Single-site, five-year experience with human eosinophil isolation by density gradient centrifugation and CD16 immunomagnetic negative separation.

OBJECTIVE: Little has been reported regarding the reliability of methods for the purification of human blood eosinophils. We retrospectively reviewed our experience with 350 consecutive eosinophil isolations. RESULTS: Between January 2014 and December 2018, we conducted 350 eosinophil purifications from 83 donors. Absolute eosinophil count (AEC), calculated from hospital complete blood counts when available (n = 289), ranged from 32 to 1352 eosinophils/µL ([Formula: see text]: 179 ± 136/µL). Eosinophil yields ranged from 0.4 to 24.4 million cells per 20 mL of blood drawn ([Formula: see text]: 3.1 ± 1.9 million eosinophils) with > 98% purity. Comparing AEC to actual yield, recovery was 87% ± 29% ([Formula: see text]) and AEC strongly correlated with yield. To explore the reproducibility of yield, a subsequent analysis was limited to those donors drawn ≥ 3 times (N = 35), and there was no difference in the average coefficient of variation for yield between allergic and non-allergic donors. Viability of isolated eosinophils was consistently > 95% and after 24 h of culture did not differ between allergic and non-allergic donors. We conclude that this immunomagnetic separation method for human eosinophil isolation from whole blood is a reliable, reproducible technique for obtaining an average of 87% yield with high purity and viability.

Comparison of microfluid sperm sorting chip and density gradient methods for use in intrauterine insemination cycles.

OBJECTIVE: To compare the effect of microfluiding sperm sorting chip and density gradient methods on ongoing pregnancy rates (PRs) of patients undergoing IUI. DESIGN: Retrospective cohort study. SETTING: Hospital IVF unit. PATIENT(S): Couples with infertility undergoing IUI cycles between 2017 and 2018. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Ongoing PRs. RESULT(S): A total of 265 patients were included in the study. Microfluid sperm sorting and density gradient were used to prepare sperm in 133 and 132 patients, respectively. Baseline spermiogram parameters, including volume, concentration, motility, and morphology, were similar between the two groups. Total motile sperm count was lower in the microfluiding sperm sorting group at baseline (35.96 ± 37.69 vs. 70.66 ± 61.65). After sperm preparation sperm motility was higher in the microfluid group (96.34 ± 7.29 vs. 84.42 ± 10.87). Pregnancy rates were 18.04% in the microfluid group and 15.15% in the density gradient group, and ongoing PRs were 15.03% and 9.09%, respectively. After using multivariable logistic regression and controling for confounding factors, there was a significant increase in ongoing PRs in the microfluid sperm sorting group. The adjusted odds ratio for ongoing pregnancy in the microfluid group compared with the density gradient group was 3.49 (95% confidence interval 1.12-10.89). CONCLUSION(S): The microfluid sperm sorting method significantly increased the ongoing PRs compared with the density gradient group in IUI cycles.

Standardized large-scale H-1PV production process with efficient quality and quantity monitoring.

The promising anticancer properties of rodent protoparvoviruses, notably H-1PV, have led to their clinical testing. This makes it necessary to produce highly pure, well-characterized virus batches in sufficient quantity. The present work focused on developing standardized production, purification, and characterization procedures as a basis for exploiting H-1PV both preclinically and in clinical trials for anticancer virotherapy. Two infection and two virus purification strategies were tested and the resulting virus preparations compared for their purity and full-, infectious-, and empty-particle contents. The adopted production process, which involves culturing and infecting NB-324K cells in 10-layer CellSTACK(®) chambers (1×10(3) infectious units per infected cell), is simple, scalable, and reproducible. Downstream processing to eliminate contaminating DNA and protein includes DNAse treatment, filtration, and two Iodixanol density-gradient centrifugations, the first gradient being a step gradient and the second, either a step (1×10(10) PFU/ml) or a continuous gradient (3×10(11) PFU/ml). A procedure was also developed for obtaining infectious particle-free preparations of empty virions for research purposes: cesium chloride density gradient centrifugation followed by UV irradiation (1×10(14) physical particles/ml). For quick, sensitive determination of physical particles (and hence, particle-to-infectivity ratios), a "Capsid-ELISA" was developed, based on a novel monoclonal antibody that specifically targets assembled capsids.

Isolation and quality control of functional mitochondria.

