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PubMed1996

Temperature variations in upright mechanical freezers.

We examined temperature deviations from the set temperature in specific locations, between-freezer temperature variability, and the effect of defrosting on temperature deviation in a group of 15 upright mechanical freezers, part of a biological sample bank of a large prospective cohort study. By using an Omega Type T Thermocouple Microcomputer thermometer with the freezers set at -80 degrees C, the internal temperature (12 locations in each freezer) ranged from -90 degrees C to -43.5 degrees C. Overall, internal temperatures tended to be appreciably warmer in the upper and front sections of the freezers. Upright front-loading mechanical freezers, which are widely used in research laboratories throughout the world, may not be optimally suited to preserve human biological samples for long-term banking in epidemiology.

Cohort Studies↗
PubMed1998

The accuracy of laboratory measurements in clinical chemistry: a study of 11 routine chemistry analytes in the College of American Pathologists Chemistry Survey with fresh frozen serum, definitive methods, and reference methods.

CONTEXT: Better procedures are needed whereby national proficiency testing survey providers can assess and improve the accuracy of laboratory measurements in clinical chemistry. SETTING: The 1994 College of American Pathologists Comprehensive Chemistry Survey. DESIGN: This study of matrix effects and the accuracy of laboratory measurements for 11 analytes linked the logistics of the Survey to definitive methods at the National Institutes of Standards and Technology, reference methods at the Centers for Disease Control and Prevention, proficiency testing materials, and a fresh frozen serum sample. The data were analyzed with a statistical model of laboratory measurements. RESULTS: (1) Matrix biases affected the results reported from 69% of the 644 peer group/survey specimen pairs evaluated. (2) Because of matrix biases, the reference value was the correct target value only 32% of the time; thus, the traceability established by definitive method and reference method value assignments on Chemistry Survey specimens did not assure accuracy on patient samples. (3) In contrast to matrix biases, the error caused by random matrix effects with proficiency testing samples was about the same as that caused by random specimen effects with fresh frozen serum, and both were less than within-run random analytic error. (4) Calibration biases occurred in 73% of the 180 peer groups evaluated, and, after matrix biases were removed, the total variance of interlaboratory measurements was due to peer group calibration bias (48%), within-peer-group random calibration error (31 %), within-run random error (14%), and random specimen effects (7%). CONCLUSIONS: An opportunity exists to improve method calibration accuracy in clinical chemistry. With improved design, national proficiency testing surveys can monitor and help reduce method calibration error by converting reported survey results to a true accuracy base that predicts accuracy on patient samples. For medical purposes, the correct target values on artificial (matrix-modified) chemistry materials are reference values adjusted for the matrix bias of each peer group. Matrix biases estimated by the use of fresh frozen serum can be used as factors to transfer the accuracy of definitive methods from artificial reference materials to patient samples.

Bias↗
PubMed1988

Cryopreservation of sperm from the marine shrimp Sicyonia ingentis.

Sperm from a marine shrimp, Sicyonia ingentis, were frozen to -196 degrees C using a variety of cooling rates and cryoprotectants. A cooling rate of 1 degree C/min resulted in minimal cell breakage. Sperm samples were frozen in solutions of known membrane stabilizers--trehalose, sucrose, proline, and glycerol. These compounds were somewhat effective but a dramatic increase in sperm viability was seen when DMSO was present in the freezing medium. Sperm viability was assessed using the in vitro acrosome reaction technique of Griffin et al. (1987). The highest sperm survival (56%) was obtained with samples frozen at 1 degrees C/min in a 5% (v/v) DMSO solution. No decrease in viability was seen in sperm samples stored in liquid nitrogen (-196 degrees C) for 1 month.

Animals↗
PubMed1987

Cryopreservation of Dictyocaulus viviparus third-stage larvae and Trichinella spiralis muscle larvae.

