New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.     

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.     

Comparison of three methods for human corneal cryopreservation that utilize dimethyl sulfoxide.

We compared endothelial cell survival in human corneas after cryopreservation by three methods that utilize dimethyl sulfoxide. Twenty-eight human cadaver corneas were cryopreserved by one of three methods, stored briefly over liquid nitrogen, thawed, cultured at 37 degrees C for 3 days, and fixed for scanning electron microscopy. Seventeen control corneas underwent identical cryoprotectant immersion and culture protocols but were not frozen. Endothelial photographs taken after 1 and 3 days of culture were analyzed. Endothelial cell losses in cryopreserved corneas by Methods 1, 2, and 3, respectively, were 36, 22, and 10% after 1 day of culture and 57, 36, and 27% after 3 days of culture. Cryopreservation by Method 3 had less cell loss than Methods 1 or 2 (P<0.02) but greater cell loss than the control corneas for Method 3 (P<0.001). No loss of cells occurred in the control corneas for Methods 1 and 3 but substantial cell loss (26%) occurred in the control corneas for Method 2. Polymegethism and pleomorphism of the endothelial cells were seen in the corneas that lost cells. The endothelial cell loss of 10% seen after 1 day of culture in human corneas cryopreserved by Method 3 is similar to the loss that occurs during organ culture storage as currently used clinically and therefore would be acceptable for clinical use. After 3 days of culture, however, the cell loss had increased significantly to 27%. This additional decrease in cell number that occurs in culture may represent latent cryodamage and must be understood and overcome in vivo before the technique can be used clinically.     

Antibody-dependent cell-mediated cytotoxicity of cryopreserved human monocytes

Human peripheral blood monocytes (PBM) modulate and participate in a variety of host defences. Cryopreservation of PBM has facilitated studies of their function. Peripheral blood samples cleared of red cells and granulocytes by centrifugation over Ficoll-Hypaque were cryopreserved at 1 degree C/min in 10% Me2SO and stored at -150 degrees C. Cryopreserved cells were thawed rapidly, diluted at a constant rate over 10 min with 9 vol of media, and washed twice prior to study. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity against anti-D-coated Rh-positive erythrocytes of both fresh and cryopreserved PBM was tested and found to be equal (52.5 vs 51%). The myeloperoxidase positive, EA-rosette-positive population in cryopreserved cells was 39% compared with 17% for fresh cells (P less than 0.0001). This difference is due to preferential recovery of cryopreserved monocytes among mononuclear cells. The proportion of cells expressing Fc receptors among the myeloperoxidase-positive mononuclear cell population increased after freezing, suggesting an alteration in membrane structure induced by cryopreservation. It is concluded that PBM can be cryopreserved in Me2SO and that ADCC function is fully retained in the cryopreserved cells. This study along with a previous study (R.S. Weiner and S.J. Norman, J. Natl. Cancer Inst. 66, 255-260, 1981) demonstrate the feasibility of using cryopreserved human PBM for functional studies.     

Characterization of cryopreserved human Langerhans cells

Epidermal Langerhans cells are potent antigen-presenting cells in the epidermis. The establishment of a cryopreservation method for human Langerhans cells would greatly contribute to our ability to successfully conduct various experiments dealing with Langerhans cells. Since Langerhans cells are known to be sensitive to cold injury, there have been no reports concerning the cryopreservation of Langerhans cells. We have investigated the effect of cryopreservation on the function and phenotype of human Langerhans cells. Langerhans cells from human foreskins were isolated with the immunomagnetic microbead method using monoclonal antibodies for CD1a. Langerhans cells were cryopreserved in the presence of dimethylsulfoxide (DMSO) 10% and fetal calf serum 90%. Cryopreserved Langerhans cells were phenotypically assessed by flowcytometry using monoclonal antibodies to HLA-DR and CD1a. The ultrastructures of the Langerhans cells were compared using electron microscopy. An autologous T cell stimulation test was performed to compare the functions of cryopreserved Langerhans cells and fresh Langerhans cells. The viability of the cryopreserved Langerhans cells was able to be maintained at more than 90%. Cryopreserved Langerhans cells expressed high levels of HLA-DR and CD1a antigens and stimulated autologous T cells to an extent almost identical to that obtained from fresh Langerhans cells. These findings indicate that the cryopreservation of human Langerhans cells could lead to a breakthrough in various experiments dealing with human Langerhans cells.     

