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PubMed1996

Andrological work-up of patients undergoing microsurgical epididymal sperm aspiration or testicular sperm extraction.

Microsurgical epididymal sperm aspiration (MESA) and testicular sperm extraction (TESE) require close cooperation between andrologists, urologists and gynaecologists. Intracytoplasmic sperm injections were established in Giessen in March 1994 and embryo transfer (ET) was performed in 342 of 375 patients (91.2%). The percentage of pregnancies and ongoing pregnancies are 35.4% per ET (32.3% per cycle) and 25.1% per ET (22.9% per cycle), respectively. Microsurgical procedures such as epididymovasostomy or vasovasostomy and cryopreservation of human semen are also established methods. The purpose of the present study was to describe the andrological work-up for patients before MESA and TESE. Experiments demonstrate that incubation of testicular tissue samples in IVF medium and treatment with 1 mg ml-1 pentoxifylline increase the number of extracted motile spermatozoa. Centrifugation of the medium results in a further concentration of sperm cells. If no motile spermatozoa can be found in the supernatant medium, they may be extracted directly from the testicular tissue samples by means of a micromanipulator.

Cell Separation↗
PubMed1997

Optimal immunocytochemical and flow cytometric detection of P-gp, MRP and LRP in childhood acute lymphoblastic leukemia.

The clinical relevance of multidrug resistance (MDR)-related proteins in childhood acute lymphoblastic leukemia (ALL) is largely unknown. The diversity of techniques, fixation methods, storage of cells (fresh or cryopreserved) etc, may contribute to discrepancies observed between several studies. We therefore optimized the detection of P-glycoprotein (P-gp), MDR-associated protein (MRP) and lung resistance-related protein (LRP) by immunocytochemistry and flow cytometry in childhood ALL cells. Thirteen fixation methods were compared using six antibodies in both immunocytochemistry and flow cytometry. The optimal fixation for P-gp (C219, MRK16), MRP (MRPr1) and LRP (LRP56) was a mixture of 2% (v/v) formaldehyde solution and acetone incubated for only 10 s at room temperature (FAc). For MRP recognized by MRPm6, the optimal fixation condition was acetone for 5 min at room temperature in immunocytochemistry, and methanol for 15 min at -20 degrees C in flow cytometry. P-gp staining by 4E3 was strongly antibody batch-dependent; on cytospins FAc fixation was optimal, but inconclusive data were obtained by flow cytometry. The optimized fixation conditions on fresh samples revealed a day-to-day variation in staining (both increasing and decreasing) in one third of the immunocytochemical tests. In flow cytometry the day-to-day variation in the fluorescence index was -1 +/- 22%. In both techniques, staining was comparable between fresh and cryopreserved cells. We recommend the use of the above mentioned fixation methods in order to study the clinical relevance of P-gp, MRP and LRP in childhood ALL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification or one-step freezing.

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at -196 degrees C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45 degrees C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.

Aesculus↗
PubMed1997

Cryopreservation of channel catfish spermatozoa: effect of cryoprotectant, straw size, and formulation of extender.

Various aspects of the cryopreservation of spermatozoa of channel catfish (Ictalurus punctatus ) were studied in relation to spermatozoa motility. The objectives were to evaluate 1) the efficacy of 5, 10 and 15% of methanol or n,n-dimethyl acetamide (DMA) as cryoprotectants; 2) the acute toxicity of 5, 10 and 15% methanol or DMA; 3) the use of 0.5-ml vs. 0.25-ml straws; 4) the efficacy of 5, 10 and 15% of methanol in Hanks' balanced salt solution (HBSS) or HBSS without glucose, and 5) the use of HBSS with or without 5% methanol. We found that use of 5% methanol as a cryoprotectant resulted in significantly higher post-thaw motility (P = 0.0001) than did 5, 10 or 15% DMA. The use of 5% of either cryoprotectant resulted in significantly higher post-thaw motility (P = 0.0001) than did 10 or 15% of the cryoprotectants. Samples containing 10 or 15% DMA had significantly lower motility (P = 0.0001) after 30 min exposure than did samples containing 5, 10 or 15% methanol. The use of 0.25-ml straws resulted in significantly higher post-thaw motility (P = 0.0001) than that of 0.5-ml straws. No difference was found in post-thaw motility between HBSS with and without glucose as the extenders. Cryopreservation in HBSS without addition of cryoprotectant resulted in post-thaw motility values of about 1%.

