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DNA amplification determines donor-cell fate in cryopreserved skin allografts.

Cryopreserved donor skin-cell survival was tested after allo- and isotransplantation by DNA amplification of male donor-cell genes which detects trace quantities of cells. Essentially, all cryopreserved allograft skin cells were rejected at the recipient site. Fine-haired, cryopreserved, belly skin of male BALB/c mice was transplanted onto the coarser-haired back of either female Swiss-Webster mice (allograft) or female BALB/c mice (isograft). Six weeks later, skin samples from the graft sites were tested by DNA polymerase chain reaction (PCR) amplification. Although allografts initially engrafted, more than 99.9 percent of male allograft skin cells were subsequently rejected, and gradually replaced by hairless host scar tissue. Clinically, all isografts, including hair follicles, engrafted permanently and maintained donor-cell SRY gene sequences in fine-haired graft site cells. Thus, cryopreservation maintained both the viability and antigenicity of mouse skin cells, because allografts were rejected and isografts survived. Furthermore, DNA amplification, quantified at multiple control dilutions and amplification cycles, can conclusively determine the fate of transplanted cells.

Animals↗

Comparative study of different procedures for the collection and banking of umbilical cord blood.

Different procedures for umbilical cord blood (CB) collection, separation, and cryopreservation were compared to evaluate the feasibility of large-scale CB banking for unrelated transplant. CB collection using an open system was associated with a 12.5% rate of bacterial contamination, whereas this rate fell to 3.3% using a closed collection system. When the umbilical cord was clamped within 30 s after delivery, on 378 occasions it was possible to collect 77 +/- 23 ml of CB without risk to mother or infant. Considering that engraftment can be obtained in recipients of 20 x 10(3) CFU-GM/kg, 86% and 28% of CB samples with a volume larger than 50 ml were found to contain a sufficient number of CFU-GM to engraft patients of 20 and 70 kg, respectively. Both Ficoll and gelatin purification procedures were associated with a recovery of 86-92% of CFU-GM, BFU-E, CFU-GEMM, and HPP-CFC, but the gelatin method appears to be more suitable for large-scale CB banking in vials. After cryopreservation, the recovery of clonogenic progenitors was similar for both CB samples stored as whole blood or as mononuclear cells separated using Ficoll or gelatin. In conclusion, large-scale CB banking seems feasible, and CB cell separation could allow storage of a large number of CB samples in a limited liquid nitrogen space.

Blood Banks↗

Cryopreservation of the bone marrow from patients with acute myeloid leukaemia leads to functional abnormalities.

We compared the performance of the stroma (SL) and haematopoietic progenitors before (group A) or following cryopreservation from patients in complete remission (CR) of acute myeloid leukaemia before autologous transplantation (AML) (group B), to similarly treated normal cells (groups A and B). From each group, fibroblastic (F) colony-forming units (CFU) and SL were established and their growth quantitated. Upon confluence of SL, 1 x 10(4)selected CD34+ cells from patient or control samples (of group A and B) were seeded on pre-formed SL from AML or control group A and B layers and adhesive precursors were scored for the formation of blastic (-b1) CFU (>20 cells). Lastly, CD34+ cells from the 4 progenitor sources were cultured in the presence of growth factors and scored for the production of granulocyte-macrophage (GM) CFU, erythroid burst forming units (BFU-E) and mixed colonies (CFU-MIX). In the patients, CFU-F numbers were decreased and by 5 wk SL failed to reach confluence. Group A AML CD34+ progenitors gave a mean of 119.2 (SD 80.4) CFU-bl/10(4) CD34+ cells, but following cryopreservation these numbers declined (83.2, SD 72.71; p = 0.009). Although AML SL prior to cryopreservation supported control CFU-bl adequately, contrary to the norm, this property decreased in group B stroma (mean 50.9/10(4)CD34+ cells, SD 49.33; p =(0.02). Group B AML samples gave significantly fewer CFU-MIX and BFU-E than pre-freezing cultures and than control samples at the first 2-growth factor combination but numbers improved following further addition of cytokines. We conclude that cryopreservation of the bone marrow of patients in CR of AML results in an inhibition of the proliferation of the CD34+ precursors. It also modifies the stroma, leading to reductions in the support of normal clones.

Acute Disease↗

Platelet cryopreservation using a reduced dimethyl sulfoxide concentration and second-messenger effectors as cryopreserving solution.