Numerous protocols are available being adapted for different cell or tissue types allowing isolation of pure mitochondria trying to preserve their "structural and functional" integrity. In this chapter we intend to provide a more general framework introducing differential isopycnic density gradient centrifugation strategy with a special focus sensitizing for the specific challenges coming along with this method and how to obtain "functional," enriched, "intact" mitochondria. Due to the fact that in any study dealing with these organelles standardized processing is mandatory. Here we describe a strategy addressing quality control of prepared intact mitochondria. The quality control should be an integrated part of all isolation processes. The underlying protocol should be seen as starting point and has to be carefully adjusted to cover different sample types used for the diverse research questions.

Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion sperm.

The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0µM, 0.1µM, 0.05µM or 0.01µM, or caffeine at 200µg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino-2-phenylindole (DAPI) and examining for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. In a second experiment, aliquots of frozen-thawed stallion sperm were subjected to DL-DGC selection - or not - and then further subjected to the two best capacitation treatments (0.1µM and 0.05µM ionomycin). The fertilising capacity of the semen was then determined as above. The DL-DGC/capacitated sperm samples showed the highest mean penetration rates: 24.16% following capacitation with 0.1µM ionomycin, and 12.21% following capacitation with 0.05µM ionomycin. The capacitated but non-DL-DGC-selected sperm returned significantly lower values: 6.26% and 7.02% for the same ionomycin treatments respectively. These findings suggest that combining DL-DGC selection with ionomycin capacitation improves the fertilising capacity of frozen-thawed stallion sperm.

The effect of repeated freezing and thawing on human sperm DNA fragmentation.

OBJECTIVE: To investigate the effects of repeated freezing and thawing on human sperm motility, vitality, and DNA integrity. DESIGN: A prospective clinical study. SETTING: Tertiary care fertility clinic. PATIENT(S): Twenty men presenting for infertility investigations. INTERVENTION(S): Each sample was subjected to three cycles of freezing and thawing both with and without washing steps and the addition of fresh cryoprotectant between each cycle. MAIN OUTCOME MEASURE(S): Percentage sperm DNA fragmentation, motility, and vitality before and following repeated freezing and thawing. RESULT(S): The percentage sperm DNA fragmentation rose significantly following each freeze-thaw cycle; however, samples that were not washed and to which fresh cryoprotectant was not added after each cycle fared significantly better than their washed counterparts. Both motility and vitality decreased steadily following each cycle but cell survival was significantly greater in the unwashed samples. CONCLUSION(S): In terms of the level of sperm DNA fragmentation, up to three cycles of freezing and thawing can be performed with a level of risk comparable to that following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original cryoprotectant and not washed or altered in any way in between, and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology.

Improved, low-cost methods for pancreatic islet purification in rats.

OBJECTIVE: Density gradient separation of islets from exocrine tissue is usually performed using Ficoll. However, this reagent significantly increases the cost of isolation. The aim of the present study was to investigate the effects on islet preparations of purification methods using Lymphoprep and Iodixanol (OptiPrep) density gradients. METHODS: Pancreata were procured from 46 Wistar rats, loaded with collagenase V (Sigma), and mechanically dissociated using standard procedures. After the digestion phase, the islets purified by 3 methods-Ficoll, Lymphoprep, and Iodixanol (OptiPrep)-were assessed for yields, purity, morphology, and in vitro function. RESULT: We expressed the yields as islet equivalents (IEQ, diameter standardizing to 150 microm), showing no significant differences. Compared with the Ficoll group, the purity was significantly higher in the Lymphoprep (P = .005) and Iodixanol (OptiPrep) groups (P = .011). While the viability was >90% in all 3 groups, the viability in the Lymphoprep Group and OptiPrep groups was significantly higher than that of the Ficoll group (P < .001). In vitro islet function did not differ among the 3 experimental groups. CONCLUSION: Lymphoprep and Iodixanol were as effective as Ficoll in terms of islet yield and in vitro function. High-purity and high-viability islet cells were obtained using improved Lymphoprep-based or Iodixanol (OptiPrep)-based density gradient methods, potential low-cost substitutes for Ficoll.

[A novel model for isolation of Walker's tumoral cells using the Ficoll-Hypaque gradient]. / Um novo modelo de isolamento do tumor de Walker utilizando o gradiente de Ficoll-Hypaque.