Journal Article↗
PubMed2004

Six-year monitoring of the donor-specific immune response to cryopreserved aortic allograft valves: implications with valve dysfunction.

BACKGROUND: The immune rejection has been anticipated as one of the major causes of allograft aortic valve (AAV) degeneration. The purpose of this study was to prospectively serially measure the magnitude and evolution of the recipient anti-HLA class I antibody response up to 6 years from AAV implant and to correlate serologic data with valve performance by means of a concurrent echocardiographic survey. METHODS: Cryopreserved AAVs were obtained from multiorgan HLA-typed donors. Nineteen patients younger than 50 years (mean age, 43.3 +/- 8 years) were prospectively studied. After successful surgery, all AAV recipient underwent at 3 and 6 months and each year postoperatively (mean follow-up, 71.9 months) concomitant serum sample collection and two-dimensional transthoracic echocardiography. The presence of anti-HLA antibodies was tested against a panel of lymphocytes obtained from 30 blood donors. RESULTS: Progressive structural valve deterioration was seen in 6 patients (31.5%) of whom 4 (21%) were reoperated. All pretransplant recipients sera were panel-reactive antibody negative. Seventeen patients (89.4%) demonstrated significant panel-reactive antibody levels, which peaked at 6 months postoperatively, declined from 6 to 24 months, and slowly decreased afterward. In 14 of 19 cases (73.6%) donor-specific HLA antibodies were identified. A strong immunization (6-year persistence of panel-reactive antibody > 70% and peak panel-reactive antibody > 80%) was detected in 31.5% and 36.8% of recipients, respectively. Strong immunization was found to be significantly associated with progressive structural deterioration. CONCLUSIONS: The immune reaction after cryopreserved AAV implantation is a peculiar long-lasting response occurring in the majority of recipients younger than 50 years of age. An association between a sustained and pronounced immunization and an aggressive AAV degeneration was observed.

Adolescent↗
PubMed2005

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone.

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.

Acrosome↗
PubMed1990

Freeze-drying foot-and-mouth disease virus antigens. I. Infectivity studies.

The ability of foot-and-mouth disease virus strains type O1 BFS 1860 and type A22 IRQ 24/64 to retain infectivity after freeze-drying with or without additives being made to virus suspensions was studied. The infectivity titres of freeze-dried antigens was assessed at intervals over a six month storage period at various temperatures and also after reconstitution to the liquid phase and storage with or without glycerination. Certain additive solutions were necessary to prevent degradation of virus during the freeze-drying procedure which reduced any loss of infectivity caused by storage of products at 4 degrees C and 20 degrees C. Additive solutions composed of 10% sucrose and 5% lactalbumin hydrolysate; 10% skimmed milk; 4% peptone and 1% gelatin; and 5% dextran, 1% sodium glutamate and 5% sucrose all prolonged the keeping qualities of virus at the elevated temperature of 37 degrees C. The results indicate that short-term storage and shipment of freeze-dried foot-and-mouth disease virus antigens is possible without the need for refrigeration, thereby reducing transportation and storage costs. Reconstituted antigens survived better after glycerination and storage at -20 degrees C than did non-glycerinated samples stored at 4 degrees C.

Animals↗
PubMed1994

Determination of oestrogen receptors by enzyme immunoassay. Technical differences between laboratories and their consequences.

When multicentre breast cancer trials are performed, receptor analyses must be comparable both over time and in the participating laboratories. However, we show for the first time a high variability for the distribution of oestradiol receptor (ER) values measured by enzyme immunoassay (EIA) from 1987 to 1991. This variability could be explained by calibration changes in the immunoassay kits. We have also analysed the influence on ER-EIA levels of technical differences between laboratories apart from the assay itself. Many steps emerged as being critical, i.e. homogenisation buffer, homogenisation procedure and cytosol dilution. Finally, we show that addition of 4-monohydroxytamoxifen increases the apparent ER content measured by EIA in 92% of cytosols. Thus, many factors must be controlled to ensure high precision with ER-EIA assays. We have to be particularly cautious with the conformational changes that could occur during cytosol preparation and that could also pre-exist in the tumour samples. Quality controls of cytosol preparation are essential.

Breast Neoplasms↗
PubMed2005

Epoxy resin as fixative during freeze-substitution.

An alternative protocol for freeze-substitution is described. Araldite/Epon embedding medium (20% in acetone) is first used as a stabilizer (as e.g., OsO(4)) and then as embedding medium. The major components of the Araldite/Epon resin formulation react with proteins and lipids and provide for an excellent preservation and reasonable visualisation of the ultrastructure. The ultrastructural appearance can be deliberately influenced with the standard freeze-substitution procedure [Van Harreveld, A., Crowell, J., 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381-386.] using OsO(4) as stabilizing agent by protocols which degrade cytoplasmic and membrane proteins. Epoxy stabilized and embedded samples may become an important tool to get information about the effects of different reagents and protocols used in freeze-substitution. We believe that an in-depth understanding of the procedures is required to correctly interpret images and to complement studies of dynamic processes by light microscopy with reliable, highly detailed ultrastructural information. The block face of epoxy stabilized samples after ultrathin sectioning is highly suited for the analysis of the ultrastructure by AFM.

Animals↗
PubMedDate not supplied

Improvement in the cervical mucus penetration test by using standard sperm control.

The objective of the present experiments was to establish an acceptable standard for the cervical mucus penetration test (CMPT) by determining the minimal progressive motile spermatozoa concentration (PMSC) that will yield the highest score for proven fertile donor sperm specimens. For this purpose, fresh and frozen-thawed samples were used. Semen was obtained from 29 fertile donors and different PMSC (8, 10, and 14 x 10(6)/mL) were prepared for each sample. The same mucus specimen was used for testing each sperm sample in the three different dilutions. No difference in the scoring of the CMPT between the fresh vs. frozen-thawed groups was found. When PMSC of 14 x 10(6)/mL was used, almost all specimens scored the highest rank. The present study revealed that only semen samples with a minimal PMSC of 14 x 10(6)/mL cells can be used in the CMPT. The information that the freeze-thaw process does not affect the CMPT results supports the concept of cryopreservation of pooled fertile donor specimens in aliquots with adequate concentration of progressive motile spermatozoa for later use as a CMPT control.

Cervix Mucus↗
PubMed1992

Role of the frozen tissue bank in molecular pathology.

The new discipline of molecular pathology requires that high-quality, intact genomic DNA, mRNA, and proteins be available from frozen tissue samples. It is now necessary for pathology laboratories to establish consistent guidelines for the preparation and storage of frozen tissue samples in order to have properly preserved tissues available for diagnostic molecular techniques. Maintaining a frozen tissue bank requires a pathologist to oversee this program and to integrate it into the routine surgical pathology activities. A member of the laboratory technical staff can serve as a tissue bank coordinator and have responsibility for preparation of tissue samples, their systematic storage and retrieval, and routine maintenance of equipment and supplies. Tissue sampling must be done as soon as possible after excision of the specimen and is the responsibility of a qualified pathologist. The samples may be snap frozen without cryoprotection at -78 degrees C or colder for subsequent use in procedures requiring the extraction of genomic DNA, mRNA, or protein. To preserve tissue architecture and cytologic features for immunohistochemistry and in situ hybridization, the tissue should be frozen at -78 degrees C or colder with a cryoprotectant such as OCT. Long-term storage of the frozen tissue is recommended at -140 degrees C or colder in a locked liquid nitrogen freezer, and the record of sample inventory can easily be kept in a computerized database. Tissues sampled and stored under these conditions have been used successfully in a wide variety of molecular techniques. In addition to malignant tumor tissue, samples from benign lesions and normal tissues should be frozen.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreservation↗
PubMed1992

Freezing and calcium chloride marination effects on beef tenderness and calpastatin activity.

Because freezing samples decreases calpastatin activity and the application of exogenous calcium activates the calpain proteolytic system, thereby improving tenderness, the objective of this study was to determine whether freezing would enhance the effects of CaCl2 marination on the tenderness of beef steaks. Longissimus steaks were obtained from 10 beef steers 6 d postmortem. One-half of the steaks were frozen at -30 degrees C for 6 wk. The remaining steaks were treated fresh; one-half were subjected to a 150 mM CaCl2 marinade for 48 h. Frozen steaks were thawed and subjected to the same treatment. Treatments consisted of 1) fresh control, 2) fresh marinated, 3) frozen control, and 4) frozen marinated. Samples were taken before and after treatment (6 and 8 d) for calpastatin activity determination and d 8 for SDS-PAGE. Warner-Bratzler shear force values were measured 8 d postmortem. Data were analyzed using a paired comparison t-test procedure. Results showed that freezing and marination significantly decreased calpastatin activity. A .35-kg improvement (P = .07) in Warner-Bratzler shear force was observed with freezing, whereas a .78-kg improvement (P less than .01) in tenderness was observed with marination. However, prior freezing enhanced the effects of marination. Therefore, the decrease in calpastatin activity seemed to allow greater proteolysis by the calpains with the application of Ca2+. The SDS-PAGE of myofibril preparations indicated that more small polypeptide fragments (28 to 32 kDa) appeared and a 95-kDa fragment was more intense in the marinated samples than in control samples, indicating that proteolysis was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Brain banks and non nervous tissues.

Nervous system diseases may not be confined to neural tissue, but also affect other organs. These organs could be involved indirectly or could be simultaneously affected by the same pathological process. A brain bank (BB) should also guarantee the storage of specific organs primarily or secondary affected other than nervous system. Tissues from patients with primary nervous system diseases without or with unknown systemic involvement should also be stored. Samples stored will be identified and registered in a BB database for an accurate distribution and utilization of the material. To guarantee the best quality of the material stored, several techniques for the collection and preservation (cryopreservation, chemical fixation and microwave irradiation) and tissue management are described.

Brain↗
PubMed1979

The survival of cryopreserved human bone marrow stem cells.

Two methods for cryopreservation of bone marrow stem cells were compared using bone marrow obtained from 36 patients. Included in this group were 21 persons with the diagnosis of leukaemia including 14 either with acute myeloid or lymphoblastic leukaemia in remission following intensive remission induction chemotherapy. After freeze-preservation and reconstitution, all marrow samples were tested for nucleated cell (NC) recovery and grown on agar to assess colony forming units (CFUC) and cluster forming units in culture (CluFUc). A slow dilution reconstitution method using freezing media containing AB negative plasma resulted in recovery of 85% of the CFUc activity of fresh marrow. This result was significantly better than the 47% CFUc recovery obtained when freezing media without plasma and a rapid dilution reconstitution technique were used. NC recoveries following slow dilution (51%) and rapid dilution (44%) were not significantly different. CluFUc were disproportionately reduced compared with CFUc although yielding similar results with both methods (26% and 32%). No correlation was found for either method between CFUc and NC recovery or between CFUc and CluFUc recovery in cryopreserved bone marrow.

Adolescent↗
PubMed2005

Experiences in deer sperm cryopreservation under practical conditions--a pilot study.

The genetic potential of the red and fallow deer populations in Hungary is well known. Conserving the variability in this excellent genetic material for game preservation is one of our most important task. The aim of the present pilot study was to test the logistical steps of a sperm processing and storing system in which deer sperm can be stored at a level that meets quality standards accepted for domestic animals. Moreover, two different semen extenders, commercially used for freezing bull semen, were compared from the viewpoint of applicability to freeze fallow deer sperm. Sperm was collected from epididymes of eight red stags (Cervus elaphus hippelaphus) and six fallow bucks (Dama dama) during the rutting season. Red deer samples were washed in Triladyl extender, while fallow deer samples were split and processed in Triladyl or Bioxcell extender. In the samples, which had a shorter time interval between the death of the animal and the sperm collection, the percentage of viable spermatozoa with intact acrosome was typically higher.

Animals↗
PubMed2000

The mechanism of cryoinjury: In vitro studies on human internal mammary arteries.

The mechanism of cryoinjury was investigated in human internal mammary arteries (IMA) by monitoring contractile responses to ET-1 and KCl. For cryopreservation segments of IMA were equilibrated for 20 min with the cryomedium (RPMI 1640 culture medium containing 1.8 M DMSO and 0.1 M sucrose), frozen at a mean cooling rate of 1.3 degrees C min(-1) to -70 degrees C and stored in liquid nitrogen. Before use, samples were thawed slowly and the cryomedium removed by dilution. Compared to unfrozen controls, ET-1 stimulated frozen/thawed IMA with similar efficacy but at 3 fold lower concentrations (P<0.001). Addition of ET-1 (100 nM) induced maximal contraction of unfrozen IMA within 10 min, declining thereafter to 25% after 90 min. In frozen/thawed IMA the ET-1-induced contraction was sustained but could be reversed if protein kinase C was blocked by staurosporine (100 nM). Responses to ET-1 of cryostored IMA were 5 fold more susceptible to blockade by nifedipine than those of controls. After cryostorage the efficacy of KCl was diminished to 40% (P<0.05) and the KCl curve was shifted to the left (2 fold, P<0. 001). In both unfrozen and cryostored IMA the KCl (60 mM) effect was sustained and equally susceptible to nifedipine. It is suggested that the smooth muscle cell of IMA is receptive to physical forces which occur during cryopreservation. These forces modify transmembrane signal transduction and intracellular pathways, that are common to pharmacological agonists thereby changing vascular responses to several contractile agonists after thawing.

Calcium↗
PubMed2005

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation.

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.

Acrosome Reaction↗
PubMed1997

Functional recovery of human mesenteric and coronary arteries after cryopreservation at -196 degrees C in a serum-free medium.

PURPOSE: Long-term patency of cryopreserved vascular grafts is determined by maintained cellular and tissue viability, which implies preservation of various biochemical, smooth muscle, and endothelial functions. Therefore, it was investigated whether the presence of fetal calf serum (FCS) in the cryomedium improves the postthaw contractile and endothelial function of human arteries. METHODS: Rings from human mesenteric (HMA) and left circumflex coronary arteries (HCA) obtained from organ donors were randomized into three groups and studied either unfrozen or after storage for 3 to 6 weeks at -196 degrees C while suspended in Krebs-Henseleit solution without or with 20% FCS as the vehicles and 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose as cryoprotecting agents. The samples were slowly frozen to -70 degrees C and then stored in liquid nitrogen. Before use, the tissues were thawed within 3 minutes in a 40 degrees C water bath. RESULTS: After thawing the sensitivity to various agonists and maximal responses to the endothelium-independent relaxing agent sodium nitroprusside were unchanged. However, after cryopreservation of HMA was performed without and with FCS, maximal contractile responses to noradrenaline were significantly reduced to 10.1 +/- 0.7 gm and 9.9 +/- 0.9 gm compared with 13.3 +/- 0.6 gm in unfrozen HMA (mean +/- SEM, n = 15). After cryopreservation of HCA was performed without and with FCS, maximal contractile responses to prostaglandin F2 alpha (6.9 +/- 0.4 gm in unfrozen HCA) were significantly reduced to 4.3 +/- 0.3 gm and 3.8 +/- 0.2 gm (mean +/- SEM, n = 6). In both types of arteries cryopreservation also attenuated significantly the endothelium-dependent relaxant responses to bradykinin during U46619 (10 nmol/L)-induced tone. In HMA the maximal bradykinin-induced relaxation (85% +/- 4%) was significantly diminished to 29% +/- 7% and 38% +/- 9% after cryopreservation without and with FCS (mean +/- SEM, n = 6). In HCA maximal bradykinin-induced relaxation (88% +/- 4%) was significantly diminished to 26% +/- 10% and 36% +/- 11% after cryopreservation without and with FCS (mean +/- SEM, n = 6). This result was reflected by a marked endothelial denudation in all groups of cryopreserved arteries. Neither functional nor morphologic preservation of the endothelial cell lining was significantly improved by FCS supplementation of the cryomedium. CONCLUSIONS: Cryopreservation diminished contractile and endothelium-dependent relaxant responses of human arteries. The presence of FCS in the cryomedium did not modify these changes.

Animals↗