Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Development of a Novel Ex-vivo 3D Model to Screen Amoebicidal Activity on Infected Tissue.

Amoebiasis is a parasitic disease that causes thousands of deaths every year, its adverse effects and resistance to conventional treatments have led to the search of new treatment options, as well as the development of novel screening methods. In this work, we implemented a 3D model of intestine and liver slices from hamsters that were infected ex vivo with virulent E. histolytica trophozoites. Results show preserved histology in both uninfected tissues as well as ulcerations, destruction of the epithelial cells, and inflammatory reaction in intestine slices and formation of micro abscesses, and the presence of amoebae in the sinusoidal spaces and in the interior of central veins in liver slices. The three chemically synthetized compounds T-001, T-011, and T-016, which act as amoebicides in vitro, were active in both infected tissues, as they decreased the number of trophozoites, and provoked death by disintegration of the amoeba, similar to metronidazole. However, compound T-011 induced signs of cytotoxicity to liver slices. Our results suggest that ex vivo cultures of precision-cut intestinal and liver slices represent a reliable 3D approach to evaluate novel amoebicidal compounds, and to simultaneously detect their toxicity, while reducing the number of experimental animals commonly required by other model systems.

[Delirium as a late onset manifestation of quetiapine intoxication]. / Delier als late complicatie van intoxicatie met quetiapine.

In this article we describe the developments of two patients who were hospitalized after a quetiapine overdose, subsequently developing delirium. Delirium occurred several hours after ingestion and lasted several days. Laboratory blood testing revealed a slower decline in quetiapine plasma concentration than expected when compared to the standardized half-life averages of therapeutic doses of quetiapine. The clinical state during the first hours after ingestion does not sufficiently predict further clinical outcome. The changes in pharmacokinetics due to the intoxication, so-called toxicokinetics, are the underlying cause.

Pessary for prevention of preterm birth in twin pregnancy with short cervix: 3-year follow-up study.

OBJECTIVE: A recent randomized clinical trial (ProTWIN) showed that a cervical pessary prevented preterm birth and improved neonatal outcome in women with multiple pregnancy and cervical length (CL) < 38 mm. In this follow-up study, the long-term developmental outcome of these children was evaluated at 3 years' corrected age. METHODS: This was a follow-up study of ProTWIN, a multicenter trial conducted between 2009 and 2012 in which asymptomatic women with a multiple pregnancy were randomized to placement of a cervical pessary or no intervention. Current follow-up and analysis were limited to mothers with a mid-trimester CL < 38 mm (78 women (157 children) in the pessary group and 55 women (111 children) in the control group). At 3 years of corrected age, surviving children were invited for a Bayley Scales of Infant and Toddler Development-third edition (Bayley-III) assessment. Death after randomization or neurodevelopmental disability (Bayley-III score of ≤ 85, 1 SD below mean) rates were compared between the pessary and control groups, according to the intention-to-treat principle and using multiple imputation for missing data. Mean Bayley-III scores in surviving children were also assessed. A linear mixed-effects model was used to adjust for correlation between children of one mother. RESULTS: From the time of entry in the ProTWIN trial until follow-up at 3 years of age, a total of 27 children had died (six (5%) in the pessary vs 21 (26%) in the control group; odds ratio (OR), 0.13; 95% CI, 0.04-0.48). Bayley-III outcomes were collected for 173/241 (72%) surviving children (114 (75%) in the pessary vs 59 (66%) in the control group). The cumulative incidence of death or survival with a neurodevelopmental disability was 12 (10%) in the pessary vs 23 (29%) in the control group (OR, 0.26; 95% CI, 0.09-0.73). No statistical or clinically relevant differences were found with respect to cognitive, language and motor development among surviving children between the groups. Comparable results were found after multiple imputation. CONCLUSION: In women with twin pregnancy and a CL < 38 mm, the use of a cervical pessary strongly improved survival of the children without affecting neurodevelopment at 3 years' corrected age. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Incidence of and risk indicators for vasa praevia: a systematic review.

