is a cryopreservation procedure that allows similar hematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in a URF manner should use HES for volume reduction. On the other hand, for CRF cryopreservation methodology volume reduction with and without HES are equally useful.     

Reduced dimethyl sulfoxide concentrations successfully cryopreserve human hematopoietic stem cells with multi-lineage long-term engraftment ability in mice

Background aimsThe cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured.MethodsCryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33⁺ myeloid, CD19⁺ B-lymphoid, CD235a⁺ erythroid and CD34⁺ progenitors; (ii) engraftment of the same four populations plus CD41⁺CD42b⁺ platelets; (iii) engraftment of CD34⁺⁺CD133⁺ cells; and (iv) engraftment of CD34⁺⁺CD38⁻ cells.ResultsHematopoietic colony-forming, CD34^++/+, CD34⁺⁺CD133⁺ and CD34⁺⁺CD38⁻ cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs.ConclusionCryopreservation of HSCs in DMSO concentrations as low as 5% is effective.     

T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (αCD3/αCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or αCD3/αCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or αCD3/αCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3⁺CD4⁺ or CD3⁺CD8⁺ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time.In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or αCD3/αCD28 as positive control.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Development of a reliable low-cost controlled cooling rate instrument for the cryopreservation of hematopoietic stem cells

Background aimsAn optimal cooling rate is one of the critical factors influencing the survival of cells during cryopreservation. We describe a novel device, called the box-in-box, that has been developed for optimal cryopreservation of human hematopoietic stem cells (HSC).MethodsThis work presents the design of the device, a mathematical formulation describing the expected temperature histories of samples during the freezing process, along with actual experimental results of thermal profile tests. In experiments, when the box-in-box device was transferred from room temperature to a ?80°C freezer, a cooling rate of ?1 to ?3.5°C/min, which has been widely used for the cryopreservation of HSC, was achieved. In order to evaluate this device further, HSC cryopreservation was compared between the box-in-box device and a commercially available controlled-rate freezer (CryoMed).ResultsThe experimental data, including total cell population and CD34⁺ hematopoietic progenitor cell recovery rates, viability and cell culture colony assays, showed that the box-in-box worked as well as the CryoMed instrument. There was no significant difference in either survival rate or the culture/colony outcome between the two devices.ConclusionsThe box-in-box device can work as a cheap, durable, reliable and maintenance-free instrument for the cryopreservation of HSC. This concept of a box-in-box may also be adapted to other cooling rates to support cryopreservation of a wide variety of tissues and cells.     

Cryopreservation of hybrid Cymbidium protocorm-like bodies by encapsulation–dehydration and vitrification

Cryopreservation can be a stable, long-term method of germplasm conservation, but successful application can be challenging for tropical material. To optimize survival and re-growth from cryopreserved tissues derived from protocorm-like bodies (PLBs) of hybrid Cymbidium Twilight Moon ‘Day Light’, the effects of explant type (intact PLBs, half-PLBs, or PLB longitudinal thin cell layers) and various explant treatments were studied. Encapsulation in alginate beads was essential, and intact PLBs were best for cryopreservation, based on survival and ability to form neo-PLBs and/or percentage re-growth. Osmotic hydration of intact PLBs in 2% sucrose for 24 h increased neo-PLB formation and re-growth, with the best responses seen when PLBs were excised from alginate beads prior to re-growth after cryopreservation. Both non-transgenic and transgenic PLBs were amenable to cryopreservation for up to 1 year using these methods. This optimized protocol will improve the viability of hybrid Cymbidium germplasm after long-term cryopreservation.     

Viability,proliferation and phenotype maintenance in cryopreserved human iliac apophyseal chondrocytes

Cryopreservation preserves cells at low temperature and creates a reserve for future use while executing the clinical translation. Unlike articular chondrocyte, cryopreservation protocol and its outcome are not described in iliac apophyseal chondrocytes, a potential source of chondrocytes in cartilage engineering. This study for the first time describes the cryopreservation of human iliac apophyseal chondrocytes. Four cartilage samples were procured from iliac crests of children undergoing hip surgery after consent. The total chondrocyte yield was divided into two groups. First group was grown as monolayer while second group was cryopreserved following the slow cooling method in the medium containing 10 % Dimethyl sulfoxide for 3 months. Group two cells were also grown as a monolayer following thawing. Viability, time to confluence, population doubling time and phenotype maintenance were compared for both the groups. Viability was 65.75 % after 3 months of cryopreservation at ?196 °C, as compared to 94.19 % for fresh chondrocytes (p = 0.001). Fresh and cryopreserved cells reached confluence on 10th and 15th day of culture respectively. Population doubling time was significantly more in fresh than cryopreserved chondrocytes on 10th (p = 0.0006) and 15th day (p = 0.0002) in culture. Both fresh and cryopreserved cells maintain their chondrocyte phenotype as assessed by immunocytochemistry. Relative gene expression by real time polymerase chain reaction showed similar upregulation of mRNA of Collagen 2, SOX 9, Aggrecan and Collagen 1 in cryopreserved chondrocyte as compared to fresh chondrocyte. Iliac apophyseal chondrocytes cryopreserved for 3 months maintained the phenotype successfully 2 weeks after thawing in culture. The viability and proliferation rates after thawing were adequate for a clinical translation of these cells.     