PURPOSE: The purpose was to evaluate a novel technique for isolation of Walker's tumoral cells using a Ficoll-Hypaque gradient and its further influence on tumor development. METHODS: Twenty male Wistar rats have been divided in 2 groups: G1= without ficoll, G2= with ficoll. Tumor was excised, homogenized and suspended in lactate ringer. A sample of the cell suspension was adjusted at a concentration of 1x10(6) cells/ml (G1). A second sample was centrifuged on a Ficoll-Hypaque gradient and the cell concentration was then adjusted (G2). Tumor was implanted by subcutaneous injection of 1.0 ml in the right armpit of rats. Tumor volume (TV) and tumor weight (TW) were compared in two groups. RESULTS: There were no differences between the two groups in TV (G1=17.9+/-3.8 cm3 vs. G2=17.2+/-4.4 cm3; p=0.190) and TW (G1=7.0+/-1.8 g vs. G2=7.3+/-2.8 g; p=0.569). The histological analysis showed similar patterns of infiltration by small-undifferentiated cells and necrosis in both groups. However, a mild to moderate granulocytic exudate was more frequent in the animals whose tumors derived from Ficoll-isolated cells. Hemorrhage from slight to moderate was only observed in this group. CONCLUSION: A Ficoll-Hypaque gradient can provide more adequate isolation of Walker's tumor and the cell suspension obtained by this technique has lower contamination by other cell types.

Um novo modelo de isolamento do tumor de Walker utilizando o gradiente de Ficoll-Hypaque / A novel model for isolation of Walker's tumoral cells using the Ficoll-Hypaque gradient

OBJETIVO: Analisar uma nova técnica de isolamento das células do tumor de Walker, utilizando o gradiente de ficoll-hypaque. MÉTODOS: Vinte ratos Wistar machos foram divididos em 2 grupos (G1, G2). O tumor do animal doador foi dissecado, triturado em solução de ringer lactato e filtrado. A suspensão celular obtida foi dividida em duas alíquotas (A1 e A2) e a alíquota A2 foi centrifugada em gradiente de ficoll-hypaque. As duas alíquotas foram ajustadas para uma concentração de 1X10(6) células/ml. Um ml da suspensão A1 ou A2 foi injetado por via subcutânea na axila direita dos ratos dos seus respectivos grupos. Os diâmetros tumorais, peso tumoral e análise histológica foram avaliados. RESULTADOS: Não houve diferença no volume tumoral (G1=17,9±3,8cm³; G2=17,2±4,4cm³; p=0,190) e no peso tumoral (G1=7,0±1,8g; G2=7,3±2,8g; p=0,569). A análise histológica evidenciou padrões semelhantes de infiltração de células pequenas indiferenciadas e de necrose nos dois grupos. Entretanto, exsudato granulocítico leve a moderado foi mais acentuado no G2 do que no grupo G1 e hemorragia leve a moderada foi observada somente no grupo com ficoll. CONCLUSÃO: O gradiente de ficoll-hypaque é uma técnica adequada de isolamento das células do tumor de Walker e fornece uma suspensão celular menos contaminada com outros tipos celulares.

Comparison of six density gradient media for selection of cryopreserved donor spermatozoa.

The aim of our study was to evaluate the efficiency of 4 density gradient media for motile cryopreserved spermatozoa selection to Percoll (Kabi Pharmacia, Uppsala, Sweden) and to Puresperm (J.C.D. International Laboratory, L'Aigle, France). Puresperm was the new medium chosen in our laboratory in 1996 as the substitute for Percoll. The solutions tested were 3 colloidal silane-coated silica particle media (Isolate, SpermGrad-100, Sil-Select Plus) and iodixanol (Optiprep). Semen parameters analyzed after selection were concentration, motility, and morphology. Semen parameters after Puresperm gradient had similar values compared to Percoll. Optiprep was less efficient with a poor concentration. Isolate had a comparatively better concentration, but the capacity of selection was not satisfactory. SpermGrad-100 and Sil-Select Plus were less effective than Puresperm. In conclusion, Puresperm could be considered a better alternative to Percoll for cryopreserved spermatozoa migration.

A clinical study comparing PureSperm and SpermFilter for density gradient separation of human spermatozoa in assisted reproduction.

OBJECTIVE: To compare a new density gradient medium, SpermFilter, for purifying spermatozoa in assisted reproduction with the more established medium, PureSperm. DESIGN: Part 1, a multicenter study on 225 semen samples purified using either PureSperm (115 semen samples) or SpermFilter (110 semen samples). Part 2, a retrospective, single center study on a total of 898 assisted reproductive cycles (245 insemination cycles using husband semen, 58 insemination cycles using donor semen and 595 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. SETTING: Part 1, three fertility clinics in Denmark (two university-affiliated fertility clinics and one private clinic). Part 2, one university-affiliated fertility clinic in Denmark. MAIN OUTCOME PARAMETERS: Part 1, purity of purified spermatozoa (% motile), motility index and recovery of motile spermatozoa. Part 2, malformation and baby take-home rates (insemination cycles), fertilization, cleavage, implantation, malformation and baby take-home rates (IVF/ICSI cycles). RESULTS: No statistical differences were observed in any of the parameters investigated. CONCLUSION: SpermFilter is a valid alternative to PureSperm in assisted reproduction technology (ART).