In cryopreservation studies with third-stage larvae of Dictyocaulus viviparus, best results were achieved by incubating larvae in 0.05% NaOCl at 37 degrees C to remove the sheath, followed by cooling at a rate of 1 degree C min per min down to about 0 degree C. After an equilibration time of 10 min at +4 degrees C with or without 4% polyethylene glycol-400 as cryoprotectant, samples were frozen at the same cooling rate to an intermediate temperature of -20 degrees C, maintained at this temperature for 10 min and finally plunged into liquid nitrogen for storage. Three groups of 3 calves were infected with the following batches of third-stage larvae: (a) fresh, sheated; (b) fresh, exsheathed; (c) exsheathed, cryopreserved for 13 weeks in liquid nitrogen and subsequently thawed. Although 62% of group (c) were regarded as viable in vitro, their infectivity to calves was low and only an average of 0.08% of the inoculated larvae (3000 per animal) developed into adult lungworms (= infectivity rate). Average infectivity rates of fresh, sheathed (a) and fresh, exsheathed (b) larvae were much higher (38.3% and 29.7%) and not significantly different from each other. Two of the calves inoculated with previously frozen larvae and all of the calves infected with fresh larvae excreted first-stage larvae in their faeces, but the latter groups in higher quantities. The results show that cryopreservation of exsheathed third-stage larvae of D. viviparus is possible, but for strain maintenance infection doses greater than 3000 larvae should be used for inoculation of calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Cryopreservation of Echinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones).

Four isolates of larval Echinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2-4 degrees C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (-196 degrees C). After storage for one week the samples were rapidly thawed at +37 degrees C for approximately 3 min, washed in fresh EMEM/A (37 degrees C) and transferred into the peritoneal cavity of Meriones for viability testing. As judged by histological examinations and metacestode weights of each 24 Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at -28 degrees C, 30 min at -80 degrees C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7 degrees C min-1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65 degrees C min-1 for step 3. These results demonstrate that metacestodes of E. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.

Animals↗
PubMed2006

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos.

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.

Animals↗
PubMed2005

Comparison between glycerol and ethylene glycol for dog semen cryopreservation.

The aim of this study was to compare the effect of ethylene glycol versus glycerol for dog semen freezing, on post-thaw longevity, motility and motility parameters, and on plasma membrane functional integrity. Semen was diluted in two steps with an egg yolk TRIS extender containing a final concentration of either 5% glycerol or 5% ethylene glycol, and frozen in 0.5 ml straws, with 100 x 10(6) spermatozoa/ml, over nitrogen vapours. Semen motility was evaluated both under a light microscope and with a Computer Assisted Motility Analyser System, immediately after thawing and then hourly till 4h of incubation. Sperm membrane functional integrity was assessed with the hypoosmotic swelling test (60 mOsm fructose solution) applied at thawing and then hourly, for 4 h, on incubated samples. Motility (light microscope) and total and progressive motility (analyser) were significantly higher in ethylene glycol frozen samples at thawing (P < 0.01); from hour 1 onwards the effect of the cryoprotectant became not significant. Semen frozen with ethylene glycol showed higher path velocity and higher straight line velocity till 3 h after thawing; however, ethylene glycol semen samples also showed higher curvilinear velocity and higher lateral head displacement, which may indicate a capacitation-like condition affecting sperm membranes and possibly reducing post-thaw longevity. Functional integrity of plasma membrane was similar in glycerol and ethylene glycol samples till 3 h after thawing, then ethylene glycol samples showed a higher decline. The strong though short-lived positive effect of ethylene glycol is worth being evaluated further.

Animals↗
PubMed2001

Seminal plasma addition attenuates the dilution effect in bovine sperm.

Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 x 10(6) sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 x 10(6) sperm/mL in EYC extender with (+SP) and without (-SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 x 10(6) sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 x 10(6)/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the -SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the -SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR- 14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the -SP samples at a concentration of 10 x 10(6) sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.

Animals↗
PubMed1995

Relationship among seminal characteristics, fertility and suitability for semen preservation in draft stallions.

