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PubMed1995

Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes.

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.

Journal Article↗
PubMed1994

Recommendations for the performance of UDS tests in vitro and in vivo.

The Working Group (WG) dealt with the harmonization of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to the existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guideline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are preferable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative result, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is in line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic grains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of positive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of in vivo UDS tests. Some fundamental recommendations given for in vitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS test, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vivo genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodology, AR is preferred rather than LSC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Evaluation of two in vitro assays to screen for potential developmental toxicants.

To evaluate two in vitro assays for their ability to detect known developmental toxicants and nontoxicants, a series of 44 coded compounds were assayed by two independent laboratories using standardized protocols. The two test systems were (1) the human embryonic palatal mesenchymal cell growth inhibition assay and (2) the mouse ovarian tumor cell attachment inhibition assay. After all compounds were tested, they were decoded and ranked according to the minimum IC50 value (the millimolar concentration of compound which inhibits growth or attachment by 50% compared to the solvent control) from either test. The in vitro test result concordance with established in vivo animal and human test results was examined over a wide range of concentration levels (above which the in vitro results were called positive and below which they were considered negative). A positive response from either test was defined as a positive in vitro response. Concordance was defined as the number of correct responses divided by the number of chemicals tested. At the 1 mM level, the concordance of data from the combined in vitro assays with the in vivo data was 66% in one laboratory and 58% in the other. The maximum agreement between the combined in vitro and in vivo data was reached at the 20 mM level, where there was a 73 and 74% concordance of results in the two laboratories. At that level there was a 16 and 10% incidence of false negative results, and a 54 and 77% incidence of false positive results. A portion of these false negative compounds may require metabolic activation. The use of either assay alone was not as accurate as using a positive result from either test. Agreement of the in vitro data at the 10 mM level with available human data was 71 and 75% for each laboratory. The data indicate that the two assays are complimentary and as such the combination of these assays could be useful as a preliminary screen to establish priorities for in vivo developmental toxicity testing.

Animals↗
PubMed1990

Effect of in vivo low-dose cadmium pretreatment on the in vitro interactions of cadmium with isolated interstitial cells of the rat testes.

Recent studies have shown that Cd-induced testicular interstitial cell (TIC) tumors can be prevented by low-dose Cd pretreatment. However, the mechanism by which low-dose Cd induces such tolerance is unclear. Thus, in this study we assessed the effects of in vivo Cd pretreatment (3 mumol/kg) on Cd uptake, cytotoxicity, metal content (Zn, K, and Ca), and low molecular weight testicular Cd-binding proteins (low Mr TC-BPs) of isolated TICs exposed to Cd in vitro. TICs were isolated by collagenase dispersion of Wistar (WF/NCr) rat testes and incubated with Cd (1.0 mM) for 15 to 60 min. In vivo Cd pretreatment decreased in vitro Cd uptake by 24% after 1 hr of incubation with Cd. In vivo Cd pretreatment also resulted in a marked reduction of in vitro Cd-induced cytotoxicity, as reflected by reduced loss of cellular K, glutamic-oxaloacetic transaminase, as well as reduced lipid peroxidation and decreased Cd-induced Ca influx into TICs in vitro. These cytotoxic effects were not attributed solely to cell death as TIC viability remained high even after 1 hr of in vitro Cd exposure. Cd-induced inhibition of intercellular enzymes, as assessed by cellular lactate dehydrogenase activity, was also reduced by low-dose Cd pretreatment. Cd pretreatment did not alter basal levels of Zn, Ca, or K. Neither low-dose in vivo Cd pretreatment nor in vitro Cd exposure appeared to greatly alter levels of the low Mr TCBPs as assessed by electrophoresis. In vivo Zn pretreatment, which also effectively inhibits Cd-induced testicular tumors, results in a similar reduction in Cd-induced cytotoxicity in TICs. This indicates that treatments which result in reduced Cd-induced TIC tumors are consistently capable of reducing Cd-induced cytotoxicity in isolated TICs in vitro.