Cryopreservation of platelets is of great interest since it could extend to years the shelf life of therapeutic platelet concentrates (PCs) and facilitate stockpiling and inventory control in blood banking. We have compared the cryopreservation of PCs by the standard method using 6% Me(2)SO as cryoprotectant with the method of freezing employing low concentrations of Me(2)SO (2%) plus ThromboSol, a mixture of second-messenger effectors that protect platelets from cold damage. PC pools were treated either with 6% Me(2)SO or with ThromboSol and 2% Me(2)SO and then placed directly in a -80 degrees C freezer or in the vapor phase of a liquid nitrogen freezer (-120 degrees C). After storage for 1 week or for 3 months, samples were removed, thawed, and analyzed. Measurements included cell recovery, biochemical parameters, membrane glycoproteins (GPs), platelet aggregation, and binding of radiolabeled von Willebrand factor (vWF) and fibrinogen. PCs cryopreserved with ThromboSol and 2% Me(2)SO displayed a platelet recovery (90%) equivalent to those frozen with 6% Me(2)SO. Following either cryopreservation procedure, platelets showed increased surface expression of P-selectin and moderate loss of GP Ibalpha in comparison to fresh platelets. The aggregatory response to ristocetin and the binding of vWF were similar in platelets frozen by either procedure. Finally, both methods promoted comparable impairment of the reactivity of platelets to thrombin, aggregation and binding of fibrinogen and vWF, compared to that of fresh platelets. In summary, cryopreservation of PCs using reduced Me(2)SO concentration and ThromboSol yields platelets with in vitro functional characteristics equivalent to those of cells frozen with the conventional method using 6% Me(2)SO.

Blood Platelets↗

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved.

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.

Acrosome↗

Ex vivo fat graft preservation: effects and implications of cryopreservation.

There are a variety of recommended methods for harvesting, treating, and utilizing autologous fat grafts. Previous work with the MTT assay illustrated that various preimplantation handling techniques had minimal effect on the viability of fat samples. This assay was used to test the viability of harvested fat samples after being stored for up to 8 days in a variety of conditions. Surprisingly, freezing the fat before assaying also had no measurable detrimental effect, which led us to study this phenomenon in greater detail. The results demonstrated that fat stored at subzero temperatures showed remarkable maintenance of their mitochondrial metabolic activity as compared with fat stored in a 32 degrees C incubator. These data suggest exciting possibilities for storage and banking of human adipose tissue, which would reduce patient cost, discomfort, and time associated with multiple grafting procedures.

Adipose Tissue↗

Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

Chromatography, Liquid↗

Sterols of Pneumocystis carinii hominis organisms isolated from human lungs.

The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C(28) and C(29) 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently, pneumocysterol (C(32)), which is essentially lanosterol with a C-24 ethylidene group, was detected in lipids extracted from a formalin-fixed human P. carinii-infected lung, and its structures were elucidated by gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human infections, it is important to determine whether the 24-alkylsterols of organisms found in rats are also present in organisms in humans. In the present study, sterol analyses of P. carinii hominis organisms isolated from cryopreserved human P. carinii-infected lungs and from bronchoalveolar lavage fluid were performed. Several of the same distinct sterols (e.g., fungisterol and methylcholest-7-ene-3beta-ol) previously identified in P. carinii carinii were also present in organisms isolated from human specimens. Pneumocysterol was detected in only some of the samples.

AIDS-Related Opportunistic Infections↗

Cryopreservation of tissue engineered constructs for bone.

The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

Bone Substitutes↗

A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI.