Seminal characteristics, fertility and the response to semen preservation (liquid storage and cryopreservation) were evaluated in 4 Draft stallions (Percheron 2, Breton 2). Seminal characteristics (gel-free volume, sperm concentration, sperm morphology, percentage of motile spermatozoa) were assessed in 5 ejaculates from each of the 4 stallions. The fertility of the stallions was calculated retrospectively as the accumulated pregnancy rate over 3 breeding seasons. Five ejaculates from each of the stallions were subjected to liquid storage at 5 degrees C. The percentage of motile spermatozoa (PMS) was assessed at 0, 24, 48 and 72 hr. Cryopreservation was done with a glucose-EDTA-lactose egg yolk extender and a skim milk-based extender containing clarified egg yolk (4 replicates per stallion). PMS and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated before and after freezing. Although semen characteristics and fertility were within a physiological range, significant differences among stallions were found for gel-free volume, percentage of morphologically normal spermatozoa, PMS, and fertility but there was no clear relationship between these parameters. The response to liquid storage of diluted semen samples differed among stallions and best results were obtained in a stallion with the highest initial motility. The freezability of semen from the 4 stallions was poor with both of the freezing protocols (PMS range 13.8%-26.3%; IPM range 19.5%-38.0%). Results of liquid storage and cryopreservation were not correlated in individual stallions and were not related to their fertility. Since predictability for the response to semen preservation is low, the suitability of individual stallions for semen preservation has to be examined carefully.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Experiments on cryopreservation of Plasmodium falciparum.

A comparative study was carried out in order to assess the effectivity of the deep-freezing conditions for P. falciparum cultures: 1. by plunging them into liquid nitrogen, 2. by use of an established freezing program for lymphocytes. After thawing in a waterbath at 37 degrees C, samples were diluted with equal volumes of 3.5% sodium chloride solution or 15% phosphate-buffered saline supplemented glucose solution. Other samples were not diluted after thawing, but the medium was replaced twice during the first day in culture. Survival was evaluated by counting the parasites in Giemsa-stained smears taken on day 3 after thawing. We obtained the best survival rate using NaCl solution, whereas replacing the medium twice resulted in the lowest level of survival. In synchronized cultures we studied survival rates of the different developmental stages of the intraerythrocytic parasites: young trophozoites (ring stages) and schizonts. The results indicate that irrespective of the freezing technique used, the survival level of young trophozoites was high (40 and 60%). The more sensitive stages (late trophozoites and schizonts) were better protected by using the freezing program designed for lymphocytes.

Animals↗
PubMedDate not supplied

In vitro sperm-binding assay to distinguish differences in populations of human sperm or damage to sperm resulting from cryopreservation.

Annually, >1.3 million men are members of couples seeking help because of infertility. Semen from many of these men contains reasonable numbers of motile and normal sperm, but for a subset of individuals, many sperm are deficient in ability to bind to the zona pellucida during in vitro fertilization. Diagnosis of this defect has been hampered by lack of a low-cost test. Molecular similarity exists between the perivitelline membrane of a hen's egg and the mammalian zona pellucida. These facts and some preliminary data led to evaluation of binding of human sperm during incubation for 60 minutes at 37 degrees C to an extract of chicken perivitelline membrane coated in microwell assay plates. The sperm-binding assay had inter- and intraassay plate variations of 21 and 12%, respectively, using washed fresh sperm. All seminal samples were normal, except a few that had 36 to 50% motile sperm with a low rate of sperm movement (if there is a low rate of movement, World Health Organization [WHO] criterion for normalcy is >50% motile). Nevertheless, this sperm-binding assay detected differences among individuals in percentage of sperm bound. Based on data for two to four ejaculates from each of eight occasional sperm donors, the coefficient of variation for ejaculates within donor averaged 31%, and means for the donors differed (P < 0.02). Percentage of sperm bound ranged from <1 to 38% for fresh semen from 57 men and from <1 to 13% for frozen-thawed semen from 34 men. Percentage of motile sperm accounted for <30% of the variation in percentage of sperm bound. In a direct comparison based on 17 ejaculates, aliquots evaluated fresh averaged 13% sperm bound, versus 2% for frozen-thawed aliquots. We concluded that the egg membrane substrate used in these microwell assay plates might serve as the basis for a diagnostic assay. However, it remains to be established whether samples of human semen with a low percentage of sperm binding indeed have relatively low fertilizing potential.

