Seasonal changes in recovery of cryopreserved murine lymphocytes resemble endogenous rhythms of unfrozen cells

Seasonal changes in the resistance of C57BL/6 mouse splenocytes to cryopreservation stress were expressed in both the recovery of viable cells and the levels of responses of T and B lymphocytes to mitogens in vitro. Single cell suspensions in 10% Me2SO were cooled at 1 degree C/min, the optimum velocity which was determined by using a range of cooling rates during January and May, the months of minimum and maximum recoveries of viable cells, respectively. After rapid thawing and washing, ethidium bromide-fluorescein diacetate staining delineated viable and nonviable cells. Cultures containing 0.5 X 10(6) viable cells were stimulated with the T lymphocyte mitogens, phytohemagglutinin and concanavalin A, and the B lymphocyte mitogen, lipopolysaccharide. Tritiated thymidine was added to each culture for the last 18 hr of the incubation period, and its incorporation by activated dividing cells was determined. Recoveries of viable cells were high from March through July and then declined to minimum levels in January and February. During the seasons of low recoveries, greater numbers of cells lysed in response to the freeze-thaw cycle. Activation of both T and B lymphocytes by mitogens was maximal in the spring and summer and then declined to only 40% of unfrozen control levels in October. The patterns of activation resembled those of the previously documented endogenous seasonal rhythms in levels of blastogenesis of unfrozen cells. These seasonal differences in cryopreservation properties of lymphocytes from inbred mice living under constant conditions reinforce the previously reported endogenous annual rhythmicity in cellular functions.     

Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of [³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.     

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.     

Leukapheresis cell concentration adjustment required for a successful recovery of HSC after cryopreservation

The recovering of an adequate number of hematopoietic stem cells after cryopreservation is considered pivotal for successful transplantation. Various factors could influence the recovery of HSC following processing and cryopreservation. Therefore, leukapheresis product from thirty patients was cryopreserved in 10% DMSO in cryopreservation bags for their autologous bone marrow transplantation, and 2 ml were cryopreserved in cryovials for post-thaw viability assessment by flow cytometry. The percentage of viable HSCs recovered post-cryopreservation in leukapheresis product was significantly influenced by the concentration of the total nucleated cells cryopreserved per volume. Patients receiving a higher rate of viable HSCs resulted in earlier engraftment of both neutrophils and platelets, so they have been discharged earlier from the hospital. Furthermore, Storage temperature and duration played a role in the recovery of these cells and for the support of the findings, age of the patient at the time of collection did not show any impact on the recovery of this HSC post-cryopreservation. In conclusion, various influencing factors must be taken into consideration during the cryopreservation of HSCs, especially for poor mobilizing patients with a low number of collected hematopoietic stem cells.     

Susceptibility of human T cells, T-cell subsets, and B cells to cryopreservation

We have studied the number of viable and functionally active T and B lymphocytes obtainable after cryopreservation to determine the best and most practical way to recover the maximal number of viable and functionally active cells. Assays were done on purified populations of human T and B cells recovered after cryopreservation. The results were compared to those obtained from similar types of cells fractionated from fresh and from cryopreserved mononuclear cells. The number of viable T cells recovered after cryopreservation was significantly lower than the number of viable T cells obtained from either fresh or cryopreserved mononuclear cells. The residual viable T cells recovered after cryopreservation showed significantly reduced blastogenic activity in response to pokeweed mitogen (PWM) stimulation. This occurred despite their normal blastogenic response to phytohemagglutinin and their normal ability to help B cells in the production of immunoglobulins following PWM stimulation. The reduction in the blastogenic responses of these T cells to PWM stimulation is attributed to the loss of a portion of the PWM responding subset of T cells. The loss in this subset of T cells was related to the exposure of cells to ammonium chloride prior to cryopreservation. The viability and functional abilities of B cells were not affected regardless of whether purification was done before or after cryopreservation. These findings indicate that extrinsic membrane damage to T cells induced prior to cryopreservation can affect the viability and responsiveness of a certain population of normal T cells. The damage can be minimized by reversing the sequence of T-cell isolation and freezing so that isolation of T cells is done after, rather than before, freezing. These results could be important in the study of T cells from patients with T-cell abnormalities, since the patients' cells could have an intrinsic membrane defect which would make them sensitive to freezing similar to that induced by extrinsic damage.     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

Changes with senescence in the fine structure of the granular convoluted tubule of the submandibular gland of the mouse