The aim of this prospective follow-up study of children born after intracytoplasmic sperm injection (ICSI) was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones in order to evaluate the safety of this new technique. The study design included karyotyping of the parents and their agreement to genetic counselling and prenatal diagnosis and it was based on a physical examination of the child at the Centre for Medical Genetics at the ages of 2 months, 1 year and at 2 years, where major and minor malformations and psychomotor evolution are recorded. Here we describe the first 57 children born from 40 ICSI pregnancies with epididymal spermatozoa (group 1), the first 50 children born from 34 ICSI pregnancies with testicular spermatozoa (group 2) and the first 58 children born from 48 pregnancies after replacement of cryopreserved ICSI embryos (group 3). Parental karyotypes were obtained from only 72/246 (29%) parents and were all normal. Prenatal karyotypes were determined for a total of 70 samples (40%): 21 in group 1, 15 in group 2 and 34 in group 3. In this last group 2 abnormal 47,XXY karyotypes (5.8%) and no structural aberrations were found. This increase in de-novo sex-chromosomal aberrations has already been described with regard to the first 877 children born after ICSI carried out at our Centre and is probably linked directly to the characteristics of the infertile men treated rather than to the ICSI procedure itself. Major malformations, defined as those causing functional impairment or requiring surgical correction, were observed in four children: two born after ICSI with epididymal spermatozoa, one after ICSI with testicular spermatozoa and one after ICSI and cryopreservation. No particular malformation was disproportionally frequent. In the follow-up examinations at 2 months (107/161 or 66.5%) and at 1 year (37/161 or 22.9%), no additional anomalies were observed. Lost for follow-up rate at 2 months was 33.5%. These observations on a limited number of children do not suggest a higher incidence of diseases linked to imprinting, nor do they suggest a higher incidence of congenital malformations. These observations are still limited in number and should be further completed by others and by collaborative efforts. In the meanwhile patients should be told about the available data before any treatment: there appears to be some risk of transmitted chromosomal aberrations, of de-novo, mainly sex-chromosomal aberrations and of transmitting fertility problems to the offspring. Patients should also be reassured that until now there seems to be no higher incidence of congenital malformations in children born after ICSI with epididymal or testicular spermatozoa or after replacement of ICSI embryos.

Adult↗

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen.

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.

Acrosome↗

Measurement of essential physical properties of vitrification solutions.

Vitrification is an "ice-free" cryopreservation method that has rapidly developed in recent years and might become the method of choice for oocyte cryopreservation. Five sources of damage should be considered when attempting to achieve successful oocyte cryopreservation by vitrification: (1) Solution effects (2) Crystallization (3) Glass fractures (4) Devitrification and recrystallization (5) Chilling injury. The probability of successful vitrification depends on three major factors: viscosity of the sample; cooling and warming rates; and sample volume. One of the problems associated with the vitrification solution is that it may contain high concentrations of cryoprotectants (CP), which can damage the cells through chemical toxicity and osmotic shock. In the present study, we examined the principal parameters associated with successful vitrification, and attempted to compose guidelines to the most important aspects of the vitrification process. The first step was the selection of a suitable and least toxic vitrification solution. We then evaluated the effects of cooling rate and volume on the probability of vitrification. Reduction of the sample volume, combined with accelerated cooling, enabled reduction of the CP concentration. However, in practice, a delicate balance must be maintained among all the factors that affect the probability of vitrification in order to prevent crystallization, devitrification, recrystallization, glass fractures and chilling injury.

Animals↗

[Fertility preservation by ovarian tissue cryopreservation].

Female fertility may be compromised after radio or chemotherapy for malignant as well as non-malignant diseases. It could be saved by cryopreservation of ovarian tissue, since it is now well established that primordial follicles can survive freezing. Many births have been obtained in rodents with thawed ovarian fragments, after grafting or in vitro folliculogenesis. Both techniques are more difficult in large animal species, and need further development to enter clinical practice. However, storing ovarian samples is already indicated, especially in younger patients who will require restoration of their fertility in a far future.

Animals↗

Direct assessment of cryopreservation of human spermatozoa using a cryomicroscope and computer-aided sperm analysis.

Use of a cryostage has enabled direct observation of human spermatozoa as they are cryopreserved and thawed. Crystallization and recrystallization events are readily observed. In combination with computer-aided semen analysis (CASA) equipment it was possible to determine the consequence of altering the cooling, freezing and thawing rates of a temperature-rate profile on sperm motility. Increasing the cooling rate to 50 degrees C/min resulted in significantly lower pre-freeze to post-thaw ratios for average path velocity (VAP, 13%), mean straight line velocity (VSL, 35%), mean linearity (LIN, 28%) and straightness (STR, 24%), while the ratio of the number of cells crossing the field of view (NCF) significantly increased (30%) compared to a standard freeze-thaw temperature rate profile. The NCF pre-freeze to post-thaw ratio was associated with the percentage of cell recovery after cryopreservation. Faster thaw rates resulted in better survival of the cells, perhaps due to the shorter time during which recrystallization occurred. The NCF ratios were significantly higher (33 and 30% for thaw rates of 50 and 100 degrees C/min respectively) than for the standard profile samples. Previous studies on cell survival have shown a link between the cooling and thaw rates. The cryostage should prove invaluable in future studies to identify the causes of cryodamage to spermatozoa. When used in combination with CASA, changes to sperm function during cryopreservation can be accurately measured.