Cryopreservation of Suspension Cell Line Derived from the Protoplast Culture of Brassica campestris var. Pekinensis

Protected by DMSO, the suspension cell line derived from the protoplast culture of Brassica campestris var. pekinensis can be stored in liquid nitrogen (-196℃) for a long term. The addition of sorbitol and mannose can increase and decrease the protection, respectively. The medium also has an effect on cryopreservation. The relative survival rates of cells are little different in different days of cryopreservation. The highest rate of relative survival of cryopreserved cells reaches 75.4%. When the cryopreserved cells are thawed and resuspended, regrowth immediately occurs after just one day of lag period. Resuspended for six days, the cells increase 300–500%. It is much better for recovery of growth to resuspend in the dark than in the light. Like the non-cryopreserved control, the cryopreserved cells can be normally digested, producing a number of viable protoplasts which can be actively divided and form calli.     

The maintenance of free-living amoebae by cryopreservation.

We have successfully cryopreserved free-living amoebae in order to maintain them feasibly under the conditions in our laboratory. The viability of trophozoites was higher when frozen by slow cooling (overall 0.7 degree C/min) than by fast cooling (overall 1.3 degrees C/min). Glycerol and dimethylsulfoxide at the final concentration of 7.5% each was used for cryopreservation of free-living amoebae trophozoites. The survival rate was 2-39% after storage in the liquid nitrogen for 60 days. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.     

Choline phospholipid metabolites of human vascular endothelial cells altered by cyclooxygenase inhibition,growth factor depletion,and paracrine factors secreted by cancer cells

Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO), reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs). We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.     

The effect of cryoprotection and cryodamage on corneal rehydration and endothelial structure

Corneal rehydration rate and endothelial ultrastructure were compared during postthaw perfusion of cryopreserved corneas and corneas that had been frozen and thawed without the benefit of cryoprotective solutions. The postthaw swelling rate of cryopreserved rabbit corneas was 171 μm/hr compared to 333 μm/hr in the cryodamaged corneas. Most of the endothelial cells were intact in the cryopreserved corneas, but the intercellular spaces were enlarged. In contrast, all of the endothelial cells in the cryodamaged corneas were disrupted and Descemet's membrane was only partially covered by the remaining cell fragments.     

Gradual Thawing Improves the Preservation of Cryopreserved Arteries

This study was designed to test a slow, controlled, automated process for the thawing of cryopreserved arteries, whereby specimen warming is synchronized with the warming of its environment. Segments of minipig iliac artery, 4-5 cm in length, were subjected to controlled, automated cryopreservation in a biological freezer at a cooling rate of 1 degrees C/min to -120 degrees C, followed by storage in liquid nitrogen at -196 degrees C for 30 days. Following storage, the arterial segments were subjected to rapid (warming rate of approximately 100 degrees C/min) or gradual (1 degrees C/min) thawing. Thawed specimens were processed for light microscopy and for scanning and transmission electron microscopy, Cell death was determined by the TUNEL method. Metalloproteinase (MMP) expression was estimated by immunohistochemical analysis. Most of the cryopreserved vessels subjected to rapid thawing showed spontaneous fractures, mainly microfractures, whereas these were absent in slowly thawed specimens. In rapidly thawed vessels, the proportion of damaged cells was double that observed in those thawed more gradually. Increased intensity and extent of MMP-2 expression was shown by rapidly thawed specimens. The slow-thawing protocol tested avoids the formation of spontaneous fractures and microfractures and the accumulation of fluid within the arterial wall tissue. This results in improved tissue preservation.     