Animals↗
PubMed1992

Redistribution of B-50/growth-associated protein 43 during differentiation and maturation of rat hippocampal neurons in vitro.

Morphologically polarized hippocampal neurons, grown in culture for two days, contain immunoreactivity of the growth-associated protein B-50 along the plasma membrane of both dendrites and axons. In mature hippocampal neurons, both in vitro and in vivo, B-50 is located in the axon. In order to assess at which stage during neuronal differentiation B-50 is selectively located in the axon, an immuno-light and electron-microscopic study was performed on rat hippocampal neurons developing in vitro. B-50 immunofluorescence was detected in the axon, dendrites and soma of two-day-old polarized neurons. Simultaneously, microtubule-associated protein 2, a marker specific to dendritic microtubules, was predominantly found in the soma, the short dendritic processes and at the base of axonal growth cones. In hippocampal neurons cultured beyond seven days in vitro, microtubule-associated protein 2 immunofluorescence is restricted to the cell soma and dendrites. The spatial distribution of B-50, however, varies. In solitary neurons maturing without interneuronal contacts, B-50 immunofluorescence is observed in axons and in the dendrosomatic domain characterized by the presence of microtubule-associated protein 2. In contrast, in high-density cell cultures B-50 immunofluorescence is absent in the cell body and dendrites, but punctate in axons running along the dendrites. Electron microscopy was carried out on hippocampal neurons of eight to 21 days in vitro to study the process of redistribution of B-50 at the subcellular level. In neurons of eight days in vitro with prominent synapses, B-50 immunoreactivity is significantly elevated at the axonal plasma membrane compared to the plasma membrane of the dendrites and the soma. In neurons from the same culture without synapses, B-50 immunoreactivity is distributed rather densely along the plasma membrane of the soma, dendrites, and on the axonal plasma membrane. A similar B-50 distribution is observed in mature neurons cultured at low cell density without interneuronal cell contacts, for 15 days in vitro. In high-density cell cultures of 21 days in vitro, B-50 is virtually absent at the plasma membrane of the soma and dendrites, and heterogenously distributed along the plasma membrane of axon and axonal varicosities. Our results indicate that selective sorting of B-50 into axons occurs after initial morphological polarization of hippocampal neurons and is correlated with the formation of synapses and with the cessation of dendritic outgrowth.

Animals↗
PubMed1990

Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.

Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts.

A longitudinal study was performed to confirm the inverse relationship between in vivo age and in vitro proliferative capacity previously observed in a cross-sectional study, and to investigate the relationship between the growth of dermal fibroblasts in vitro and a physiological function (i.e., wound repair) that is known to decline with age in vivo. Fibroblast cultures were generated from skin punch biopsies from 12 male hamsters beginning at 1 month of age and at 6-months interval thereafter until the natural death of the animal. All cultures from all individuals exhibited finite proliferative capacity, and an inverse relationship was observed between donor age and maximum in vitro proliferative capacity. In addition, a direct correlation between the in vitro proliferative capacity of the dermal fibroblasts in vitro and the repair efficiency of the biopsy site was observed. However, these changes in the in vitro proliferative capacity and in vivo wound repair efficiency were not progressive beyond 12-18 months of age and were not indicative at any age of an individual's ultimate lifespan. This study provides evidence that in vitro proliferative capacity of dermal fibroblasts and in vivo wound repair may be comparable phenomena that share a common mechanism. However, the nonprogressive nature and the lack of correlation between these phenomena and the individual's ultimate lifespan indicate that their use as biological markers of aging is limited to animals younger than the mean lifespan of the species.

Aging↗
PubMed2006

The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro.