BACKGROUND: Vasa praevia (VP) is a rare phenomenon that is assumed to increase the risk of severe complications, including fetal death. Critical data on its incidence are lacking, so there is no rational basis for prenatal screening. OBJECTIVES: To review the literature on the incidence and risk indicators for VP. SEARCH STRATEGY: We searched OVID MEDLINE, OVID EMBASE, the Cochrane Library and PubMed for case-control and cohort studies on incidence and risk indicators for VP. SELECTION CRITERIA: Two reviewers selected studies and scored their methodological quality. DATA COLLECTION AND ANALYSIS: We calculated the mean incidence of VP. We constructed 2 × 2 tables cross-classifying potential risk indicators against the incidence of VP to calculate common odds ratios and 95% confidence intervals, using the Mantel-Haenszel method. MAIN RESULTS: We included 13 studies (two prospective cohort studies, ten retrospective cohort studies and one case-control study) reporting on 569 410 patients with 325 cases of VP. Based on ten included cohort studies providing information on the incidence, the mean incidence of VP was 0.60 per 1000 pregnancies. We identified five different risk indicators and markers for VP: second-trimester placenta praevia, conception by assisted reproductive technologies, a bilobed or succenturiate placenta, umbilical cord insertion in the lower third part of the uterus at first-trimester ultrasound and velamentous cord insertion. Almost 83% of the cases of VP had one or more risk indicators. AUTHORS' CONCLUSIONS: In view of the low incidence, screening for VP in an unselected population is not advised. Targeted screening of women with one or more risk indicators as a part of routine mid-gestation scanning should be considered. TWEETABLE ABSTRACT: Vasa praevia is more common in placenta praevia, conception by ART, velamentous cord insertion and bilobed placenta.

Second-trimester cervical length as risk indicator for Cesarean delivery in women with twin pregnancy.

OBJECTIVE: To determine whether second-trimester cervical length (CL) in women with a twin pregnancy is associated with the risk of emergency Cesarean section. METHODS: This was a secondary analysis of two randomized trials conducted in 57 hospitals in The Netherlands. We assessed the univariable association between risk indicators, including second-trimester CL in quartiles, and emergency Cesarean delivery using a logistic regression model. For multivariable analysis, we assessed whether adjustment for other risk indicators altered the associations found in univariable (unadjusted) analysis. Separate analyses were performed for suspected fetal distress and failure to progress in labor as indications for Cesarean section. RESULTS: In total, 311 women with a twin pregnancy attempted vaginal delivery after 34 weeks' gestation. Emergency Cesarean delivery was performed in 111 (36%) women, of which 67 (60%) were performed owing to arrest of labor. There was no relationship between second-trimester CL and Cesarean delivery (adjusted odds ratio (aOR): 0.97 for CL 26(th) -50(th) percentiles; 0.71 for CL 51(st) - 75(th) percentiles; and 0.92 for CL > 75(th) percentile, using CL ≤ 25(th) percentile as reference). In multivariable analysis, the only variables associated with emergency Cesarean delivery were maternal age (aOR, 1.07 (95% CI, 1.00-1.13)), body mass index (BMI) (aOR, 3.99 (95% CI, 1.07-14.9) for BMI 20-23 kg/m(2) ; 5.04 (95% CI, 1.34-19.03) for BMI 24-28 kg/m(2) ; and 3.1 (95% CI, 0.65-14.78) for BMI > 28 kg/m(2) ) and induction of labor (aOR, 1.92 (95% CI, 1.05-3.5)). CONCLUSION: In nulliparous women with a twin pregnancy, second-trimester CL is not associated with risk of emergency Cesarean delivery.

Systematic review of accuracy of ultrasound in the diagnosis of vasa previa.