Effects of cryopreservation on the immunogenicity of porcine arterial allografts in early stages of transplant vasculopathy

BACKGROUND: The number of revascularization procedures including coronary and lower extremity bypass, have increased greatly in the last decade. It suggests a growing need for vascular grafts. Cryopreserved allografts could represent a viable alternative but their immunologic reactivity remains controversial. METHODS: 71 pigs (40 recipients and 31 donors) were used. Two femoral grafts per recipient animal were implanted for 3, 7, and 30 days. Types of grafts: fresh autograft as a control graft (n=19), fresh allograft (n=31) and cryopreserved allograft (n=30). Histological and immunohistochemical studies were performed. RESULTS: Fresh allografts compared to autografts showed intimal inflammatory infiltration at 3 days (328 vs. 0 macrophages/mm2; P<0.05) and 7 days (962 vs. 139 T lymphocytes/mm2; P<0.05) post-transplantation. At 30 days, there was a loss of endothelial cells, presence of luminal thrombus and aneurismal lesions (total area=15.8 vs. 8.4 mm2; P<0.05). Cryopreservation did not reduce these lesions nor modify endothelial nitric oxide synthase (eNOS) expression nor modify the number of animals that developed anti-SLA antibodies. Moreover, at 7 days, cryopreserved allografts compared to fresh allografts showed a higher expression of P-selectin (5 out of 5 vs. 1 out of 5; P<0.05) and, at 30 days, a greater inflammatory reactivity (2692 vs. 1107 T lymphocytes/mm2 in media; P<0.05) with a trend towards a higher presence of multinucleated giant cells than in the fresh ones. CONCLUSIONS: The cryopreservation method used maintained immunogenicity of allografts and increased the inflammatory reactivity found in fresh allografts up to 30 days of vascular transplantation.     

The effect of liquid nitrogen submersion on cryopreserved human heart valves

Cryopreservation and storage of human heart valves have become an accepted means of maintaining a usable supply of heart valves for outflow track reconstructive surgery. Valves are typically stored at the vapor phase temperature of liquid nitrogen, -130 degrees C and below, to reduce the chance of recrystallization within the tissues. Concern over the effects of submersion of the valves in liquid nitrogen, i.e., plunging to -196 degrees C, prompted this study. Cryopreserved valves were plunged into liquid nitrogen, held for 5 min, and then processed (thawed) by standardized protocols. The thawed valves were then assessed using scanning electron microscopy and the more traditional histology at the light microscope level. Cuspal tissues plunged into liquid nitrogen appear to have numerous microfractures over both surfaces of the tissue, penetrating into the collagen/proteoglycan matrix. Control cryopreserved valves do not exhibit these microfractures. Histologically, the submerged valves appear normal. The clinical use of valves which have been submerged in liquid nitrogen is discussed.     

Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.)

A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15–21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.Abbreviations DMSO dimethyl sulfoxide - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtylacetic acid - FDA fluorescein diacetate     

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10⁻⁷m) for 24 h. The concentrations of PGE₂ and PGF_2α in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE₂ and PGF_2α release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.     

Restoring the endothelium of cryopreserved arterial grafts: co-culture of venous and arterial endothelial cells