Understanding mechanisms responsible for meiotic resumption in mammalian oocytes is critical for the identification of strategies to enhance developmental competence of in vitro-matured oocytes. Improvement of in vitro oocyte maturation systems is dependent on a better understanding of mechanisms that regulate oocyte maturation both in vivo and in vitro as well as on the identification of methods to manipulate the meiotic progression of oocytes matured in vitro in a physiological manner. The purpose of this review is two-fold: first, to examine the mechanisms that underlie the acquisition of oocyte developmental competence and regulation of oocyte maturation in vivo and in vitro; second, to present data examining the role of transcription in mediating the ability of EGF and FSH to induce oocyte maturation in vitro. Results presented support the conclusions that (1) EGF-induced oocyte maturation does not require nascent gene transcription in both mice and domestic cats; (2) FSH requires gene transcription to induce oocyte maturation in both species; (3) EGF must be present in the maturation medium to optimize the effectiveness of FSH to promote oocyte maturation; (4) the mechanism used by FSH to induce oocyte maturation in vitro appears to predominate over that used by EGF when both EGF and FSH are present in maturation medium used for either murine or feline cumulus oocyte complexes.

Animals↗
PubMed2006

Multiple hormonal activities of UV filters and comparison of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish.

UV filters have been detected in surface water, wastewater and fish, and some of them are estrogenic in fish. At present, little is known about their additional hormonal activities in different hormonal receptor systems despite their increasing use and environmental persistence. Besides estrogenic activity, UV filters may have additional activities, both agonistic and antagonistic in aquatic organisms. In our study, we investigate a series of UV filters for multiple hormonal activities in vitro in human receptor systems and evaluate the predictive value of these findings for the activity in fish in vitro and in vivo. First we systematically analysed the estrogenic, antiestrogenic, androgenic, and antiandrogenic activity of 18 UV filters and one metabolite in vitro at non-cytotoxic concentrations with recombinant yeast systems carrying either a human estrogen (hERalpha) or androgen receptor (hAR). All 19 compounds elicited hormonal activities, surprisingly most of them multiple activities. We found 10 UV-filters having agonistic effects towards the hERalpha. Surprisingly, we identified for the first time six UV filters with androgenic activities and many of them having pronounced antiestrogenic and antiandrogenic activities. As much as 17 compounds inhibited 4,5-dihydrotestosterone activity in the hAR assay, while 14 compounds inhibited estradiol activity in the hERalpha assay, indicating antiandrogenic and antiestrogenic activity, respectively. In particular, the antiandrogenic activities of phenyl- and benzyl salicylate, benzophenone-1 and -2, and of 4-hydroxybenzophenone were higher than that of flutamide, a known hAR antagonist. In a second series of experiments, we investigated the predictive power of the hERalpha assay for aquatic organisms by further investigating the estrogenic UV filter ethyl 4-aminobenzoate (Et-PABA) in vitro and in vivo in fish. Et-PABA showed estrogenic activity in a recombinant yeast system carrying the rainbow trout estrogen receptor (rtERalpha) with higher activity than in the hERalpha assay. In addition, Et-PABA induced vitellogenin after 14 days of exposure in juvenile fathead minnows at 4394mug/L. Our study shows estrogenic activity of this UV filter in fish both in vitro and in vivo. In conjunction with in vitro human receptor-based systems our results give a more detailed picture about distinct hormonal activities of UV filters occurring in aquatic systems. We conclude that receptor-based assays are important for in vitro assessment of UV-filters prior to or concurrently with in vivo assays, which ultimately provide data for the environmental risk assessment of these important personal care products.

Androgen Antagonists↗
PubMed2006

Retention of KOH-soluble fluoride on enamel and dentine under erosive conditions--A comparison of in vitro and in situ results.