OBJECTIVE: Vasa previa is an obstetric complication in which the fetal blood vessels lie outside the chorionic plate in close proximity to the internal cervical os. In women with vasa previa, the risk of rupture of these vessels is increased, thus potentially causing fetal death or serious morbidity. Our objective was to assess the accuracy of ultrasound in the prenatal diagnosis of vasa previa. METHODS: We searched MEDLINE, EMBASE, the Cochrane Library and PubMed for studies on vasa previa. Two reviewers independently selected studies on the accuracy of ultrasound in the diagnosis of vasa previa. The studies were scored on methodological quality using the Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2). Data on sensitivity and specificity were subsequently extracted. RESULTS: The literature search revealed 583 articles, of which two prospective and six retrospective cohort studies were eligible for inclusion in the qualitative analysis. All studies documented methods suitable for the prenatal diagnosis of vasa previa. Four out of the eight studies used transvaginal ultrasound (TVS) for primary evaluation, while the remaining four studies used transabdominal ultrasound and performed a subsequent TVS when vasa previa was suspected. The QUADAS-2 tool reflected poor methodology in six of the eight included studies, and prenatal detection rates varied from 53% (10/19) to 100% (total of 442,633 patients, including 138 cases of vasa previa). In the two prospective studies (n = 33,795, including 11 cases of vasa previa), transvaginal color Doppler performed during the second trimester detected all cases of vasa previa (sensitivity, 100%) with a specificity of 99.0-99.8%. CONCLUSION: The accuracy of ultrasound in the diagnosis of vasa previa is high when performed transvaginally in combination with color Doppler.

Sonographic measurement of lower uterine segment thickness to predict uterine rupture during a trial of labor in women with previous Cesarean section: a meta-analysis.

OBJECTIVE: To evaluate the accuracy of antenatal sonographic measurement of lower uterine segment (LUS) thickness in the prediction of risk of uterine rupture during a trial of labor (TOL) in women with a previous Cesarean section (CS). METHODS: PubMed and EMBASE were searched to identify articles published on the subject of sonographic LUS measurement and occurrence of a uterine defect after delivery. Four independent researchers performed identification of papers and data extraction. Selected studies were scored on methodological quality, and sensitivity and specificity of measurement of LUS thickness in the prediction of a uterine defect were calculated. We performed bivariate meta-analysis to estimate summary receiver-operating characteristics (sROC) curves. RESULTS: We included 21 studies with a total of 2776 analyzed patients. The quality of included studies was good, although comparison was difficult because of heterogeneity. The estimated sROC curves showed that measurement of LUS thickness seems promising in the prediction of occurrence of uterine defects (dehiscence and rupture) in the uterine wall. The pooled sensitivity and specificity of myometrial LUS thickness for cut-offs between 0.6 and 2.0 mm was 0.76 (95% CI, 0.60-0.87) and 0.92 (95% CI, 0.82-0.97); cut-offs between 2.1 and 4.0 mm reached a sensitivity and specificity of 0.94 (95% CI, 0.81-0.98) and 0.64 (95% CI, 0.26-0.90). The pooled sensitivity and specificity of full LUS thickness for cut-offs between 2.0 and 3.0 mm was 0.61 (95% CI, 0.42-0.77) and 0.91 (95% CI, 0.80-0.96); cut-offs between 3.1 and 5.1 mm reached a sensitivity and specificity of 0.96 (95% CI, 0.89-0.98) and 0.63 (95% CI, 0.30-0.87). CONCLUSIONS: This meta-analysis provides support for the use of antenatal LUS measurements in the prediction of a uterine defect during TOL. Clinical applicability should be assessed in prospective observational studies using a standardized method of measurement.

Effect of 17-alpha hydroxyprogesterone caproate on cervical length in twin pregnancies.