Cells of the granular convoluted tubules (GCTs) of the submandibular gland of senescent male mice show structural changes indicative of functional decline. In order to define the nature of these age-related changes more clearly, the fine structure of GCT cells of 12- and 28-month-old males was compared. In old mice, there was cell-to-cell variation in the extent of these changes, with some cells of senescent males appearing no different from those of young adults. In affected cells the most striking alterations were seen in secretion granules and lysosomal elements. Secretion granules varied greatly in size, with some GCT cells having only very fine apical granules. Secondary lysosomes and large lipofuscin granules were frequent in the basal cytoplasm. Very large dense bodies (3-5 micron) occurred in many cells. These possibly represent intracellular pools of released secretory materials, as they were occasionally seen in continuity with the luminal contents. Structures whose appearance was intermediate between the very large dense bodies and lipofuscin granules were common, suggesting crinophagic activity. There was an apparent decrease in numbers of polysomes and in the extent of the Golgi apparatus. These fine structural changes are consistent with impairments with advanced age in synthesis and posttranslational processing of secretory products by affected GCT cells. In addition to cell-to-cell variation in any one male, there was also interanimal variation in the degree and extent of these senescent changes.     

Functional assessment of cryopreserved human femoral arteries for pharmaceutical research

An established method for cryopreservation that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. HFAs were harvested from multiorgan donors and stored at 4 degrees C in saline solution before cryostorage. Thirty HFA rings were isolated and randomly assigned to one control group of unfrozen HFAs (eight rings) and one group of cryopreserved HFAs (22 rings).Cryopreservation was performed in RPMI solution containing dimethylsulfoxide (DMSO) and the rate of cooling was -1 degrees C/min until -40 degrees C and faster rates until -150 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs.After thawing, the maximal contractile responses to the contracting agonist tested (noradrenaline) were in the range of 43% of the responses in unfrozen HFAs. The endothelium-independent responses to sodium nitroprusside were not altered whereas the endothelium-dependent relaxant responses to acetylcholine were weakly altered.The cryopreservation method used provided a limited preservation of contractility of HFAs, a good preservation of the endothelium-independent relaxant responses, and a good preservation of endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of DMSO, might allow better post-thaw functional recovery.     

Age-related alterations in superoxide anion generation in mouse peritoneal macrophages studied by repeated stimulations and heat shock treatment.

The ability of thioglycollate-elicited peritoneal macrophages (PM) from young and senescent mice to generate superoxide anions (O2-) under repeated stimulation or thermal stress was studied using either zymosan, opsonized zymosan (OZ), or phorbol myristate acetate (PMA). A diminished capacity to recover from repeated stimulation was found with aging. When stimulated for a second time 24 hours after the primary stimulation, PM from young animals generated 80% of the initial O2- responses to either zymosan, or OZ. Under the same conditions, PM from senescent mice generated 62% of the initial O2- produced in response to zymosan, and 45% in response to OZ. In both age groups the response to a second PMA stimulation comprised only 10% of the primary response. A considerably diminished capacity to generate O2- was also demonstrated in PM from senescent mice after recovery from exposure to thermal stress. Exposure to 42.5 degrees C for 20 minutes was found to be the threshold temperature for irreversible loss of activity in senescent PM, whereas at this temperature, PM from young animals recovered up to 70% of their O2- generating activity. Since NADPH oxidase and superoxide dismutase activities were only mildly affected by the hyperthermia in all age groups, they could not account for the age-related decline in the recovery from stress. Age-related alterations in signal transduction or receptor alterations could possibly play a primary role in this decline.     

Biochemical and ultrastructural examination of cryopreserved hepatocytes in rat

We investigated the ultrastructure and metabolic capabilities of isolated rat hepatocytes after cryopreservation using 1.5 M Me₂SO as protectant and a slow cool/fast thaw regime. Ultrastructural assessment of the cryopreserved population revealed only approximately 10% of cells with normal morphology. Conjugation of bilirubin by the cryopreserved cells was reduced to 20% of that seen in unfrozen hepatocytes and there was a net loss of glycogen measured in cryopreserved cells incubated in conditions which stimulated glycogen synthesis by unfrozen cells. These results are in contrast to other reports in which cryopreserved hepatocytes have been successfully used for transplantation to reverse hepatic insufficiency.     

Ageing,opioid analgesia and the pineal gland

The effects of ageing on day-night rhythms of analgesia was examined with young (1–2 months), mature (8–12 months) and old (20–30 months) mice. Significant age-related declines were observed both in the absolute levels and diel rhythms of morphine analgesia, with the most pronounced changes occuring at night. Administration of the pineal hormone, melatonin, augmented day-time levels of analgesia in all age classes and reversed the age-related decline in nocturnal morphine analgesia in old mice. Inhibition of pineal function in young mice by either exposure to light pulses or treatment with benserazide mimicked the effects of ageing on nocturnal morphine analgesia. These findings suggest that the pineal gland and melatonin are involved in modulating diel rhythms of analgesia and have an influential role on age-related changes in opioid responses.     

