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.     

Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocytes

Controlled ovarian stimulation during an in vitro fertilization cycle usually produces large numbers of oocytes and, consequently, it is likely that more embryos will be generated than can be transferred in a given cycle. It is desirable to freeze-bank surplus oocytes before insemination to avoid the ethical and legal complications of disposing of or storing embryos. Although many attempts have been made to cryopreserve human oocytes, to date, post-thaw survival has been poor, and viable pregnancies after in vitro fertilization have been rare. A possible explanation for the lack of success is that the freezing methods have been adapted from animal studies but have not been optimized for the human oocyte. In this study, video microscopy was used to determine the volumetric responses of mature human oocytes to changes in osmolarity during preparation for freezing. A Boyle van't Hoff plot of data collected in static experiments with fresh human oocytes gave a value of 0.19 +/- 0.01 (mean +/- SEM) for the osmotically inactive volume. Dynamic measurements during exposure to dimethyl sulphoxide at room temperature (22 degrees C) were analysed by a two-parameter transport model and produced values of 1.30 x 10(-6) cm atm-1 s-1 for the hydraulic conductivity of the plasma membrane and 3.15 x 10(-5) cm s-1 for dimethyl sulphoxide permeability (chi-squared = 0.43, df = 20) of fresh human oocytes. Oocytes that had failed to fertilize had a slightly lower hydraulic conductivity and dimethyl sulphoxide permeability and, after exposure to 1.5 mol dimethyl sulphoxide l-1, these cells appeared to become permeable to normally impermeable solutes. These permeability properties have been used to design a protocol for the addition and removal of dimethyl sulphoxide to control the magnitude of volumetric changes.     

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.     

Characteristics of hematopoietic stem cells of umbilical cord blood

Umbilical cord blood collected from the postpartum placenta and cord is a rich source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow transplantation. In this review we wanted to describe the differences (in phenotype, cytokine production, quantity and quality of cells) between stem cells from umbilical cord blood, bone marrow and peripheral blood. HSCs present in cord blood are more primitive than their counterparts in bone marrow or peripheral blood, and have several advantages including high proliferation. With using proper cytokine combination, HSCs can be effectively developed into different cell lines. This process is used in medicine, especially in hematology.     

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.     

Hyperbaric oxygen treatment effects on in vitro cultured umbilical cord blood CD34+ cells

Background aims

Umbilical cord blood (UCB) provides an alternative source for hematopoietic stem/progenitor cells (HSPCs) in the treatment of hematological malignancies. However, clinical usage is limited due to the low quantity of HSPCs in each unit of cord blood and defects in bone marrow homing. Hyperbaric oxygen (HBO) is among the more recently explored methods used to improve UCB homing and engraftment. HBO works by lowering the host erythropoietin before UCB infusion to facilitate UCB HSPC homing, because such UCB cells are not directly exposed to HBO. In this study, we examined how direct treatment of UCB-CD34⁺ cells with HBO influences their differentiation, proliferation and in vitro transmigration.

Factors affecting retrovirus-mediated gene transfer to human CD34+ cells

BACKGROUND: Retrovirus-mediated gene transfer is a useful technology in studying the biology of hematopoietic stem cells (HSCs) as well as in developing gene therapy products for a variety of human diseases. One of the most important factors determining the success of these studies is the number of HSCs receiving the gene of interest. METHODS: We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on a literature survey, three medium formulations of CD34+ cells have been compared for their effects on gene delivery efficiency and differentiation of them. We also tested whether FBS, used in the medium formulation, could be replaced with human serum or synthetic material. RESULTS: Formulation A, consisting of stem cell factor, Flt-3 ligand, thrombopoietin, and IL-3, provided optimum results in that it maintained the highest percentage of CD34+ cells during the culture as well as produced the highest gene delivery efficiency. It was found that the synthetic serum substitute containing bovine serum albumin, insulin and human transferrin could replace the fetal bovine serum present in the original formulation A without compromising gene transfer efficiency. When the transduction procedure was repeated three times, the gene could be delivered in up to 60% of the cell population. Gene delivery efficiency was comparable between CD34+ cells derived from bone marrow and mobilized peripheral blood. CONCLUSIONS: Our data could be useful in designing a procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs.     