Cell Adhesion↗
PubMed1996

Technical report: effect of cryopreservation on chromosomal aberration yield in irradiated lymphocytes.

Cryopreservation is the usual method to store cells before analysis or use, for instance for biological dosimetry purposes. Some investigations have shown that thawing following freezing may induce cell injury but few studies have been made of the effect of cryopreservation on cells containing radiation-induced unstable chromosomal aberrations. In this work, lymphocytes were irradiated with 1 to 4 Gy gamma rays and stored in liquid nitrogen. The dicentric and centric ring yields were analysed after storage periods of 1 week, 1 month and 3 months. No difference in aberration frequency from control, unfrozen samples was observed over this period. Lymphocytes stored at -196 degrees C for up to at least 3 months may therefore be used for chromosome aberration scoring when over-exposure to ionizing radiation is suspected.

Chromosome Aberrations↗
PubMedDate not supplied

Kinematic changes during the cryopreservation of boar spermatozoa.

The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P > .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P > .05), sP2 and sP3 varied significantly (P < .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.

Animals↗
PubMed1989

A cytogenetic investigation of the effects of cryopreservation on human sperm.

The effects of cryopreservation on the frequency and type of chromosome abnormalities in human sperm have been investigated for the first time. With a technique which enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 13 normal men were examined before and after being frozen in liquid nitrogen. The overall abnormality frequencies of 17.8% for fresh semen and 13.4% for previously frozen semen were not significantly different (chi 2(1) df = 3.04, p = 0.08). When specific abnormality types were analyzed, only the category of hypohaploidy was significantly different (chi 2(1) df = 6.75, p = 0.009) before (7.5%) and after (3.4%) freezing. Hypohaploidy was significantly higher than hyperhaploidy both prefreeze and postfreeze, and chromosome loss was random. Because the observed excess of hypohaploid cells may be attributable to technical artifact, the aneuploidy levels were estimated by doubling the number of hyperhaploid cells. Neither the adjusted numerical abnormality frequencies (1% prefreeze vs. 0% postfreeze) nor the overall abnormality frequencies (11.8% prefreeze vs. 10.4% postfreeze) were significantly different. The types and distributions of karyotypically abnormal sperm complements (numerical, structural, or combined) observed before and after freezing were not different. Interdonor variability in sperm chromosome abnormality frequencies and a possible donor-dependent response to cryopreservation were suggested by the data. The sex ratios were not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the type or frequencies of chromosome abnormalities or alter the sex ratio in human sperm.

Aneuploidy↗
PubMed1994

Elevated frequencies of hprt mutant lymphocytes in cigarette-smoking mothers and their newborns.

Maternal cigarette smoking during pregnancy has been associated with increased perinatal mortality and low birth weight. Several epidemiological studies have demonstrated an association between smoking during pregnancy and an elevated risk of hematopoietic cancer in the child, but other studies have failed to confirm this association. We have used an assay for somatic cell mutation to evaluate the in utero effects of exposure to maternal cigarette smoking. Cord blood samples were obtained from 10 newborns whose mothers smoked cigarettes during pregnancy and 10 newborns of non-smoking mothers. Blood samples were also obtained from 5 of the smoking and 5 of the non-smoking mothers. Smoking status was confirmed in all samples by testing the blood plasma for cotinine. The frequency of lymphocytes containing mutations at the hypoxanthine phosphoribosyltransferase (hprt) locus was determined with an autoradiographic assay using cells that had been cryopreserved. The mothers who were smokers had a mean frequency (+/- SE) of 3.08 (+/- 0.55) variant (mutant) cells per 10(6) evaluatable lymphocytes. The frequency (Vf) in non-smokers was 1.07 (+/- 0.17) x 10(-6). The Vf of newborns of smokers was 2.17 (+/- 0.24) x 10(-6), and newborns of non-smokers had a Vf of 0.77 (+/- 0.13) x 10(-6). In both mothers and newborns the difference in Vf between smokers and non-smokers was statistically significant (p < 0.05). Maternal and newborn Vfs were significantly correlated (r = 0.88; p < 0.004), and there was a positive association (r = 0.86; p < 0.001) between the reported number of cigarettes smoked per day and the Vfs. This study provides further evidence that maternal smoking may be hazardous to the future health of children exposed in utero to mutagenic agents in cigarette smoke.