The null cell compartment of the mouse spleen

Null lymphocytes were defined as lymphocytes without detectable T- or B-cell markers using a battery of techniques. The null cell compartment was divided into pre-T cells, pre-B cells, and other null cells based upon their acquisition of membrane markers when incubated with ubiquitin. The null cell subpopulations were remarkably consistent in spleen cell suspensions from young adult mice of various strains. Commitment to T- or B-cell differentiation took place at the null cell stage and did not require thymic input. Pre-T cells, but not pre-B cells, were steroid sensitive. Pre-T cells accumulate with congenital thymic deficiency. This differed from senescent thymodeprivation where the outstanding finding was an accumulation of uninducible null cells. Neonatal mouse spleens were deficient in pre-T and pre-B cells but had an accumulation of uninducible cells.     

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.     

Cryopreservation of swine colostrum-derived cells

Mesenchymal stromal cells (MSCs) have been demonstrated to possess anti-inflammatory and antimicrobial properties and are of interest in biotechnologies that will require cryopreservation. Recently, MSC-like cells were isolated from colostrum and milk. We used an interrupted slow freezing procedure to examine cryoinjury incurred during slow cooling and rapid cooling of MSC-like cells from swine colostrum. Cells were loaded with either dimethyl sulfoxide (Me₂SO) or glycerol, cooled to a nucleation temperature, ice-nucleated, and further cooled at 1 °C/min. At several temperatures along the cooling path, cells were either thawed directly, or plunged into liquid nitrogen for storage and later thawed. The pattern of direct-thaw and plunge-thaw responses was used to guide optimization of cryopreservation protocol parameters. We found that both 5% Me₂SO (0.65 M, loaded for 15 min on ice) or 5% glycerol (0.55 M, loaded for 1 h at room temperature) yielded cells with high post-thaw membrane integrity when cells were cooled to at least −30 °C before being plunged into, and stored in, liquid nitrogen. Cells cultured post-thaw exhibited osteogenic differentiation similar to fresh unfrozen control. Fresh and cryopreserved MSC-like cells demonstrated antimicrobial activity against S. aureus. Also, the antimicrobial activity of cell-conditioned media was higher when both fresh and cryopreserved MSC-like cells were pre-exposed to S. aureus. Thus, we were able to demonstrate cryopreservation of colostrum-derived MSC-like cells using Me₂SO or glycerol, and show that both cryoprotectants yield highly viable cells with osteogenic potential, but that cells cryopreserved with glycerol retain higher antimicrobial activity post-thaw.     

Splenic and inguinal lymph node T cells of aged mice respond differently to polyclonal and antigen-specific stimuli.

Numerous changes have been reported to occur in T cell responsiveness of mice with increasing age. However, most of these studies have examined polyclonal stimulation of spleen cells from a limited number of mouse strains. This study investigated the influence of genetic background, source of lymphocytes, and type of stimulus on age-associated changes in T cells response. Con A-induced proliferation and IL-2 and IFN-gamma production by splenic lymphocytes (SL) was significantly greater in CBA/Ca mice compared to C57BL/6 mice, regardless of age. SL of both strains exhibited the predicted age-dependent decline in proliferative response and an increase in IFN-gamma production in response to Con A. In contrast, however, only SL from C57BL/6 mice demonstrated the predicted age-dependent decline in Con A-induced IL-2 production; Con A-induced SL of young and aged CBA/Ca mice produced comparable amounts of IL-2. Differences in age-associated responses to Con A were also observed between SL and inguinal lymph node (ILN) cells of CBA/Ca mice. In contrast to SL, ILN cells demonstrated an increased proliferative response to Con A. However, lymphokine production by Con A-stimulated ILN cells from aged CBA/Ca mice was similar to that of Con A-stimulated SL from aged CBA/Ca mice. To determine if aged ILN T cells respond similarly to polyclonal and antigen-specific stimuli, keyhole limpet hemocyanin (KLH) responses of T cells isolated from ILN of aged and young CBA/Ca mice were examined. KLH-specific T cells from aged mice cultured with KLH-pulsed macrophages (M phi) from aged mice were significantly reduced in their ability to proliferate compared to KLH-specific T cells of young mice cultured with young KLH-pulsed M phi. In contrast to the expected results, the defect was not at the level of the T cells; proliferation of young T cells cultured with aged KLH-pulsed M phi was equivalent to the proliferation of aged T cells cultured with aged M phi. These results suggest that aging has differential effects on polyclonal and antigen-specific T cell proliferation and on polyclonal stimulation of T cells isolated from different lymphoid organs and from different strains of mice.     