The use of arterial homografts in clinical practice is becoming increasingly common, yet there is an urgent need to address one of the most well-established problems associated with their use: the loss of integrity of the endothelium following cryopreservation. The partial lack of endothelium causes contact between the extracellular matrix and blood flow, which, in turn, often gives rise to thrombosis and/or restenosis. Our objective was first to attempt to replace the arterial endothelial cells lost during the cryopreservation process by seeding autologous venous endothelial cells, and to evaluate the behaviour of venous and arterial endothelial cells in co-culture. The idea was to establish whether venous endothelial cells would be accepted by arterial endothelial cells and could therefore be used to restore the endothelial lining for the subsequent use of these vessels in in vivo grafting procedures. For the co-culture experiments, endothelial cells were obtained from the jugular vein and both iliac arteries of the minipig by treatment with 0.1% type I collagenase. The venous endothelial cells were fluorescently labelled with the membrane intercalating dye PKH26. Equal numbers of venous and arterial endothelial cells were mixed and co-cultured for 24h, 48h or 4 days. Cell viability, determined by 2% trypan blue staining and the TUNEL method, was established before and after fluorescence labelling. Cellular activity was determined by estimating PGI2 levels in the cultures. The proliferation index was established by [H(3)]thymidine (1muCi/ml) in the cell culture medium. For the in vivo tests, 5 cm length segments of minipig iliac artery were used to establish the groups: control (n = 6), fresh arterial segments; group I (n = 16), cryopreserved arterial segments and group II (n = 16), cryopreserved arterial segments seeded with autologous venous endothelial cells. The cryopreserved vessels in group II were seeded by flooding with a labelled venous endothelial cell suspension. Once seeded, the arterial segments were included in an in vitro flow circuit. All the specimens were processed for fluorescence and light microscopy, and scanning electron microscopy. The denuded endothelial surface was determined in each group. Cell death was evaluated by the TUNEL method. We confirmed the existence of intercellular PECAM1-type junctions between venous (PKH26+) and arterial cells in co-culture and the functional activity of the cells. The cryopreserved arterial segments showed a well-preserved wall structure. However, different size areas of marked endothelial denudation were detected. After seeding with labelled cells (PKH26+), these denuded areas of the cryopreserved artery were entirely covered by fluorescent cells. After seeding, a drop in the proportion of damaged endothelial cells was recorded. Despite some loss of seeded cells after inclusion in the in vitro flow circuit, the endothelial cell count was not significantly different to those recorded for control, non-cryopreserved specimens. In conclusion arterial and venous endothelial cells growing in co-culture modify their behaviour to form multilayers. The two cell populations form normal PECAM1 junctions and preserve their functional properties. Seeding autologous venous endothelial cells on the luminal surface of cryopreserved arterial segments serves to restore the integrity of the endothelial layer.     

Optimization of a Method for the Cryopreservation of Rabbit Corneas: Attempted Application to Human Corneas

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 ±300 viable cellsmm² when using dimethylsulfoxide; 3403 ± 245 viable cellsmm² when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1°C/min and thawed at 37°C (2003 ± 372 viable cells/mm² when using dimethylsulfoxide and 1357 ± 667 viable cells/mm² when using 1,2-propanediol). Cryopreserved human corneas had 753 ± 542 viable cells/mm² when using dimethylsulfoxide and 56 ± 56 viable cells/mm² when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.     

Cell cultures from cryopreserved human lung tissue.

To assess gene induction in primary human fibroblasts, we have developed a method for cryopreservation of lung biopsies in liquid nitrogen. Fresh biopsies (n = 10) were chopped into 5 x 5 mm pieces and transferred into an ice-cold freezing medium. Biopsies were kept on ice for 15 min, followed by further cooling of the tissue to -70 degrees C. With this method, lung biopsies were preserved for more than 1 year before they were used for generating cell cultures. There was no significant difference in the biological responsiveness of fibroblasts generated from immediately cultured lung biopsies compared with those from cryopreserved tissue. The doubling rate of fibroblasts from fresh tissue was 23.6 +/- 1.1 hr; compared to 23.5 +/- 1.5 hr for fibroblasts generated from cryopreserved tissue. PDGF-BB enhanced de novo synthesis of DNA 100 times, in both the immediately cultured fibroblasts and those generated from cryopreserved biopsies. Macrophages, dendritic cells and endothelial cells could also be recovered from cryopreserved lung tissue. This method permits long-term storage of lung tissue and the possibility of establishing primary cell lines from the same tissue at later times without appreciable changes in their cellular biological characteristics.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.     

Endothelial cells from hematopoietic stem cells are functionally different from those of human umbilical vein

Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth suplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12-15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow-cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM-1 9.65 ± 0.2% and CD106/VCAM 7.73±0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.