OBJECTIVES: Previous studies on singleton pregnancies have indicated that progestogens may reduce the rate of cervical shortening during pregnancy. The aim of this study was to investigate whether treatment with 17-alpha hydroxyprogesterone caproate (17-OHPC) has an effect on cervical shortening in twin pregnancies. METHODS: This was a secondary analysis of patients who had participated in a multicenter randomized clinical trial on the effectiveness of 17-OHPC in preventing preterm birth in multiple pregnancies (the AMPHIA-trial). We included all trial participants with a twin gestation who had undergone repeat cervical length measurements during pregnancy. We performed a separate analysis of women with repeat measurements in centers where this was standard protocol for multiple pregnancies. The rate of cervical shortening for both the 17-OHPC group and the placebo group was analyzed using a linear mixed model. RESULTS: Of the 671 patients who participated in the trial, 282 (42%) had a twin pregnancy and underwent two or more cervical length measurements. Of these women, 140 were monitored in centers where repeat measurements were standard protocol. We observed an overall reduction of cervical length from 44.3 mm at 14-18 weeks to 30.0 mm at 30-34 weeks' gestation. In the 17-OHPC group, cervical length decreased by 1.04 mm each gestational week, while this was 1.11 mm per week for the placebo group (P = 0.6). For the overall group, each 10% decrease in cervical length led to an increase in the risk of preterm birth (hazard ratio, 1.14; 95% CI, 1.08-1.21). CONCLUSION: In women with a twin pregnancy, there is progressive shortening of the cervix during pregnancy, regardless of 17-OHPC use.

Techniques for wound closure at caesarean section: a randomized clinical trial.

OBJECTIVE: It is unclear which technique for skin closure should be used at caesarean section (CS) in order to get the best cosmetic result. STUDY DESIGN: We conducted a randomized controlled trial to assess the cosmetic result of different techniques for skin closure after CS. A two-center single-blind randomized controlled trial was performed in The Netherlands. Women undergoing their first CS were eligible for the trial. In a factorial design, women were randomly allocated to (1) closure of the fat layer versus non-closure and (2) staples or intracutaneous stitches for skin closure. The cosmetic result was assessed using the Patient and Observer Scar Assessment Scale (POSAS). RESULTS: We included 124 women. In the stitches group 63% [39/62] women judged the scar as satisfactory, versus 63% [38/60] in the staples group (RR 1.01; 95% CI 0.64-1.6). When the subcutaneous fat layer was closed, 52% [33/63] of the women scored the scar as satisfactory, versus 75% [44/59] of the women in whom the fat layer was not separately closed (RR 0.53; 95% CI 0.32-0.89). This effect was independent of the subcutaneous thickness (p-value for interaction 0.64). Of the secondary outcomes, subcutaneous closure of the fat layer was associated with a longer admission time (median 4 days; IQR 3-5 versus 3 days; IQR 3-5, p-value 0.023). CONCLUSIONS: The choice of staples or stitches does not affect the cosmetic result after a caesarean section. Closing of the subcutaneous fat layer, however, negatively affects the cosmetic result and is associated with a longer admission time.

Induction of phase I and II drug metabolism in rat small intestine and colon in vitro.

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon.

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.

Cryopreservation of precision-cut tissue slices for application in drug metabolism research.

Cryopreservation of tissue slices greatly facilitates their use in drug metabolism research, leading to efficient use of human organ material and a decrease of laboratory animal use. In the present review, various mechanisms of cryopreservation such as equilibrium slow freezing, rapid freezing and vitrification, and their application to cryopreservation of tissue slices are discussed as well as the viability parameters often used to evaluate the success of cryopreservation. Equilibrium freezing prevents intracellular ice formation by inducing cellular dehydration, but (large) ice crystals are still formed in the interstitial space of the slices. Upon rapid freezing, (small) intra- and extracellular ice crystals are formed which slices from some tissues can resist. Vitrification prevents the formation of both intra- and extracellular ice crystals while an amorphous glass is formed of the slice liquid constituents. To vitrify, however, high molarity solutions of cryoprotectants are required that may be toxic to the slices. The use of mixtures of high molarity of cryoprotectants overcomes this problem. We conclude that vitrification is the approach that most likely will lead to the development of universal cryopreservation methods for tissue slices of various organs from various animal species. In the future this may lead to the formation of a tissue slice bank from which slices can be derived at any desirable time point for in vitro experimentation.