Structure and functional alterations in lymphocytes induced by cryopreservation

Surface markers, Con A-induced capping, blastogenic transformation stimulated by PHA and allogeneic mononuclear cells, and natural killer activity of Ficoll — Hypaque-separated lymphocytes were studied before and after varying periods of cryopreservation. An increase was observed in the relative number of E rosetteforming cells and in the incorporation of [³H]thymidine into DNA, by the unstimulated cryopreserved cells after thawing. On the other hand, a substantial drop occurred in the Con A-induced capping and the natural killer activity of cryopreserved cells. The possible causes for the variation in the effects of cryopreservation on lymphocyte functions as reported by different investigators were discussed. It was concluded that until universally accepted, standardized procedures for the assessment of lymphocyte functions in vitro become available, each laboratory should establish the changes induced by cryopreservation in lymphocyte function with the methods employed locally to allow the observations made on cryopreserved lymphocytes to be meaningful.     

人衰老成纤维细胞经紫外线损伤后的DNA修复和细胞周期调控

 以体外培养的不同代龄的人胚肺二倍体成纤维细胞（２ Ｂ Ｓ）为对象,紫外线诱导 Ｄ Ｎ Ａ 损伤后,观察细胞形态、增殖特性、细胞周期、 Ｄ Ｎ Ａ 修复变化等细胞应答以及 ｇａｄｄ１５３、ｐ２１ Ｗ Ａ Ｆ１／ Ｃ Ｉ Ｐ１／ Ｓ Ｄ Ｉ１、ｐ５３ 等基因的转录水平的表达变化．结果显示：紫外线诱导 Ｄ Ｎ Ａ 损伤后,衰老（＞ ５５ 代）２ Ｂ Ｓ细胞形态及增殖能力的改变不如年轻细胞（＜ ３０ 代）显著;不同代龄的细胞损伤后均出现 Ｇ１ 期阻滞现象,年轻细胞 Ｇ１ 期阻滞率明显高于衰老细胞（ Ｐ＜ ００５）;衰老细胞总的修复能力较年轻细胞明显下降（ Ｐ＜ ００１）;同时,ｇａｄｄ１５３、ｐ２１、ｐ５３ 等的可诱导性均低于年轻 ２ Ｂ Ｓ细胞．由此,分别在细胞水平与基因水平反映了衰老细胞经紫外线照射损伤后的细胞应答变化与修复机能减退的关系．     

Serum-free solutions for cryopreservation of cells

With the development of cell-based assays and therapies, the purity of reagents used to grow and maintain cells has become much more important. In particular, the use of fetal calf serum for culturing cells presents a direct path for potential contamination of cell cultures. In recent years, much research has focused on the development of serum-free culturing systems, not only to alleviate difficulties due to availability and cost of fetal calf serum but also to prevent the transmission of potentially fatal diseases to human patients. Additionally, methods need to be developed for long-term storage of cell stocks that also reduce the risk of exposure to harmful diseases. As most methods employ fetal calf serum in their freezing formulations, solutions that avoid the use of fetal calf serum while providing equivalent or better recovery of cells upon thawing would be ideal. In this study, two vascular cell lines have been cryopreserved as adherent cell populations in two widely used cryoprotectants, dimethyl sulfoxide and 1,2-propanediol, and two vehicle solutions, Euro-Collins and Unisol-cryoprotectant vehicle specifically formulated for the maintenance of cell homeostasis at temperatures below 37° C. The addition of serum to these formulations was also evaluated to determine if its presence provided any additional benefit to the cells during cryopreservation. The results demonstrated that using vehicle solutions designed for lower temperatures produced viable cells that retained cell population viability values up to 75% of unfrozen controls. These results also demonstrated that including serum in the formulation provided no additional benefit to the cells and in some cases actually produced lower cell viability after cryopreservation. In conclusion, the development of solutions designed for low-temperature storage of cells provides a viable alternative to more conventional cryopreservation protocols and eliminates the necessity of including serum in these formulations.     

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.     

Functional assessment of human femoral arteries after cryopreservation

An established method for the cryopreservation of human femoral arteries for subsequent transplantation as allografts has been studied with particular attention to preservation of smooth muscle and endothelium. Human femoral arteries (HFAs) were harvested from multi-organ donors. Two groups were established; a control group of unfrozen HFAs and a group of cryopreserved HFAs. Cryopreservation was performed using RPMI solution containing dimethyl sulfoxide and the rate of cooling was 1 degrees C/min to -40 degrees C and faster thereafter until -150 degrees C was reached. The contraction and relaxation responses of unfrozen and frozen/thawed arteries were assessed by measurement of the isometric force generated by the HFAs in an organ bath. After thawing (warming was at 15 degrees C/min) the maximal contractile response to noradrenaline was 43% of the response of unfrozen HFAs. The endothelium-independent response to sodium nitroprusside was not altered, whereas the endothelium-dependent relaxation response to acetylcholine was slightly altered. The cryopreservation method used provided limited preservation of the contractility of human femoral arteries, and good preservation of both endothelium-independent and endothelium-dependent relaxation responses.