Computers↗

Successful sperm retrieval by percutaneous epididymal and testicular sperm aspiration.

PURPOSE: We evaluated the safety and efficacy of percutaneous sperm aspiration from the epididymis or testicle as a diagnostic technique to confirm sperm production and as a therapeutic technique to harvest sperm for use in intracytoplasmic sperm injection. MATERIALS AND METHODS: We present our experience with 69 sperm aspiration procedures in men considered to have nonreconstructible obstructive azoospermia. This short outpatient procedure was performed using a butterfly needle with the patient under intravenous sedation and local anesthesia. RESULTS: Of the 32 diagnostic aspirations 20 demonstrated mature and motile sperm, 9 maturation arrest and 3 germ cell aplasia. In 35 of 37 therapeutic sperm aspirations (95%) adequate samples of sperm after processing (mean of 5.4 million) were obtained. Of 24 epididymal aspirations 13 (54%) had sufficient residual sperm for cryopreservation of 1 to 5 vials (mean 2.5) containing an average of 170,000 sperm per vial. In the 32 intracytoplasmic sperm injection cycles 221 of 392 eggs (56.3%) fertilized (2PN) and 6 resulted in ongoing pregnancies (21.4% per transfer). There have been no acute or chronic complications in this patient population. Ten men underwent a second successful aspiration procedure for intracytoplasmic sperm injection and 3 underwent a third aspiration without added difficulty. CONCLUSIONS: Percutaneous epididymal or testis sperm aspiration is a minimally invasive sperm retrieval technique and appears to be an effective alternative to microsurgical epididymal sperm aspiration, which is more invasive, costly and technically difficult.

Adult↗

Venous allografts prepared from stripped long saphenous vein. Is there a need for antibiotic sterilisation?

AIM: Can useful lengths of vein be retrieved from varicose vein stripping procedures; is it necessary to sterilise this tissue prior to use as vein allografts? METHOD: Stripped long saphenous vein was retrieved at operation. Vein samples were cultured using direct plate inoculation and enrichment culture. Further samples were immersed in two low concentration antibiotic solutions and recultured. Smooth muscle viability was assessed after antibiotic immersion and storage by cryopreservation. RESULTS: High quality vein could be retrieved by vein stripping. Vein segments grew skin commensals on enrichment culture despite negative cultures with standard media plate inoculation (Chi-squared = 53.34 1 d.f. p < 0.001). Low concentration antibiotic solutions sterilised processed vein. Smooth muscle cell viability was reduced by cryopreservation, Mann-Whitney p = 0.008 (control 98% S.E. 0.93 vs. cryopreserved 64% S.E. 6.58), but prior exposure to antibiotics did not compound this effect. CONCLUSION: Useful lengths of vein grafts can be retrieved from varicose vein stripping procedures. Venous segments are frequently contaminated by skin commensals. Enrichment culture is required to detect contamination. Low concentration antibiotics sterilise venous-tissue without affecting smooth muscle cell viability.

Anti-Bacterial Agents↗

[Cryopreservation of circulating stem cells in large quantities].

We have collected peripheral blood mononuclear cells in large quantities by leukapheresis from children with malignant disorders. The cells were fractionated on discontinuous gradients of Percoll for enrichment of hematopoietic stem cells and reduction of sample volume. They were subsequently frozen in a programmed freezer and stored in liquid nitrogen. This procedure allows a reduced DMSO volume to be infused into patients and increases storage space. The recovery rates of progenitors evaluated by freeze-thaw analysis in small aliquots were 89% for granulocyte-macrophage colony-forming units (CFU-GM) and 106% for granulocyte, erythroid, macrophage, megakaryocyte colony-forming units (CFU-GEMM). Hence, we conclude that circulating stem cells can be collected and cryopreserved in large quantities without any loss of capacity. Further studies to evaluate the relevance of performing rescue surgery with these stem cells following marrow-ablative chemotherapy are underway.

Freezing↗