OBJECTIVES: Aim of the study was to investigate the stability of CaF(2)-like precipitates on enamel and dentine under neutral or acidic conditions and to compare in vitro and in situ results. METHODS: Human enamel and dentine specimens were fluoridated (Elmex fluid, 5 min) and subjected to erosive demineralisation (Sprite light: 3/day each; 30 s in vitro, 2 min in situ) or stored under neutral conditions for 4 (in vitro) or 7 (in situ) days. KOH-soluble fluoride was determined using an ion selective electrode. Between the acid attacks, specimens were stored in remineralisation solution (in vitro) or retained in the oral cavity (four volunteers for enamel and dentine each). RESULTS: Initially, high amounts of KOH-soluble fluoride were gained (between 77.9+/-12.3 and 96.0+/-46.4 microg/cm2). Under neutral conditions in vitro, a significant decrease on enamel (16.2+/-5.0 microg/cm2) and dentine (18.6+/-10.5 microg/cm2) was found, which was more severe under acidic conditions (6.3+/-3.0 and 5.1+/-2.1 microg/cm2, respectively). Under in situ conditions, KOH-soluble fluoride was strikingly stable on enamel under neutral (42.3+/-12.6 microg/cm2) as well as under acidic conditions (54.1+/-17.4 microg/cm2). On dentine, the dissolution kinetics of KOH-soluble fluoride was similar to the in vitro conditions, but the loss was less severe (45.3+/-12.9 microg/cm2 under neutral and 8.8+/-6.4 microg/cm2 under acidic conditions). CONCLUSIONS: In vitro, more KOH-soluble fluoride was lost under erosive compared to neutral conditions. The intra-oral environment was considerably protective for CaF2-like precipitates especially on enamel.

Amines↗
PubMed2005

In vivo and in vitro studies of fetal hemoglobin induction by hydroxyurea in beta-thalassemia/hemoglobin E patients.

OBJECTIVE: Some, but not all, beta-thalassemia/hemoglobin E (beta-thal/HbE) patients respond to hydroxyurea treatment. It would be helpful if patient responses to hydroxyurea could be screened in vitro to identify responders and nonresponders before beginning in vivo treatment. MATERIALS AND METHODS: Thirteen beta-Thal/HbE patients were treated with hydroxyurea orally for 2 years at a starting dose of 5 mg/kg/day for 5 days/week with escalation to a maximum of 10 mg/kg/day. For comparison, erythroid cells obtained from peripheral blood of the same patients 1 year after they had stopped hydroxyurea treatment were treated with hydroxyurea in vitro. The gamma-globin mRNA was measured by real-time reverse-transcription polymerase chain reaction, fetal hemoglobin (HbF) by high-performance liquid chromatography, (G)gamma- and (A)gamma-globin chains by Triton X-100 acid urea polyacrylamide gel electrophoresis. RESULTS: Treatment of cells in primary culture with 30 microM hydroxyurea for 96 hours significantly increased the fractional HbF content in beta-Thal/HbE patients. The (G)gamma:(A)gamma-globin mRNA was induced 0.30- to 8-fold in vitro and 0.30- to 6-fold in vivo (r(2) = 0.51, p = 0.16 by paired t-test); the fractional HbF content was induced 0.50- to 19-fold in vitro and 0.30- to 12-fold in vivo (r(2) = 0.61, p = 0.20) and the (G)gamma:(A)gamma-globin chain ratio was increased 0.80- to 1.40-fold in vitro and 1- to 1.20-fold in vivo (r(2) = 0.62, p = 0.13). CONCLUSION: The correlation of in vivo and in vitro results of HbF synthesis and globin mRNA suggest that in vitro testing may predict the in vivo response.

Adult↗
PubMed2004

In vitro and in vivo correlation of buserelin release from biodegradable implants using statistical moment analysis.

Here we investigated the possibility to develop different levels of correlation between in vitro drug release profiles and in vivo pharmacokinetic parameters for three Buserelin implant formulations. The in vitro and in vivo data were analyzed using model-independent and model-dependent methods. Since diffusion, dissolution and erosion effects influence drug release in most cases a simple kinetic model is unlikely to explain the overall in vivo release behavior. Thus the in vitro drug release curves were analyzed according to the theoretical models of Higuchi and Korsmeyer-Peppas. For the formulation with predominant diffusion controlled release level A IVIVC could be established (R2=0.986). Independent on drug release mechanism, a level B correlation between the mean in vitro dissolution time (MDT) and mean in vivo residence time (MRT) was obtained with a correlation coefficient of 0.983. Finally, level C correlation were observed when single in vitro parameters, e.g. T50% (time required to release 50% of drug in vitro) where compared with single in vivo parameters like AUC. This study suggests that a level B correlation could be achieved even when drug release occurs by a combination of diffusion and erosion processes. More sophisticated in vitro models mimicking drug release under in vivo conditions are clearly desirable for parenteral depot formulations.