A simple centrifugation method for harvesting myeloblasts.

Myeloblast-rich samples, required for investigation of myeloid malignancies, can be obtained only during the untreated stage of leukemia. Existing methods for myeloblast enrichment have various prerequisites that limit their application. In this new method, a mixture of peripheral blood (Mixed PB) from an acute myeloid leukemia (AML) patient and from a healthy control containing 5% myeloblasts was subjected to density gradient centrifugation using a 14.5% metrizamide solution. Both high purity (86.3% +/- 1.5%) and high recovery of viable myeloblasts were achieved. Close to 100-fold blast enrichment, even from Mixed PB containing only 0.15% myeloblasts, was achieved. Similarly, this method highly enriched myeloblasts from unprocessed samples, including marrow cells, from patients with AML, myelodysplastic syndromes, and chronic myeloid leukemia (purity: 2.7% +/- 2.0% before separation, 56.6% +/- 28.3% after separation) (n = 22). The enriched blasts were suitable for various analyses, eg, flow cytometry, immunocytochemistry, cytochemistry, fluorescence in situ hybridization, and gene analysis.

Separating X-bearing human spermatozoa through a discontinuous Percoll density gradient proved to be inefficient by double-label fluorescent in situ hybridization.

PURPOSE: Double-label fluorescence in situ hybridization (FISH) was used to evaluate the efficiency of separating X- and Y-chromosome-bearing spermatozoa through 12-step discontinuous Percoll gradients. METHODS: Liquefied normal semen samples from 10 healthy donors were overlaid onto 25% Percoll and centrifuged. Parts of the sperm pellet were saved as control, while the remaining portion was separated by 12-step Percoll gradient. After centrifugation, the spermatozoa in the 80% Percoll layer were collected. The X:Y ratio of the control and separated spermatozoa was verified by double-label FISH (CEP SOX/SGY probes) and scored blindly by one observer. Differences in the X:Y ratios between matched groups were analyzed by paired t tests. RESULTS: The overall average labeling efficiency was 99.2%. A significant enrichment (P = 0.02) of X-bearing spermatozoa was obtained in Percoll separated fractions (mean X:Y ratio = 52.2:46.4) compared with the control group (X:Y ratio = 49.5:48.4). Discontinuous Percoll gradients also decreased the proportion of aneuploid spermatozoa (from 1.0 to 0.8%), but the differences were nonsignificant. CONCLUSIONS: Discontinuous Percoll separation did increase the X:Y ratio significantly, but the enrichment of X-bearing spermatozoa is insufficient for clinical use in preconceptional sex selection.

Quantitative and qualitative comparative analysis of gradient-separated hematopoietic cells from cord blood and chemotherapy-mobilized peripheral blood.

Some of the uncertainty regarding the use of cord blood (CB) in transplant settings includes the suspected relative rarity of hematopoietic stem cells (SC) in CB and the feasibility of incorporating a cell separation protocol to remove red blood cells, which may result in an unacceptable loss of SC. To address this uncertainty, we isolated a SC fraction by Percoll or Ficoll gradients from CB and peripheral blood (PB), which had been mobilized by chemotherapy. The frequencies of mononuclear cells (MNC), CD34+ cells, colony-forming units granulocyte-macrophage (CFU-GM), and the output of colony-forming cells (CFC) after five weeks in long-term culture (LTC) assay were then evaluated. The mean numbers of these cells per ml of CB sample before gradient separation were, respectively, 4.9 x 10(6), 13.8 x 10(4), 3.0 x 10(3) and 681 (n = 37). In the recovery phase of PB, these numbers were, respectively, 2.0 x 10(6), 14.9 x 10(4), 3.5 x 10(3) and 270 per ml of processed blood at apheresis (n = 35). After Percoll separation, the recovery rates of these cells were, respectively, 29%, 92%, 97% and 95% in CB, and 65%, 87%, 123% and 102% in PB. After Ficoll separation of CB, the rates were, respectively, 55%, 107%, 92% and 105%. These results suggest that CB contains an adequate number of more immature progenitors which can be retained after cell separation with Percoll or Ficoll, thereby making it feasible to incorporate a cell processing procedure into a CB transplant protocol. Percoll separation provided a greater enrichment of cells than Ficoll.

Selection of human spermatozoa by a hyperosmotic two-layer Percoll gradient.

OBJECTIVE: To investigate whether a hyperosmotic Percoll solution improves the spermatozoal recovery rate of a two-layer Percoll gradient. DESIGN: A total of 49 semen samples were prepared by both the conventional two-layer Percoll gradient and a hyperosmotic Percoll gradient. SETTING: In vitro fertilization laboratory of a tertiary care, university-affiliated hospital. PATIENTS: Semen samples were obtained from patients attending semen analysis. MAIN OUTCOME MEASURES: The number of spermatozoa recovered, percentage motility, percentage normal morphology, and their survival at 24 hours were assessed after preparation by both Percoll gradients. RESULTS: The hyperosmotic Percoll gradient resulted in a significantly higher total and motile sperm recovery rate. The degree of increase in total sperm recovery was significantly higher in abnormal semen samples compared with normal semen samples. Percentage normal morphology of sperm samples prepared by hyperosmotic Percoll was improved compared with conventional Percoll but was only significant in abnormal semen samples. However, percentage motility of sperm samples prepared by the hyperosmotic Percoll was significantly lower than those prepared by conventional Percoll gradients. CONCLUSIONS: The hyperosmotic two-layer Percoll gradient improved motile sperm recovery but also recovered more immotile sperm, leading to a decrease in percentage motility. This technique may allow us to recover more spermatozoa when we come across samples of low sperm concentration.

Insemination of HIV-negative women with processed semen of HIV-positive partners.

Many HIV-discordant couples want to have children so much that they are willing to abandon condom-protected sexual intercourse irrespective of the risks. Previous testing in our laboratory showed that gradient centrifugation followed by a swim-up procedure effectively removed HIV-1-infected cells from the semen of HIV-seropositive men. 85 HIV-discordant couples were screened for fertility; 29 women were found suitable for a timed insemination course with the processed semen of their HIV-seropositive partner. None of the inseminated women seroconverted, and 17 pregnancies were achieved in 15 women. All 10 babies born to these mothers remain HIV seronegative. The findings may help in the counselling of such couples and also give them hope of having healthy babies.

Relative enrichment of hematopoietic progenitor cells: efficiency of a repeated density centrifugation.

Low-density cells were prepared from 11 bone marrow samples by centrifugation on Ficoll-sodium diatrizoate. Repeated density gradient centrifugation of the cells collected from the interface of the first gradient removed most nonnucleated erythroid cells. A mean of 47.9% (19.4% to 76.0%) of the mononuclear cells collected after the initial centrifugation were recovered from the interface of the second gradient, whereas 13.3% (3.7% to 34.9%) of the MNCs were counted in the high-density pellet and 38.9% (3.8% to 65.7%) of the MNCs were lost unspecifically. In contrast, a mean of 71.7% (43.0% to 91.3%) of the colony-forming units were recovered from the interface after the second centrifugation (as determined by colony formation assays), whereas only 3.2% (0.5% to 7.0%) were found in the high-density pellet. The unspecific loss of colony-forming units was 25.1% (1.7% to 51.4%). The results demonstrate a relative enrichment of colony-forming units in the culture assay by 1.7 times (average). The method is recommended as an additional preparative step before fluorescence-activated sorting of viable cells, because removal of most erythrocytes and late normoblasts strongly reduces the time required for sorting.

Purification of adrenal chromaffin cells on Renografin gradients.

Bovine adrenal medullary cells purified on discontinuous gradients of the radiopaque contrast agent Renografin contained about 20% more catecholamine than those prepared by centrifugation on Percoll gradients, a standard method for chromaffin cell purification. Catecholamine recoveries by the two methods were similar, suggesting increased purity in the Renografin preparation. Neutral red staining indicated that the cells prepared on Renografin gradients were approximately 90% chromaffin cells, compared to an average of 75% chromaffin cell purity obtained following centrifugation on self-generated gradients of Percoll. Cells purified on Renografin gradients were equivalent to Percoll-prepared cells by the criteria of maintenance in culture and secretory activity. Thus, density gradient centrifugation in Renografin is a rapid technique for producing adrenal medullary chromaffin cells of high purity and is superior to Percoll gradient centrifugation for this purpose.