134 Cryopreservation of Chlamydomonas Reinhardtii (Chlorophyceae): A Cause of Low Viability at High Cell Density

Cryopreservation provides a convenient method for long term storage of living organisms. Current protocols allow the successful cryopreservation of a wide range of algae, although many strains remain recalcitrant to cryopreservation. Chlamydomonas reinhardtii , a species utilized in many molecular and biochemical studies, survives cryopreservation best at low cell density. We show that reduced viability at higher cell densities is caused by the accumulation of a substance released from C. reinhardtii into the culture medium during cryopreservation. A mutant strain of C. reinhardtii (cw10) with a greatly reduced cell wall did not release a substance inhibitory to wild type or cw10 C. reinhardtii during cryopreservation, and could be cryopreserved with the same viability regardless of cell density. The inhibitory substance is small (mw<1300), polar, heat-stable and organic. Chlamydomonas moewusii Gerloff and Chlamydomonas zebra Korschikov ex Pascher both produce substances that reduce the viability of cryopreserved C. reinhardtii . However, neither is affected by the inhibitory substance produced by themselves or C. rienhardtii. Pandorina morum (Müller) Bory and Volvox carteri f. nagariensis Iyengar are colonial Volvocalean algae related to C. reinhardtii that cannot be successfully cryopreserved. They both generate substances that inhibit C. reinhardtii during cryopreservation. The identification of the substance inhibitory to C. reinhardtii during cryopreservation should explain why this alga cryopreserves best at a low cell density, and may lead to protocols that facilitate the more successful cryopreservation of C. reinhardtii and related algae.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Smooth muscle cell injury after cryopreservation of human thoracic aortas

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.     

Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts

Background aimsThe use of effective methods for the cryopreservation of hematopoietic stem cells (HSCs) is vital to retain the maximum engraftment activity of cord blood units (CBUs). Current protocols entail the use of dimethyl sulfoxide (DMSO) as intracellular cryoprotective agent (CPA) and dextran and plasma proteins as extracellular CPAs, but DMSO is known to be cytotoxic, and its infusion in patients is associated with mild to moderate side effects. However, new, commercially available, DMSO-free cryopreservation solutions have been developed, but their capacity to protect HSCs remains poorly investigated.MethodsHerein the authors compared the capacity of four DMSO-free freezing media to cryopreserve cord blood (CB) HSCs: CryoProtectPureSTEM (CPP-STEM), CryoScarless (CSL), CryoNovo P24 (CN) and Pentaisomaltose (PIM). Clinical-grade DMSO/dextran solution was used as control.ResultsOf the four cryopreservation solutions tested, the best post-thaw cell viability, recovery of viable CD45+ and CD34+ cells and potency were achieved with CPP-STEM, which was equal or superior to that seen with the control DMSO. CSL provided the second best post-thaw results followed by PIM, whereas CN was associated with modest viability and potency. Further work with CPP-STEM revealed that CB CD34-enriched HSCs and progenitors cryopreserved with CPP-STEM maintained high viability and growth expansion activity. In line with this, a pilot transplantation assay confirmed that CPP-STEM-protected CB grafts supported normal short- and long-term engraftment kinetics.ConclusionsThe authors’ results suggest that new, valuable alternatives to DMSO are now available for the cryopreservation of HSCs and grafts, including CBUs.     

Non-cryopreserved hematopoietic stem cells in autograft patients with lymphoma: a matched-pair analysis comparing a single center experience with the use of cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry

Background aimsAround 50 000 autologous stem cell transplantations are done each year worldwide using cryopreserved peripheral blood stem cells (PBSCs). Cryopreservation is time-consuming and expensive. Since 2007, several retrospective studies have shown that PBSCs can be stored at 4°C for 2–3 days, allowing autologous stem cell transplantation in patients with multiple myeloma receiving high-dose melphalan. Data with non-cryopreserved PBSCs in patients autografted for lymphoma following longer pre-conditioning regimens are limited. In addition, no controlled comparison has been able to detect unforeseen differences.MethodsThe authors compared outcomes of 94 consecutive adult patients with lymphoma (66 with Hodgkin lymphoma) autografted in our department in Oran (Algeria) using PBSCs stored at 4°C, from 2009 to 2018, with patients receiving cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry. Patients autografted in Oran were matched with patients receiving cryopreserved PBSCs in the registry (four controls per patient in Oran).ResultsNeutrophil engraftment was significantly faster with cryopreserved PBSCs (P = 0.003). By day 10, only 17% of patients receiving non-cryopreserved PBSCs engrafted versus 48% for cryopreserved PBSCs. Likewise, platelet recovery to 20 000/mm³ was significantly faster in patients receiving cryopreserved PBSCs (P = 0.01). However, all patients in both groups had recovered by day 20. There were no significant differences in non-relapse mortality (9% versus 7%, P = 0.4), relapse incidence (22% versus 32%, P = 0.13), progression-free survival (70% versus 61%, P = 0.4) or overall survival (85% versus 75%, P = 0.3).ConclusionsThis analysis suggests that, in patients with lymphoma receiving pre-transplant regimens such as carmustine, etoposide, cytarabine and melphalan, PBSCs stored at 4°C for up to 6 days can be used safely in centers with no cryopreservation facility. However, the kinetics of hematopoietic recovery showed a significant, albeit small, delay in engraftment for both neutrophils and platelets, which favors the use of cryopreservation if available.