Adult↗
PubMed2004

Anatomical methods for voxelation of the mammalian brain.

Voxelation allows high-throughput acquisition of three-dimensional gene expression patterns in the brain through analysis of spatially registered voxels (cubes). The method results in multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging techniques. An important issue for voxelation is the development of approaches to anchor correctly harvested voxels to the underlying anatomy. Here, we describe experiments to identify fixation and cryopreservation protocols for improved registration of harvested voxels with neuroanatomical structures. Paraformaldehyde fixation greatly reduced RNA recovery as judged by ribosomal RNA abundance. However, gene expression signals from paraformaldehyde-fixed samples were not appreciably diminished as judged by average signal-noise ratios from microarrays, highlighting the difficulties of accurate quantitation of cross-linked RNA. Additional use of cryoprotection helped to improve further RNA recovery and signal from fixed tissue. It appears that the best protocol to provide the necessary resolution of neuroanatomical information in voxelation entails a controlled dose of fixation and thorough cryoprotection, complemented by histological staining.

Animals↗
PubMed1988

Homograft valve preparation and predicting viability at implantation.

An experimental study was performed using C14 proline uptake in order to: (1) assess the effects of current sterilization and storage methods on fibroblast viability, and (2) establish a control tissue that could be used to determine viability of each homograft valve at the time of implantation in the clinical setting. The results were expressed as disintegrations per minute per milligram of tissue (DPM/mg). Swine aortic (AV), pulmonary (PV), and tricuspid leaflets (TV), and adjacent AV and PV arterial wall were procured sterile and subjected to routine sterilization and storage. Thirty samples of AV were analyzed for incorporation of labeled proline at procurement (208 +/- 7 DPM/mg), following 48-hour antibiotic exposure (87 +/- 6 DPM/mg, P less than .0001), and following controlled rate cryopreservation and storage for 12 days at -80 degrees C (78 +/- 8 DPM/mg, P = .42). Proline uptake of the other tissues at the same intervals disclosed that only the TV resulted in the same degree of viability at implantation (AV 78 +/- 8, PV 68 +/- 3, TV 75 +/- 2, P = NS). The homograft valves were obtained under sterile conditions from brain dead, multiorgan donors (homovital). It has been postulated that these valves are sterile and ready for implantation. Of 17 homovital valves cultured at procurement, 9 had positive cultures within 48 hours (53%). We conclude that: (1) the TV can be processed as a control tissue with each homograft and then utilized to predict viability at the time of implantation in the clinical setting; (2) antibiotic exposure is an essential step in the preparation of all homografts, however, modification of the antibiotic solution is necessary.

Carbon Radioisotopes↗
PubMed1999

Production of ex vivo expanded hematopoietic cells and progenitors in a closed bioreactor, starting with a small volume marrow collection: A feasibility study in patients with poor-risk breast cancer and receiving high-doses of cyclophosphamide.

We report a clinical pilot study conducted in 6 women with poor-prognosis breast cancer. The goal was to evaluate the feasibility and safety of producing hematopoietic progenitors and cells from a small marrow sample, for clinical use after high-dose cyclophosphamide. A small volume marrow collection was obtained, using local anesthesia and conscious sedation, before the first of two chemotherapy cycles. Cells were cryopreserved, and later thawed to inoculate two Aastrom Biosciences Inc Replicell bioreactors, on time to reinfuse ex vivo expanded cells after the second chemotherapy cycle. Patients recovered neutrophils and platelets at similar times after the first and second chemotherapy cycles, and showed comparable clinical events. This pilot study prepares future randomized trials, designed to evaluate clinical benefits associated with the use of ex vivo expanded cells in the setting of multicycle high-dose chemotherapy.

Aged↗