Incubation at 37 degrees C prior to cryopreservation decreases viability of liver slices after cryopreservation by rapid freezing.

Precision-cut liver slices are to some extent resistant to ice formation induced by rapid freezing. Susceptibility to rapid freezing damage has been shown to be (partly) dependent on intrinsic properties of cells. In the present study an attempt was made to decrease the susceptibility of rat liver slices for rapid freezing damage: the slices were pre-incubated at 37 degrees C under oxygen, prior to cryopreservation to recover from low ATP levels, impaired ion regulation and cell swelling induced by their preparation. It was shown that, unexpectedly, recovery of cellular homeostasis prior to the cryopreservation procedure by the 37 degrees C pre-incubation markedly decreased viability of rapidly frozen slices (in which ice was formed), but not of vitrified slices (in which no ice was formed), in a time- and temperature-dependent manner. UW was found to protect slices from this 'warm pre-incubation phenomenon.' Apparently, pre-incubation prior to freezing causes certain cellular alterations that render slices more susceptible to rapid freezing damage.

Comparison of in vitro preparations for semi-quantitative prediction of in vivo drug metabolism.

Various in vitro preparations were compared with respect to their ability to mimic in vivo metabolism. For this purpose, S9-liver homogenate, microsomes, cryopreserved hepatocytes, cryopreserved liver slices and fresh liver, lung, kidney, and intestinal slices were incubated with three drugs in development, which are metabolized in vivo by a wide range of biotransformation pathways. Metabolites were identified and quantified with liquid chromatography-mass spectometry/UV from the in vitro incubations and compared with metabolite patterns in feces, urine, and bile of dosed rats. In vitro systems with intact liver cells produced the same metabolites as the rat in vivo and are a valuable tool to study drug metabolism. Phase I metabolites were almost all conjugated in intact cells, whereas S9-homogenate only conjugated by sulfation and N-acetylation. Microsomes and S9-homogenate are useful to study phase I metabolism but not for the prediction of in vivo metabolism. Extra-hepatic organ slices did not form any metabolites that were not produced by liver cells, but the relative amounts of the various metabolites differed considerably. Small intestinal slices were more active than liver slices in the formation of the N-glucuronide of compound C, which is the major metabolite in vivo. When the relative contribution of liver and small intestinal slices to the metabolism of this compound was taken into account, it appeared that the in vivo metabolite pattern could be well predicted. Results indicate that for adequate prediction of in vivo metabolism, fresh or cryopreserved liver slices or hepatocytes in combination with slices of the small intestines should be used.

Water crystallization within rat precision-cut liver slices in relation to their viability.

This study examined whether tissue vitrification, promoted by partitioning within the tissue, could be the mechanism explaining the high viability of rat liver slices, rapidly frozen after preincubation with 18% Me2SO or VS4 (a 7.5 M mixture of Me2SO, 1,2-propanediol, and formamide with weight ratio 21.5:15:2.4). To achieve this, we first determined the extent to which crystallization or vitrification occurred in cryoprotectant solutions (Me2SO and VS4) and within liver slices impregnated with these solutions. Second, we determined how these events were related to survival of slices after thawing. Water crystallization was evaluated by differential scanning calorimetry and viability was determined by histomorphological examination of the slices after culturing at 37 degrees C for 4 h. VS4-preincubated liver slices indeed behaved differently from bulk VS4 solution, because, when vitrified, they had a lower tendency to devitrify. Vitrified VS4-preincubated slices that were warmed sufficiently rapid to prevent devitrification had a high viability. When VS4 was diluted (to 75%) or if warming was not fast enough to prevent ice formation, slices had a low viability. With 45% Me2SO, low viability of cryopreserved slices was caused by cryoprotectant toxicity. Surprisingly, liver slices preincubated with 18% Me2SO or 50% VS4 had a high viability despite the formation of ice within the slice. In conclusion, tissue vitrification provides a mechanism that explains the high viability of VS4-preincubated slices after ultrarapid freezing and thawing (>800 degrees C/min). Slices that are preincubated with moderately concentrated cryoprotectant solutions (18% Me2SO, 50% VS4) and cooled rapidly (100 degrees C/min) survive cryopreservation despite the formation of ice crystals within the slice.

Genetic analysis of fetal nucleated red blood cells from CVS washings.

Chorionic villus sampling (CVS) is an established invasive prenatal diagnostic method for the detection of fetal chromosome aberrations. In 1-2% the karyotype result of CVS is inconclusive and follow-up confirmation will be required. To avoid another invasive procedure we examined fetal nucleated red blood cells (NRBCs) from CVS washings for genetic analysis. We analysed the washings of 20 chorionic villi samples of male fetuses. Fetal NRBCs were immunostained by an antibody against embryonic haemoglobin (HbE). FISH was performed with probes specific for the X and Y chromosome and the nucleus was counterstained with DAPI. Cells positive for the antibody, as well as for DAPI, were collected and stored by a semi-automated microscope. An operator reviewed those cells for their FISH signals. In 19 out of 20 CVS washings we found nucleated cells positive for HbE together with XY FISH signals. In none of the washings HbE positive cells with two X signals were found. Our results indicate that anti-HbE is a very specific antibody for identifying fetal NRBCs. NRBCs from CVS washings can be used as an additional fetal tissue for first trimester prenatal diagnosis.

The influence of fetal position on nuchal translucency thickness.

OBJECTIVE: To determine whether nuchal translucency thickness is influenced by the fetal position at ultrasound examination. SUBJECTS: Transabdominal ultrasound examination for pregnancy dating and measurement of nuchal translucency thickness was performed at 10-14 weeks' gestation in all women attending the antenatal clinic of our hospital. During the examination special attention was paid to a change in fetal position from prone to supine or vice versa. METHODS: For each fetus the nuchal translucency measurement was repeated when a positional change from prone to supine or vice versa was recorded. All measurements were recorded on hard copy. An image-scoring method was used and evaluated by three independent reviewers. RESULTS: Eighty-five fetuses were included in this study. The mean nuchal translucency for supine fetuses was 1.91 mm compared with 1.93 mm for prone fetuses. The mean quality-score was 6.54 for supine fetuses and 6.55 for prone fetuses. This difference was not statistically significant. CONCLUSION: Fetal position has no influence on the measurement of nuchal translucency.

Increased post-thaw viability and phase I and II biotransformation activity in cryopreserved rat liver slices after improvement of a fast-freezing method.

An existing cryopreservation method for liver slices applies 12% dimethylsulfoxide and rapid freezing. We found that cells in rat liver slices cryopreserved in this manner deteriorated rapidly upon culturing. To improve this cryopreservation method, we varied the dimethylsulfoxide concentration (0, 12, 18, and 30%), the cryopreservation medium (Williams medium E, fetal calf serum, and University of Wisconsin medium), slice thickness, and the storage period at 4 degrees C during slice preparation before cryopreservation. After thawing, slices were cultured for 4 h at 37 degrees C before their viability was evaluated by their potassium content and the number of intact cells determined histomorphologically. The biotransformation capacity of liver slices cryopreserved by the improved method was assessed by testosterone oxidation, hydroxycoumarin sulfation, and glucuronidation. Best results were obtained with 18% dimethylsulfoxide in Williams medium E: the potassium content of cryopreserved slices was higher than 65%, and the number of intact cells was higher than 60% of that in fresh slices; with 12% dimethylsulfoxide, potassium content was less than 40%, and the number of intact cells was less than 30%. Results did not differ between the three cryopreservation media. Viability of thin slices (8-10 cell layers) was better maintained than that of thicker slices (>14 cell layers). Storage at 4 degrees C of slices before cryopreservation decreased viability after cryopreservation. Both oxidative and conjugation activities were better than 60% of fresh values. Although results varied, slices cryopreserved with this improved method and cultured for 4 h retained viability between 50 and 80%, and biotransformation activity between 60 and 90% of fresh slices.

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices.

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.