Optimization of cryopreservation condition for hematopoietic stem cells from umbilical cord blood

The conditions for cryopreservation of CD34⁺ hematopoietic stem cells (HSC) from umbilical cord blood (UCB) were optimized with a new cryo-medium containing 10% ethylene glycol (EG) and 2% dimethyl sulfoxide (Me₂SO) using a controlled-rate freezing (CRF) method. After the cryopreservation of mononuclear cells (MNC) from UCB, recoveries of MNC, CD34⁺ cells, and total colony-forming units (CFU) were significantly improved compared to those in the control cryo-medium containing 10% Me₂SO and 2% Dextran-40 (P < 0.05). This study shows that the new cryo-medium and CRF method provide better recoveries of MNC, HSC and total CFU than the control cryo-medium and isopropylalcohol freezing (IPA) method. Therefore, this cryo-medium, combined with the CRF method, is valuable for optimizing cryopreservation conditions for HSC from UCB to obtain satisfactory HSC recovery.     

A novel high-yield volume-reduction method for the cryopreservation of UC blood units

BACKGROUND: For the application of umbilical cord blood (UCB) units as hematopoietic grafts, a dose of 3.7 x 10(7) nucleated cells (NC)/kg body weight is required. NC can be lost during volume-reduction processing and during thawing. A novel modification of the double-processing protocol with the aim of minimizing NC loss is described and evaluated. METHODS: One-hundred and fifty UCB were collected. The volume was reduced by a centrifugation step following double-processing in the presence of 2% HES 200/0.5. Pre- and post-processing cell counts and platelet parameters were measured with an automatic counter. The number of viable CD34+ hemopoietic stem cells was measured by flow cytometry. In 25 of the samples, colony-forming units (CFU) were also determined. The same samples were thawed 6 months after cryopreservation and re-evaluated. RESULTS: The volume was reduced to 6 +/- 1.5 mL. The recovery of NC, MNC, CD34+ hemopoietic stem cells, RBC depletion and CFU following double-processing was 93.6 +/- 3.2%, 95.8 +/- 2.2%, 98.4 +/- 1.5%, 96.8 +/- 1.1% and 107.1 +/- 6.1% (for 25 samples), respectively. The post-thaw recoveries of NC, MNC, CD34+ hemopoietic stem cells and CFU (for 25 samples) were 78.6 +/- 5.4%, 90.8 +/- 4.4%, 96.4 +/- 2.5%, 89.1 +/- 4.1%, respectively. No post-thaw cell aggregation was observed. A significant (P<0.05) post-thaw loss of platelets and signs of platelet activation was observed. DISCUSSION: The protocol uses non-expensive equipment and clinically approved materials and results in samples that can be used in patients with a mean weight of 32.7 kg.     

Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO), but at 37 °C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me₂SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me₂SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.     

Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB

BACKGROUND: Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40, saline or albumin. However, the process of freezing and thawing results in loss of HSC numbers and/or function. METHODS: This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol, volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40, 10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. RESULTS: Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001), CD34+ recovery, viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). Discussion: These results suggest that improved HSC recovery, viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.     

不同降温速率对脐血干细胞冷冻复苏后生物学特性的影响

考察了不同降温速率对脐血造血干细胞各种生物学特性的影响。在4℃～-40℃的降温范围内,分别选择-0.5℃/min, -1℃/min, -5℃/min的降温速率进行降温,对复苏后的脐血单个核细胞的回收率、活性和CD34+含量的变化以及BFU-E、CFUGM和CFU-MK集落的回收率进行了考察,发现在-1℃/min的降温速率下,脐血MNC回收率可达93.3%±1.8%,活性可达95.0%±3.9%, CD34^＋细胞回收率达80.0%±17.9%,BFUE回收率为87.1%±5.5%,CFUGM回收率达88.5%±8.9%,CFUMK的回收率也达到86.2%±7.4%。并且对复苏后的细胞进一步进行体外培养,发现在-1℃/min的降温速率下复苏的细胞仍然具有与未经冷冻细胞相似的扩增能力,而-0.5℃/min和-5℃/min这两种降温速率条件下复苏的细胞与未经冷冻的细胞相比差距较大。因而-1℃/min的降温速率对冻存脐血干细胞比较合适。     

Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system

The performance of a small-scale automated cryopreservation and storage system (Mini-BioArchive system) used in the banking of umbilical cord blood (UCB) units was evaluated. After thawing the units, the viability and recovery of cells, as well as the recovery rate of hematopoietic progenitor cells (HPCs) such as CD34⁺ cells, colony-forming unit-granulocyte-macrophage (CFU-GM), and total CFU were analyzed. Twenty UCB units cryopreserved using the automated system and stored for a median of 34 days were analyzed. Mean CD34⁺ cell viabilities before freezing were 99.8 ± 0.5% and after thawing were 99.8 ± 0.4% in the large bag compartments and 99.7 ± 0.5% in the small compartments. The mean recovery values for total nucleated cells, CD34⁺ cells, CFU-GM, and total CFU were 94.8 ± 16.0%, 99.3 ± 18.6%, 103.9 ± 20.6%, and 94.3 ± 12.5%, respectively in the large compartments, and 95.8 ± 25.9%, 106.8 ± 23.9%, 101.3 ± 23.3%, and 93.8 ± 19.2%, respectively in the small compartments. A small-scale automated cryopreservation and storage system did not impair the clonogenic capacity of UCB HPCs. This cryopreservation system could provide cellular products adequate for UCB banking and HPC transplantation.     

Carbohydrate-mediated inhibition of ice recrystallization in cryopreserved human umbilical cord blood

Cryopreservation of human umbilical cord blood (UCB) typically involves the cryoprotectant dimethylsulfoxide (DMSO), however, infusional toxicity and reductions in cell viability remain a concern. Ice recrystallization (IR) is an important source of cryopreservation-induced cellular injury and limits the stem cell dose in UCB units. Carbohydrates have wide-ranging intrinsic IR inhibition (IRI) activity related to structural properties. We investigated the impact of carbohydrate IRI on cell viability, induction of apoptosis and hematopoietic progenitor function in cryopreserved UCB. Mononuclear cells (MNCs) from UCB were cryopreserved in storage media containing specific carbohydrates (200 mM) and compared to 5% DMSO. Samples were analyzed under conditions of high IR (‘slow’ thaw) and low IR (‘fast’ thaw). Thawed samples were analyzed for viability and apoptosis by flow cytometry and hematopoietic function using colony-forming unit (CFU) assays. IRI of carbohydrate solutions was determined using the ‘splat cooling’ assay. Greater IRI capacity of carbohydrates correlated with increased yield of viable MNCs (r² = 0.92, p = 0.004) and CD34(+) cells (r² = 0.96, p = 0.019) after thawing under conditions of high IR. The correlations were less apparent under conditions of low IR. Carbohydrates with greater IRI modulate the induction of early apoptosis during thawing, especially in CD34+ cells (r² = 0.96, p = 0.0001) as compared to total mononuclear cells (p = 0.006), and preserve CFU capacity in vitro (r² = 0.92, p = <0.0001). Our results suggest that carbohydrates with potent IRI increase the yield of non-apoptotic and functional hematopoietic progenitors and provide a foundation for the development of novel synthetic carbohydrates with enhanced IRI properties to improve cryopreservation of UCB.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

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.     

Banking strategies for improving the hematopoietic stem cell content of umbilical cord blood units for transplantation

Umbilical cord blood (UCB) has become an alternative source of hematopoietic progenitors (HSC) for transplantation. Although most CB transplants have been performed in children, unrelated donor-cord blood transplants in adults have been growing steadily in recent years. HSC content of CB units influence significantly the transplantation outcome, as shown by many clinical studies. UCB banks are fundamental to support this increasing clinical activity and one of their main goals must be to store good quality units. Strategies for increasing HSC content of UCB units are reviewed and also its influence on transplantation outcome. Our bank selected the UCB units for cryopreservation on the basis of their total nucleated cells (TNC) and CD34(+) cells content. We also reviewed the results of our UCB bank program.     

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.     

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.     

Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture

Background

Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.

Methods and Findings

UCB-derived mononuclear cells (MNC) or selected CD34⁺ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34⁺ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin⁺, CD133⁺ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.

Conclusions

This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.     

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate. We have established addition and elution protocols that prevent osmotic damage and have used these to investigate the effect of multimolar concentrations of Me(2)SO on membrane integrity and functional recovery. We have investigated the effect of freezing and thawing over a range of cooling rates and cryoprotectant concentrations. CD34(+) cells tolerate up to 60 min exposure to 25% w/w (3.2M) Me(2)SO at +2 degrees C with no significant loss in clonogenic capacity. Exposure at +20 degrees C for a similar period of time induced significant damage. CD34(+) cells showed an optimal cooling range between 1 degrees C and 2.5 degrees C/min. At or above 1 degrees C/min, increasing the Me(2)SO concentration above 10% w/w provided little extra protection. At the lowest cooling rate tested (0.1 degrees C/min), increasing the Me(2)SO concentration had a statistically significant beneficial effect on functional recovery of progenitor cells. Our findings support the conclusion that optimal recovery of CD34(+) cells requires serial addition of Me(2)SO, slow cooling at rates between 1 degrees C and 2.5 degrees C/min and serial elution of the cryoprotectant after thawing. A concentration of 10% w/w Me(2)SO is optimal. At this concentration, equilibration temperature is unlikely to be of practical importance with regard to chemical toxicity.     