Cytokine-pretreatment of CD34(+) cord blood stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice

Stem cell homing, engraftment and organ regeneration are controlled by cytokines, chemokines and cell-cell interactions. In this paper, cytokine effects on homing- and engraftment-related characteristics of CD34(+) cord blood cells were examined. Untreated CD34(+) cells were mainly in the G(0)/G(1) cell cycle phase, expressed adhesion receptors on a low level, were positive for vimentin, and negative for the epithelial marker cytokeratin 8/18. Treatment with stem cell factor (SCF) stimulated cell proliferation, increased the number of cells in S and G(2)/M cell cycle phase as well as the expression of adhesion receptors. The expression of cytokeratin 8/18 was increased and that of vimentin remained unchanged. Hepatocyte growth factor (HGF) did not stimulate cell proliferation and expression of adhesion receptors, but increased expression of cytokeratin 8/18. In NOD/SCID mice, kinetics of stem cell distribution revealed a fast elimination of human cells from blood. An increase in the number of engrafted cells was observed in different mouse organs in a time-dependent manner, preferentially in bone marrow, spleen and liver. Pretreatment with SCF resulted in reduction of long-term engraftment in bone marrow. HGF pretreatment of cord blood cells showed no significant effects on long-term engraftment capacity in mouse organs compared to untreated cells. Our data provide in vivo evidence that pretreatment of CD34(+) cells with SCF reduces long-term cell engraftment in NOD/SCID mice.     

Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells

The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC.     

Characterization of cryobiological responses in TF-1 cells using interrupted freezing procedures

Cryopreservation currently is the only method for long-term preservation of cellular viability and function for uses in cellular therapies. Characterizing the cryobiological response of a cell type is essential in the approach to designing and optimizing cryopreservation protocols. For cells used in therapies, there is significant interest in designing cryopreservation protocols that do not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant, since this cryoprotectant has been shown to have adverse effects on hematopoietic stem cell (HSC) transplant patients. This study characterized the cryobiological responses of the human erythroleukemic stem cell line TF-1, as a model for HSC. We measured the osmotic parameters of TF-1 cells, including the osmotically-inactive fraction, temperature-dependent membrane hydraulic conductivity and the membrane permeability to 1 M Me₂SO. A two-step freezing procedure (interrupted rapid cooling with hold time) and a graded freezing procedure (interrupted slow cooling without hold time) were used to characterize TF-1 cell recovery during various phases of the cooling process. One outcome of these experiments was high recovery of TF-1 cells cryopreserved in the absence of traditional cryoprotectants. The results of this study of the cryobiology of TF-1 cells will be critical for future understanding of the cryobiology of HSC, and to the design of cryopreservation protocols with specific design criteria for applications in cellular therapies.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C

Based on previous evidence suggesting positive effects of fever on in vivo hematopoiesis, we tested the effect of hyperthermia on megakaryopoiesis (MK) in ex vivo cultures of CD34-enriched cord blood (CB) cells. The cells were cultured at 37 degrees C or 39 degrees C for 14 days in cytokine conditions optimized for megakaryocyte development and analyzed periodically. Compared to 37 degrees C, the cultures maintained at 39 degrees C produced significantly more (up to 10-fold) total cells, myeloid and MK progenitors, and total MKs, and showed accelerated and enhanced MK maturation with increased yields of proplatelets and platelets. This observation could facilitate clinical applications requiring ex vivo expansion of hematopoietic cells.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

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.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

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.     

Expansion of CD34~+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold. Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1±5.5)-fold and (57.0±19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 2     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.