Absorbable Implants↗
PubMed2002

Evaluation of preconditioning treatments to protect near-pure cortical neuronal cultures from in vitro ischemia induced acute and delayed neuronal death.

We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen--glucose deprivation, hypoxia, hypothermia and TNFalpha preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22--24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen--glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFalpha and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.

Animals↗
PubMed1998

Characterization and applications of human epidermis reconstructed in vitro on de-epidermized derma.

In recent years in vitro models have been developed to avoid the use of animals in cutaneous toxicological studies. Submerged human keratinocyte cultures in vitro could be so far employed as an alternative to animal testing and a good correlation between skin irritation and cytotoxicity has been demonstrated. Nevertheless, these submerged cultures are lacking in the stratum corneum which acts as a barrier to chemical toxicity, so that this type of culture is far from the in vivo situation. A better alternative method seems to be the use of in vitro reconstructed skin at the air-liquid interface that closely resembles the in vivo situation. In this work, in a first step we have characterized human epidermis reconstructed in vitro on de-epidermized derma (DED) after a two-week air exposure. Human skin reconstituted in vitro on DED was histologically similar to the in vivo skin. A stratified epidermis including the stratum corneum was obtained. The presence of basal lamina as well as of various important markers for epidermal differentiation (involucrin, K10 keratin, and filaggrin) were revealed. In a second step we have tested the cytotoxic and morphological effects of four surfactants on our model. A good rank correlation has been shown to exist between the irritation potency of surfactants on our model and reported ocular irritancy in vivo. From our results, in vitro reconstituted human skin could represent an attractive model for irritancy testing and could be an in vitro replacement for animal testing.

Child, Preschool↗
PubMed2002

Pharmacological concentrations of follicle-stimulating hormone and testosterone improve the efficacy of in vitro germ cell differentiation in men with maturation arrest.

OBJECTIVE: To examine whether in vitro differentiation of germ cells from men with maturation arrest is improved by augmenting FSH and T concentrations above the values effective in samples from men with normal spermatogenesis. DESIGN: Prospective, controlled in vitro study. SETTING: Private assisted reproduction centers and a university department. PATIENT(S): Men with meiotic or postmeiotic maturation arrest. INTERVENTION(S): Testicular spermatid extraction, in vitro culture of testicular biopsy samples, intraoocyte injection of elongated spermatids, embryo culture and transfer. MAIN OUTCOME MEASURE(S): Progression of in vitro germ cell differentiation, fertilization, and pregnancy outcomes with in vitro cultured germ cells. RESULT(S): In some cases of meiotic and postmeiotic maturation arrest, more advanced germ cell stages were achieved by in vitro culture in the presence of 500 IU/L FSH as compared with 50 IU/L FSH. The beneficial effect of 500 IU/L FSH was further potentiated by a simultaneous increase of T concentration from 1 to 10 microM. Fertilizations with germ cells recovered after incubation with these pharmacological hormone concentrations gave rise to viable embryos and the births of five healthy babies. CONCLUSION(S): Pharmacological concentrations of FSH and T are beneficial for in vitro maturation of germ cells from some men with in vivo maturation arrest.

Biopsy↗
PubMed2003

On the accuracy of some in vitro models for mechanical studies of maxillary removable partial dentures.