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products

Background aimsThe question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.MethodsCryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003–14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay.ResultsSignificant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = −0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration.ConclusionsOnce frozen, HPC products were stable for up to 14.6 years at <−150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.     

Peripheral blood and BM CD34+ CD38- cells show better resistance to cryopreservation than CD34+ CD38+ cells in autologous stem cell transplantation

BACKGROUND: We and others have shown a critical role for CD34+ CD38- cells in hematopoietic recovery after autologous stem cell transplantation (ASCT), in particular for platelet reconstitution. Thus a routine assessment of CD34+ CD38- cells in freezing-thawing procedures for autografting could represent an important tool for predicting poor engraftment. METHODS: To compare the impact of cryopreservation on CD34+ CD38+ and CD34+ CD38- hematopoietic stem cell subsets, 193 autograft products collected in 84 patients with malignancies were assessed before controlled-rate cryopreservation in 10% DMSO and after thawing for autografting. RESULTS: Cell counts after thawing were significantly different from the pre-freezing counts for total CD34+ (P<0.0001) and CD34+ CD38+ (P<0.0001) cells, but not for CD34+ CD38- cells (P=0.252). Median losses for CD34+, CD34+ CD38+ and CD34+ CD38- cells were, respectively, 11.8%, 11.4% and 0.0%. The magnitude of fresh/post-thawing percentage cell variation was significantly different when comparing between the CD34+ CD38+ and CD34+ CD38- cell subsets (P<0.001). Moreover, CD34+ CD38- cells exhibited recovery values > or =100% in 85/160 graft products, compared with 51/193 in CD34+ CD38+ cells (P<0.0001). Also, recovery values > or =90% were significantly better in the CD34+ CD38- (98/160 grafts) than in the CD34+ CD38+ subsets (89/193 grafts) (P<0.01). DISCUSSION: In this work we have demonstrated that CD34+ cells that do not express the CD38 Ag show a significantly better resistance to cryopreservation. This could represent another example of the particular ability of less committed progenitor cells to overcome environmental injuries. Moreover, we consider routine assessment of CD34+ CD38- cells before freezing as clinically relevant, but post-thawing controls may be avoided because of their good resistance to freezing.     

Feasibility of cord blood collection for autologous cell therapy applications in extremely preterm infants

Background aimsUmbilical cord blood (UCB)-derived cells show strong promise as a treatment for neonatal brain injury in pre-clinical models and early-phase clinical trials. Feasibility of UCB collection and autologous administration is reported for term infants, but data are limited for preterm infants. Here the authors assessed the feasibility of UCB-derived cell collection for autologous use in extremely preterm infants born at less than 28 weeks, a population with a high incidence of brain injury and subsequent neurodisability.MethodsIn a prospective study at a tertiary hospital in Melbourne, Australia, UCB was collected from infants born at less than 28 weeks and processed to obtain total nucleated cells (TNCs), CD34+ cells, mononuclear cells and cell viability via fluorescence-activated cell sorting prior to cryopreservation. Feasibility was pre-defined as volume adequate for cryopreservation (>9 mL UCB collected) and >25 × 10⁶ TNCs/kg retrieved.ResultsThirty-eight infants (21 male, 17 female) were included in the study. Twenty-four (63.1%) were delivered via cesarean section, 30 (78.9%) received delayed cord clamping before collection and 11 (28.9%) were a multiple birth. Median (interquartile range [IQR]) gestational age was 26.0 weeks (24.5–27.5) and mean (standard deviation) birth weight was 761.5 g (221.5). Median (IQR) UCB volume collected was 19.1 mL/kg (10.5–23.5), median (IQR) TNC count was 105.2 × 10⁶/kg (57.4–174.4), median (IQR) CD34+ cell count was 1.5 × 10⁶/kg (0.6–2.1) and median (IQR) cell viability pre-cryopreservation was 95% (92.1–96.0). Feasibility of collection volume and cell count suitable for cell cryopreservation was achieved in 27 (71%) and 28 (73.6%) infants, respectively.ConclusionsUCB-derived cell collection adequate for cryopreservation and subsequent autologous reinfusion was achieved in 70% of extremely preterm infants. Extremely preterm UCB demonstrated a higher CD34+:TNC ratio compared with published full-term values. Recruitment to demonstrate safety of UCB cell administration in extremely premature infants is ongoing in the CORD-SAFE study (trial registration no. ACTRN12619001637134).     

An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB)

The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei).