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PubMed2003

Immune function of cryopreserved avian peripheral white blood cells: potential biomarkers of contaminant effects in wild birds.

Contaminants can cause detrimental effects in wild birds. However, these effects are difficult to measure in all but the most severe cases. Immune function is a sensitive and meaningful biological marker of contaminant-induced effects in captive birds but has more limitations in wild birds due in part to the lack of a proven blood preservation method. We developed methods to assess ex vivo immune function in wild birds using cryopreserved peripheral white blood cells (WBCs). We assessed the effects of cryopreservation on WBC viability and functionality in two immunoassays (concavalin A-induced T lymphocyte proliferation and macrophage phagocytosis) in domestic chickens (Gallus spp.: white Wyandottes and Dominiques) and validated this approach on cryopreserved WBC samples from wild American coots (Fulicia americana). Cryopreservation of chicken WBCs caused a slight but significant decrease in cell viability (99% +/- 0.2 SE for fresh cells versus 84% +/- 2 SE for cryopreserved cells, p = 0.001, Mann-Whitney U, n = 8). No difference was detected in viability between cells that were cryopreserved for less than 10 days (88% +/- 3.7 SE) and more than 50 days (89% +/- 1.3 SE) (n = 6). Overall, there was no statistical difference in the performance of cryopreserved cells compared to fresh cells. Across multiple experiments, cryopreserved T lymphocytes exhibited 200-900% stimulated proliferation above nonstimulated cells, and 40-80% of cryopreserved macrophages ingested yeast. 9,10,Dimethyl-1,2-benz-anthracene (DMBA) reduced proliferation and phagocytosis in cryopreserved cells over an ex vivo exposure range of 0-170 microM DMBA. Tests of immune function on American coot WBCs cryopreserved for up to 10 months (viability of 72% +/- 2.5 SE, n = 24) were similar to the cryopreserved chicken WBCs. This study will facilitate greater use of ex vivo immune function assays as tools to study effects of contaminant exposure in wildlife by demonstrating the viability and functionality of cryopreserved avian cells.

Animals↗
PubMed2004

Cryopreservation alters antigenicity of allografts in a porcine model of transplant vasculopathy.

UNLABELLED: The need for arterial grafts in coronary surgery to complement autologous vessels has generated interest in cryopreservation of small diameter allografts. We evaluated functional and histologic changes occurring in cryopreserved allografts 3 months after porcine femoral artery transplants. METHODS: Twenty recipient and 15 donor pigs included a control group of 16 fresh and 12 cryopreserved nonimplant arteries were used. Fresh (n=5) and cryopreserved (n=5) autografts were implanted to assess cryopreservation effects in the absence of rejection. Fresh allografts with or without treatment with cyclosporine (CsA) (n=6 of 8) and cryopreserved allografts with or without treatment with CsA (n=6 of 10) were performed to study the antigenicity of cryopreserved allografts. Arteries were stained with hematoxylin and eosin, Masson's trichrome, and orcein for morphometric analyses and immunostained to identify endothelial cells, smooth muscle cells, T lymphocytes, and macrophages. RESULTS: Among nonimplant arteries, cryopreservation reduced alpha-actin expression and increased the luminal area. All implanted autografts were patent. Cryopreserved autografts showed reduced alpha-actin expression and developed intimal hyperplasia compared to fresh autografts. Treatment with CsA improved the patency of fresh allografts from 0% to 83% (P <.01) and of cryopreserved allografts from 40% to 100% (P <.05). Cryopreserved allografts showed substantial intimal hyperplasia, and fresh allografts had more T lymphocyte infiltration in the intimal layer with aneurysmal dilatation. CONCLUSIONS: Cryopreservation reduces the deposition of inflammatory cells and prevents the thrombosis or aneurysmal lesions observed in fresh allografts. Therefore, cryopreservation modifies the antigenicity of vascular allografts.

Animals↗
PubMed1997

Mechanisms underlying degeneration of cryopreserved vascular homografts.

OBJECTIVE: To analyze the mechanism(s) underlying homograft degeneration, we designed an experimental model in which the behavior of cryopreserved autografts and homografts, as well as fresh autografts, implanted in the same animal was compared. METHODS: A cryopreserved homograft was implanted in the aorta of 14 sheep. The excised aortic autologous segment was then subjected to cryopreservation, and 1 to 8 weeks later it was implanted 1 to 2 cm below the cryopreserved homograft. The intermediate segment of the native aorta, the fresh autograft, was dissected at this point. Animals were put to death at different times and the implanted segments were harvested together with a portion of native aorta. Histologic and immunohistochemical analyses, as well as cell viability assessments, were then performed on the explanted segments. Similar studies were also conducted on fragments of cryopreserved autografts and homografts before implantation. RESULTS: With the exception of a partial loss of the endothelium, cryopreserved specimens retained cell viability and morphologic integrity before implantation. Explanted cryopreserved homografts showed profound changes affecting all strata, as well as a decline in cell viability. Lymphocyte infiltrates were found up to 12 months after implantation. Endothelium was always absent in cryopreserved homografts. However, a reendothelialization of the cryopreserved autografts was observed. After an initial period of neuronal degeneration, reenervation of the cryopreserved autograft segment occurred 6 to 12 months after the operation. Findings regarding the fresh autografts were similar to those of the cryopreserved autografts. CONCLUSION: Our results suggest that the immunologic reaction rather than the cryopreservation process is responsible for the degenerative process occurring in cryopreserved homografts.

Animals↗
PubMed2001

Transplantation of cryopreserved cardiomyocytes.

BACKGROUND: The present study examined the survival and rate of contraction of (1) cardiomyocytes cultured from cryopreserved fetal rat myocardium and (2) cryopreserved cultured cardiomyocytes. In addition, the effects of transplantation of cryopreserved fetal cardiomyocytes were evaluated. METHODS: Segments of fetal rat myocardial tissue (0.2, 2.0, and 6.0 mm(3) mince size) and cultured cardiomyocytes were cryopreserved in liquid nitrogen for 1, 2, and 4 weeks. After cryopreservation, the tissue samples and cultured cardiomyocytes were thawed at 37 degrees C and cultured, and cell proliferation and rate of contraction were determined. Cultured cryopreserved (n = 5) and noncryopreserved (control, n = 5) fetal cardiomyocytes were transplanted into the subcutaneous tissue and into a transmural left ventricular free wall scar of Sprague-Dawley rats (n = 3). The survival and rate of contraction of these transplanted cells were also examined. RESULTS: Cryopreservation of cultured fetal cardiomyocytes resulted in viable and functional cardiomyocytes although the cell number and percentage of beating cells were diminished. Survival of cardiomyocytes isolated from cryopreserved fetal myocardium was a function of tissue size before cryopreservation; the lowest survival was recorded in tissues with the largest mince size (6.0 mm(3)). The subcutaneous transplants contracted spontaneously and regularly with an idioventricular rhythm. In addition, the transplanted cardiomyocytes were elongated and formed a myocardium-like pattern with blood vessels present within the contractile tissue. In the transmural left ventricular scar, both control and experimental fetal cardiomyocyte transplants formed myocardium-like tissue. CONCLUSIONS: The present study uncovers the following key observations: (1) cryopreservation of fetal cardiomyocytes and cardiomyocytes isolated from cryopreserved myocardial tissue results in viable and functional cells, (2) cryopreserved fetal cardiomyocytes can be successfully transplanted into subcutaneous and myocardial scar tissue, and (3) improvements in cryopreservation techniques are required to augment the rates of cardiomyocyte survival observed in the study.

Animals↗
PubMed2000

Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction.

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

Animals↗
PubMedDate not supplied

Transplantation of retinal pigment epithelium using viable cryopreserved cells.

Transplantation of retinal pigment epithelium (RPE) may have potential clinical application for the surgical treatment of RPE-specific retinal degeneration, including age-related macular degeneration. The feasibility of an RPE storage bank has been investigated by experimenting with transplantation using viable, cryopreserved RPE cells. Fresh and cultured fetal human and bovine RPE cells were cryopreserved in 90% fetal bovine serum containing 10% dimethyl sulfoxide. The viability of the cells before and after cryopreservation was evaluated by trypan blue dye exclusion test, microculture tetrazolium assay (MTA), tissue culture, and transplantation after cryopreservation. The origin of RPE cells before and after cryopreservation was assessed by immunocytochemistry, immunoblotting, and indirect ELISA of RPE-marker protein using cytokeratin for cultured fetal human RPE cells and by immunocytochemistry of cellular retinaldehyde-binding protein (CR-ALBP) for cultured bovine RPE cells. Freshly isolated and cryopreserved uncultured bovine RPE cells were transplanted by posterior transscleral approach into the subretinal spaces of adult albino rabbits and 23-day-old Royal College of Surgeons (RCS) rats with a 33 gauge Hamilton syringe. Following surgery, artificial retinal blebs were confirmed by fundus examination. Morphologic examination was performed postoperatively by light and electron microscopy in albino rabbits and by light microscopy in RCS rats up to 3 mo. Control subretinal injections using vehicle solution also were performed in RCS rats. Cultured fetal human and bovine RPE cells after cryopreservation were found to be viable, based on the results of trypan blue dye exclusion test, MTA, tissue culture, and transplantation. Expression and reexpression of cytokeratin intermediate filaments in cultured fetal human RPE were demonstrated by immunocytochemistry, immunoblotting, and indirect ELISA before and after cryopreservation. Immunocytochemistry of CRALBP before and after cryopreservation in uncultured bovine RPE cells disclosed expression and reexpression of RPE cell marker protein. No uncultured fetal human RPE cells showed proliferation in tissue culture after cryopreservation. In rabbits, light and electron microscopy disclosed xenografted RPE cells residing on Bruch's membrane of the host retina. No sign of graft vs. host reaction was observed. No morphologic difference was noted between the fresh and 10-day-old cryopreserved RPE cells in situ following transplantation at day 25. In RCS rats, subretinal injection of 3-wk-old cryopreserved bovine RPE cells partially rescued photoreceptor cells locally at the transplanted area observed at 3 mo postoperatively. The retinal photoreceptors at the inferior hemisphere of the transplanted eye and the eye injected with vehicle solution showed no rescue effect. We found that cryopreserved cultured fetal human RPE cells and uncultured and cultured bovine RPE cells can be used for RPE transplantation studies. The ability to create an RPE storage bank as a source of donor cells may result in several clinical advantages.

Animals↗
PubMed2006

Analysis of the effects of cryopreservation on rat hepatocytes using SELDI-TOF mass spectrometry.

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.

Animals↗
PubMed2005

Sperm cryopreservation before cancer chemotherapy helps in the emotional battle against cancer.

BACKGROUND: Sperm cryopreservation before cancer chemotherapy is available for young patients with cancer. However, few patients bank their sperm. The authors evaluated by questionnaire the psychological impact of sperm cryopreservation. METHODS: The authors cryopreserved the sperm of 111 patients with cancer for free at the Yokohama City University (Yokohama, Japan). For the current study, questionnaires were mailed to 66 patients whose sperm had been cryopreserved. Fifty-one patients (77.3%) with testicular carcinomas (n = 24), leukemia or malignant lymphoma (n = 19), or other cancers (n = 8) answered the questionnaire. The average age at collection and the period of sperm cryopreservation were 30.1 +/- 6.0 (mean +/- standard deviation) and 3.3 +/- 2.2 years, respectively. RESULTS: Many patients were informed of the deleterious effects of cancer chemotherapy and worried about infertility in the future. However, only half of the patients banked their sperm on their own initiative. Other patients followed their physician's instruction. Eighty percent of patients replied that sperm cryopreservation helped in the battle against cancer. Sperm banking especially was found to encourage every patient who banked sperm on their own initiative. After cancer chemotherapy, 70% of patients wanted to have a child. However, 60% of patients were worried about infertility in spite of having their sperm cryopreserved. No patients wanted to use cryopreserved sperm for fathering children if their spermatogenesis was restored. Sperm cryopreservation invigorated many patients with cancer after cancer treatments. The majority of patients recommended sperm cryopreservation to other cancer patients. CONCLUSIONS: Sperm cryopreservation encouraged young patients with cancer during and after cancer treatment. It should be recommended for all young patients with cancer. However, sperm cryopreservation did not eliminate their fear of infertility.

Adult↗
PubMed1993

Ca2+ regulation by cryopreserved bull spermatozoa in response to A23187.

The regulation of intracellular Ca2+ by fresh and cryopreserved bull spermatozoa from the same ejaculates (n = 5) was investigated using the fluorescent Ca2+ indicator, indo-1. Relative internal Ca2+ levels of the spermatozoa were monitored for 30 min prior to the addition of phosphate-buffered saline (PBS), 1 mM Ca2+, the Ca2+ ionophore A23187 (0.1 microM), or Ca2+ + A23187; during the additions the levels of intracellular Ca2+ were observed in detail. After these additions, changes in internal Ca2+ levels were monitored for 120 min. The initial intracellular Ca2+ levels in the cryopreserved spermatozoa were greater than those in fresh (1.05 +/- 0.03 vs 0.97 +/- 0.03 Ca2+ units, P = 0.0001). The addition of Ca2+ + A23187 induced elevated cellular Ca2+ in both fresh (P = 0.0177) and cryopreserved spermatozoa (P < or = 0.0588) within 5 s. Ca2+ alone did not differ from Ca2+ + A23187 in increasing Ca2+ in cryopreserved spermatozoa (P = 0.2225); fresh spermatozoa were slower to respond to exogenous Ca2+ alone (P = 0.0438). At the start of the post-treatment 120 min, cryopreserved spermatozoa had more internal Ca2+ than the corresponding fresh samples (P < or = 0.0088) except for those exposed to both Ca2+ + A23187 (P = 0.2918). All spermatozoa increased internal Ca2+ over the post-treatment time except for the cryopreserved PBS controls, and these cryopreserved controls differed from the fresh controls in accumulation of Ca2+ (P = 0.0620). None of the Ca2+/A23187 treatments induced different rates of change in internal Ca2+ over time in fresh cells (P = 0.3142). The rate of Ca2+ accumulation by cryopreserved spermatozoa in the presence of exogenous Ca2+ exceeded controls (23.12 +/- 6.75 vs 8.11 +/- 6.75, P = 0.0005), but A23187, with or without Ca2+, had no effect (P > or = 0.0907). Following Ca2+ measurements, the viability of the cryopreserved samples was reduced (P < or = 0.015) in all but the Ca(2+)-treated spermatozoa (P = 0.1474); no viability differences were noted for fresh spermatozoa (P > or = 0.2298). The cryopreservation process did not affect acrosomal morphology of the indo-1-exposed spermatozoa (P > or = 0.1147). These data indicate that Ca2+ regulation by Ca(2+)- and A23187-challenged bull spermatozoa differs following cryopreservation procedures, possibly relating to the reduced fertilization capacity of commercially cryopreserved semen.

Animals↗
PubMed2001

Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression.

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.

Cartilage, Articular↗
PubMed2003

Immune and non-immune factors in cryopreserved tissues.

BACKGROUND: Although cryopreserved tissues are used clinically, the effects of cryopreservation on antigenicity leading to immune and non-immune responses are not well known. METHODS: We investigated the change of inflammatory effects of cryopreserved tissue by using spleen and aortic allografts from Class I antigen-disparate B6.C-H-2(bm1) (bm1; K(bm1), IA(b), D(b)), Class II antigen-disparate B6.C-H-2(bm12) (bm12; K(b), IA(bm12), D(b)) and Class I and Class II antigen-disparate (bm1 x bm12)F1 (K(bm1 x b), IA(b x bm12), D(b)) mice against C57BL/6 Cr Slc (B6; H-2(b)) mice. Cryopreservation was done in a programmed freezer and cryopreserved tissues were kept in the vapor phase of liquid nitrogen for 2 weeks and thawed at room temperature. RESULTS: Cryopreserved B6 spleen cells expressed almost the same levels of Class I (K(b) and D(b)) and Class II (IA(b)) antigens as observed in fresh B6 spleen cells. Cryopreserved bm1 and bm12 spleen cells had the same stimulator activities in mixed-lymphocyte reaction (MLR) and cytotoxic T-lymphocyte (CTL) assays compared with fresh bm1 and bm12 spleen cells, respectively. To elucidate the effects of cryopreserved tissues on immune response of recipients, descending aortas of (bm1 x bm12)F1 mice were implanted into the right common carotid artery of B6 (H-2(b)) mice with the cuff technique and the reactivities of recipient B6 mice against Class I antigen-disparate bm1 antigens and Class II antigen-disparate bm12 antigens were examined 4 weeks after implantation. In both MLR and CTL assays against bm1 or bm12 antigens, anti-donor reactivities were augmented and there was no significant difference between B6 mice grafted with fresh aortic allografts and those grafted with cryopreserved ones. Histologic analysis showed that mild infiltration of mononuclear cells into the adventitia was observed in both fresh and cryopreserved aortic allografts. The fibrous change was observed more strongly in cryopreserved aortic allografts compared with fresh aortic allografts. CONCLUSIONS: Cryopreservation has no effect on eliciting immune responses to Class I or Class II alloantigens, but has some effect on promoting fibrous change.

Animals↗
PubMed1997

Transfusion of autologous, hydroxyethyl starch-cryopreserved red blood cells.

UNLABELLED: In this prospective, randomized study, we investigated the safety and efficacy of the transfusion of hydroxyethyl starch (HES) cryopreserved red blood cells (RBC) compared with the transfusion of liquid-stored RBC in patients undergoing major orthopedic or urologic surgery. Thirty-six patients donated autologous blood 35 +/- 6 days before elective surgery. Only the first of 3.5 +/- 1.3 donated units of RBC was randomly assigned to be stored in the liquid state at 4 degrees C in phosphate/adenine/guanosine/glucose/saline-Mannitol or frozen below -130 degrees C by means of liquid nitrogen after the addition of HES (molecular weight 200,000 Dalton, degree of substitution 0.5, final concentration 11.5% wt/wt) as a cryoprotectant. After induction of anesthesia, patients donated 900 mL of autologous blood before they received one unit of liquid-stored RBC in Group 1. In Group 2, one unit of cryopreserved autologous RBC was transfused after removal of the cryoprotectant HES. In Group 3, patients received one unit of cryopreserved RBC without any manipulation after thawing. Patients in Groups 1 and 2 received additional 500 mL of 10% HES. Hemodynamic variables, arterial blood gases, plasma hemoglobin, and arterial lactate concentrations were measured after the induction of anesthesia, after hemodilution, and at 10-min intervals after transfusion of the respective RBC concentrate over a period of 40 min. Skeletal muscle tissue oxygen tension was measured in the quadriceps muscle using an automatically stepwise-driven oxygen partial pressure electrode. We found no differences among groups concerning demographics, arterial blood gas values, and lactate concentrations and observed no adverse reactions after transfusion of the conventionally stored or cryopreserved RBC. Hemodynamic variables did not differ among groups, with the exception of an increased mean arterial blood pressure after the transfusion of cryopreserved unwashed RBC. In all groups, the skeletal muscle tissue oxygen tension remained constant after hemodilution and increased after transfusion of either washed or unwashed cryopreserved RBC. Although the free plasma hemoglobin concentration remained constant after the transfusion of liquid-stored RBC (26 +/- 8 mg/dL), the plasma hemoglobin concentration increased twofold after the transfusion of cryopreserved washed RBC (60 +/- 12 mg/dL) and threefold after transfusion of cryopreserved unwashed RBC (98 +/- 20 mg/dL). The authors conclude that transfusion of one unit of RBC after cryopreservation with HES is safe and well tolerated by patients. Intravascular volume replacement and skeletal muscle oxygenation characteristics by erythrocytes did not differ between liquid-stored and cryopreserved RBC. IMPLICATIONS: This study examined whether a colloid should be used to store blood. Our data suggest that the transfusion of one unit of red blood cells after cryopreservation with hydroxyethyl starch is safe and well tolerated by patients. The effects of intravascular volume replacement and skeletal muscle oxygenation provided by red blood cells after liquid storage or cryopreservation were not different.

Adult↗
PubMed1999

Modified cryopreservation and xenotransplantation of human parathyroid tissue.

INTRODUCTION: A modified cryopreservation technique for human parathyroid tissue was compared with the standard method using a programmed freezer. METHODS: Total parathyroidectomy was performed in three groups of 6-week-old Rowett nude rats. Group I (control) underwent no transplantation of parathyroid tissue (n=9). After 10 days, the rats of groups II (n=15) and III (n=15) underwent xenotransplantation of 20 mg cryopreserved human parathyroid tissue, which had been stored in liquid nitrogen at -196 degrees C for 1-22 months prior to xenotransplantation. The parathyroid tissue was derived from 15 parathyroidectomized patients with renal hyperparathyroidism. Two tissue samples were obtained from every patient. One sample from every patient was cryopreserved by means of the technique of programmed cryopreservation: programmed freezing of human parathyroid tissue at a controlled rate of -1 degrees C/min to -80 degrees C prior to transfer to liquid nitrogen (group II). The second sample from every patient was cryopreserved by means of a modified cryopreservation technique: immediate placement of human parathyroid tissue in a freezer at -20 degrees C and transfer to liquid nitrogen after 2 h (group III). Calcium and intact parathyroid hormone concentrations in serum were determined over a period of 60 days. Furthermore, individual differences in the calcium concentrations were assessed for each rat on the basis of the calcium levels recorded preoperatively and at day 60. RESULTS: All animals in the control group developed hypocalcemia. Serum calcium concentrations returned to normal levels 60 days after xenotransplantation of parathyroid tissue, which had been cryopreserved using the modified technique (group III) in 12 of 15 rats (80%). At day 60, serum calcium had normalized in 10 of 15 rats (67%) after xenotransplantation of parathyroid tissue cryopreserved using programmed freezing (group II). After modified cryopreservation (group III), the median individual difference in the calcium concentrations was -0.16 mmol/l after programmed freezing (group II). At the end of the study, a median level of human intact parathyroid hormone of 3.5 pg/ml and 2.4 pg/ml was estimated for groups II and III, respectively. There was no statistical significant difference of the individual differences in the calcium concentrations or of the levels of human intact parathyroid hormone between groups II and III. CONCLUSION: Human parathyroid tissue was successfully xenografted in the present experimental study. The results obtained after cryopreservation using the described modified technique were equivalent to those recorded after controlled freezing in a programmed freezer. The simplified cryopreservation technique therefore appears to be suitable for human parathyroid tissue.

Animals↗
PubMed1995

Histologic modification by cryopreservation in rat aortic allografts.

Histologic changes after the cryopreserved rat aortic transplantation were studied, and the influences of the cryopreservation and of the allografting on the histology were examined. Four groups of Brown Norway (RT1n) and Lewis rats (RT1(1)) were used (n = 4 at each examined period in each group): the cryopreservation-allograft group (from Brown Norway to Lewis with cryopreservation), the cryopreservation-isograft group (from Lewis to Lewis with cryopreservation), the fresh allograft group (from Brown Norway to Lewis without cryopreservation), and the fresh isograft group (from Lewis to Lewis without cryopreservation). The graft was harvested from a descending thoracic aorta of a donor rat, implanted to an infrarenal abdominal aorta of a recipient rat, and extracted at 10 days, 1, 3, 6, and 12 months after the operation. The intimal thickening, cellular loss in the media, and cellular infiltration in the adventitia were observed, which were the same phenomena seen in chronic rejection of human organ allografts. Although the degree of intimal thickening and cellular loss in the media were higher in the cryopreserved groups than in the fresh groups, the cryopreservation procedure suppressed cellular infiltration in the adventitia after allotransplantation. The immunologic attack against the graft might be diminished by cryopreservation.

Animals↗
PubMed2006

Readily available porcine aortic valve matrices for use in tissue valve engineering. Is cryopreservation an option?

The clinical use of acellular biological valves as scaffolds in tissue valve engineering would require them to be readily available. This study examines the feasibility of cryopreserving porcine aortic valve matrices for use in tissue valve engineering. Matrices prepared using an enzymatic-detergent decellularization protocol were examined before and after cryopreservation. The biochemical status of tissues were evaluated by collagen and uronic acid (proteoglycan) determination and their mechanical properties were determined using a burst test. The histological and ultrastructural properties were evaluated by light and electron microscopy. Cryopreservation did not significantly affect the collagen and uronic acid content of aortic leaflet matrices. Histological and ultrastructural sections, however, confirmed extensive disruption of the extracellular collagen matrix and inter-fibrillar proteoglycan associations following cryopreservation. Although neither the breakage force nor the maximum force at failure was significantly different in matrices before and after cryopreservation, the strain observed in matrices was significantly higher after cryopreservation. To our knowledge this is the first study to investigate the effects of cryopreservation on aortic valve matrices. Cryopreservation did not significantly alter the biochemical properties of porcine aortic valve matrices. Nevertheless, cryopreservation had significant adverse effects on the structural and mechanical properties of matrices. Cryopreserved matrices showed significantly higher strain when stressed compared to non cryopreserved matrices. While, theoretically matrices are only expected to be functional for a limited time until regenerated in vivo, further mechanical testing is necessary to evaluate the effects of these changes on the durability of porcine aortic valve matrices for use in tissue valve engineering.

Animals↗
PubMed2004

Relationship between immunological rejection and matrix GLA protein in cryopreserved vascular allografts.

INTRODUCTION: Cryopreserved tissue allografts used for cardiovascular diseases become calcified as a late complication after transplantation, probably caused by immunological rejection. Recent attention has been focused on the inhibitory effect of matrix Gla protein (MGP) on ectopic vascular calcification, but the behavior of MGP in cryopreserved allografts is uncertain. In this study we examined the relationship between immunological rejection and MGP in cryopreserved rat aortic grafts after transplantation. METHODS: Cryopreserved rat aortae were isografted or allografted intraperitoneally. Fresh isografts were also tested. The grafts were retrieved 9 days after transplantation and the intragraft MGP mRNA was measured by a real-time quantitative PCR method. The effect of daily administration of FK506 on MGP mRNA levels in cryopreserved isografts and allografts after transplantation was also evaluated. RESULTS: There was no significant difference in intragraft MGP mRNA levels between fresh and cryopreserved isografts 9 days after transplantation. MGP expression levels in cryopreserved allografts were significantly lower as compared to those in cryopreserved isografts (P < .01). Daily administration of FK506 enhanced intragraft MGP mRNA (ninefold) in cryopreserved allografts (P < .01), but not in cryopreserved isografts. CONCLUSIONS: Immunological rejection is likely to inhibit MGP expression in cryopreserved vascular allografts, resulting in late-onset calcification.

Animals↗
PubMed2001

Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts.

BACKGROUND: Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models. METHODS: The tracheas were harvested from Lewis rats. The grafts were frozen to -80 degrees C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196 degrees C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 muCi/mL of Na2 35SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients. RESULTS: The average 35S incorporation in the cartilage before cryopreservation was 224 +/- 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups. CONCLUSIONS: The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.

Animals↗
PubMed2003

Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing.

Cryopreservation of peripheral blood mononuclear cells (PBMC) from animal model studies and clinical trials is utilized as a primary method for long-term storage of PBMC for future in vitro and in vivo applications. The objective of this study was to define the mechanistic pathways involved in cryopreservation-induced apoptosis of CD4+ T-cells in PBMC, and to evaluate a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved PBMC. Using cryopreserved PBMC samples isolated from naïve and Simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, frozen PBMC showed significantly increased levels of apoptosis-induced CD4+ T-cell death compared to fresh PBMC over a 5-day culture period as detected by Annexin V/PI and trypan blue staining. Mechanistic studies using a broad-spectrum caspase inhibitor z-VAD demonstrated a crucial involvement of caspases in cryopreservation-induced apoptosis of CD4+ T-cells. Furthermore, the ability of z-VAD to inhibit both mitochondrial membrane perturbation and apoptotic cell death implicated the involvement of caspase-mediated mitochondrial membrane damage in cryopreservation-induced apoptosis of CD4+ T-cells. Due to their known properties to promote T-cell survival and inhibit apoptosis, we evaluated the ability of IL-2, IL-4, and IL-7 combination cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4+ T-cells. The cytokine treatment resulted in a significant inhibition (p<0.01) of apoptosis-induced cell death and rescued CD4+ T-cell survival (p<0.01) in the cryopreserved cells. Efficient rescue of cryopreserved CD4+ T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.

Animals↗