OBJECTIVES: Comparative in vitro/in vivo studies show wide differences in functional strain magnitudes and patterns of functional strain distribution in prosthetic devices. The aim of this study was to evaluate the accuracy of three in vitro models for biomechanical studies of prosthetic devices. METHODS: Strain gages were attached to the test RPDs of six subjects. In vitro maxillary models with simple (model A), intermediate (model B) and advanced (model C) features for the anatomical simulation of supporting structures were manufactured for each subject. The dentures were subjected to two separate series of standardized functional and laboratorial loading tests. The principal maximum strain value (epsilon (1)) obtained for each gage and experiment was used to calculate the intra-experiment variance and inter-experiment variance for the in vivo and in vitro experiments. The integrated strain gage difference value between the in vitro and in vivo data (Sigma(diff)) was calculated for every loading test and compared to the in vivo inter-experiment variance with ANOVA and Scheffés test. RESULTS: The bite forces recorded were similar for each subject and loading position and between the two loading sessions. The inter- and intra-experiment variance was found to be higher for in vivo loadings than for in vitro. Results showed that the (Sigma(diff)) values for model B and model C were significantly different from the in vivo strain inter-experiment variance (p<0.05). SIGNIFICANCE: This study showed that the anatomical simulation of in vitro models is insufficient to allow for accurate mechanical analyses of maxillary RPDs and that only simple verifications of the strain levels in prosthetic appliances can be attained in vitro.

Aged↗
PubMed1998

A comparison of low volume, draize and in vitro eye irritation test data. II. Oil/water emulsions.

The second phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro eye irritation test protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 18 representative oil/water based personal-care formulations. In general, these formulations were minimally to mildly irritating; only three were classified as moderate eye irritants. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.85; LVET-MAS values were typically about half the corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and each in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower 95% prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Sixteen endpoints were shown to have the greatest agreement with the LVET (all but two were selected for modelling when compared with the Draize procedure). While the lower maximum average scores values (compared with the Draize test) in the LVET led to lower variability in LVET-MAS compared to MAS, the upper and lower bounds on predicted LVET-MAS values conditional on observed in vitro scores were still wide. Because there was overlap in the range of scores determined by the prediction bounds for many formulations, each of the selected endpoints was frequently unable to distinguish between test formulations in terms of statistically different predicted LVET-MAS values. In summary, none of the in vitro endpoints evaluated were able to reliably predict values of LVET-MAS among the set of oil/water emulsions considered here.

Animal Testing Alternatives↗
PubMed1999

Mutations associated with in vivo aflatoxin B1-induced carcinogenesis need not be present in the in vitro transformations by this toxin.

Ingestion of aflatoxin B1 is implicated in the high incidence of human liver cancers in several developing countries. An association has been detected between human exposure to aflatoxins, and mutations in the third base of codon 249 of the p53 gene in hepatomas. In vitro experiments using human cell line cells and aflatoxin B1 have demonstrated the induction of p53 mutations in codon 249 and adjacent codons. It was therefore of interest to see if this correlation between the in vivo and in vitro situations held for other species. The present study examined a rat liver-derived cell line, transformed in vitro with aflatoxin B1, for the presence of mutations associated with in vivo aflatoxin-induced hepatocarcinogenesis. In an in vivo rodent model systems using the aflatoxin B1-sensitive male F344 rat, previous studies have shown that hepatocarcinogenesis is accompanied by significant incidences of codon 12 mutations in K-ras and codon 13 mutations in N-ras genes, but in contrast to the human, apparently not by mutations in codon 243 of the p53 gene (which corresponds to codon 249 in the human gene). In contrast to the situation in humans, mutation in the third base of codon 243 in the rat would not result in any changes in amino acid sequence, but mutations in codon 250, as seen in in vitro human systems, would be expressed in the rat p53 protein. In the present study, an immortalised, non-transformed liver epithelial cell line derived from a male F344 rat was transformed in vitro by aflatoxin B1 as demonstrated by tumour formation in nude mice. The transformation was dependent on metabolic activation of the aflatoxin B1. Transfection of DNA, extracted from these tumours, into NIH 3T3 fibroblasts conferred a stable, malignant transforming capacity. However, no mutations in codon 12 of the K-ras or codon 13 of the N-ras genes were detected in any of these tumours. These results indicate that in vitro transformation does not necessarily involve the same mutations, as those observed in vivo. Also, no mutations in codon 243 or adjacent codons of the p53 gene, paralleling those observed in the human cell line treated with aflatoxin B1, were detected. The results serve to emphasise the in vivo and in vitro variation in the oncogene activation in the same target organ or cell lines derived from that organ, even when using a single carcinogen activated by a known metabolic pathway.

3T3 Cells↗