Cryopreservation of Precision-cut Tissue Slices for Application in Drug Metabolism Research

The research described in this thesis had two important aims. The first was to determine whether tissue slices could be used as an in vitro tool to predict the in vivo metabolism of new drugs. The second aim was to find a manner to store tissue slices for longer time periods by cryopreservation. It was found that liver slices better than liver homogenate and microsomes predicted the in vivo metabolite pattern of drugs. One of the three drugs studied in this thesis, however, was found to be metabolized extra-hepatically and studying metabolism with liver slices alone gave an incomplete view of in vivo metabolism. The most important in vivo metabolite of the latter compound was found to be formed in great amounts by intestinal slices. We proposed a rough manner to combine the metabolite pattern produced by both liver and extra-hepatical slices to be able to use tissue slices in a prospective study for the prediction of in vivo drug metabolism. It was shown that in this manner, the in vivo metabolite pattern of the tested drugs was rightfully predicted. For cryopreservation of tissue slices, both classical equilibrium freezing and vitrification methods were used. Both these methods tend to prevent the formation of potentially damaging intracellular ice. Besides, slices were frozen by rapid freezing. To test the viability of cryopreserved slices, several end-points were used. It was found that slice histomorphology, potassium and ATP content and phase II biotransformation were very sensitive to cryopreservation damage. Viability of liver slices, determined with these end-points, was successfully preserved by rapid freezing, despite of the fact that intra- and extracellular ice formation was not prevented. The liver slices did not survive equilibrium slow freezing. It was suggested that by rapid freezing and thawing small ice crystals are formed that can be resisted by the cells under certain conditions. This hypothesis was supported by the fact that when the rapidly frozen liver slices were warmed slowly, allowing small ice crystals to grow, the slices lost viability. With equilibrium slow freezing, intracellular ice crystal formation was probably prevented, but large ice crystals were allowed to form between the cells in the slices, damaging the cells from the outside. Rapid freezing did not preserve viability of kidney and intestinal slices. Vitrification prevents the formation of damaging intra- and extracellular ice crystals by inducing the formation of a glass instead of ice. However, the toxicity of the highly concentrated CPAs required for vitrification was found to be a major concern. We showed that these problems could be overcome for a great part by using mixtures of CPAs like VM3 and VS4 and by adequately designing protocols for impregnation and out-washing of these compounds. Kidney and liver slices impregnated with VM3 and vitrified by slow cooling were found to survive cryopreservation. Therefore, a major conclusion of this thesis was that from a future perspective, vitrification as a starting point offers the highest chance to develop universal cryopreservation methods for tissue slices. In conclusion, the results of the research described in this thesis indicate that organ slices can be vitrified to form a tissue slice bank from which they can be derived for in vitro drug metabolism studies, serving the reduction of laboratory animals use for drug safety studies and facilitating the use of human organ material

Early recovery after endoscopic totally extraperitoneal (TEP) hernia repair in athletes with inguinal disruption:A prospective cohort study

Background Groin pain is a common problem in athletes which results in loss of playing time. Moreover, it can be for the cause of athletic career termination. A common cause of groin pain in athletes is inguinal disruption; pain in the groin area near the pubic tubercle were no obvious other pathology exists to explain the symptoms. Aim of this study was to evaluate the effect of endoscopic totally extraperitoneal (TEP) hernia repair in athletes with inguinal disruption. Methods Thirty-one athletes with chronic groin pain due to inguinal disruption, who had undergone conservative therapy without any effect, were included in this prospective cohort study. Prior to surgery patients were assessed by clinical examination, ultrasound of the inguinal region, x-ray and a radionuclide bone scan with single photon-emission computed tomography and CT (SPECT-CT). TEP hernia repair was performed and a lightweight polypropylene mesh was placed pre-peritoneally. Additionally the athletes' perception about their groin disability was assessed preoperatively and 6 weeks postoperatively by means of the Hip and Groin Outcome Score (HAGOS). The HAGOS consists of six subscales: Pain, Symptoms, Physical function in daily living, Physical function in Sport and Recreation, Participation in Physical Activities, and hip and/or groin-related Quality of Life. Results No complications occurred during and after surgery. After six weeks patients improved in all the separate subscales of the Hip and Groin Outcome Score (HAGOS). Within 6 weeks of surgery, 26 patients (84%) returned to sports activities with no or less groin pain. Conclusions This study showed that endoscopic totally extraperitoneal (TEP) hernia repair is an effective surgical treatment of inguinal disruption in athletes with chronic groin pain

Precision-cut intestinal slices:alternative model for drug transport, metabolism, and toxicology research

INTRODUCTION: The absorption, distribution, metabolism, excretion and toxicity (ADME-tox) processes of drugs are of importance and require preclinical investigation intestine in addition to the liver. Various models have been developed for prediction of ADME-tox in the intestine. In this review, precision-cut intestinal slices (PCIS) are discussed and highlighted as model for ADME-tox studies. Areas covered: This review provides an overview of the applications and an update of the most recent research on PCIS as an ex vivo model to study the transport, metabolism and toxicology of drugs and other xenobiotics. The unique features of PCIS and the differences with other models as well as the translational aspects are also discussed. Expert opinion: PCIS are a simple, fast, and reliable ex vivo model for drug ADME-tox research. Therefore, PCIS are expected to become an indispensable link in the in vitro-ex vivo-in vivo extrapolation, and a bridge in translation of animal data to the human situation. In the future, this model may be helpful to study the effects of interorgan interactions, intestinal bacteria, excipients and drug formulations on the ADME-tox properties of drugs. The optimization of culture medium and the development of a (cryo)preservation technique require more research

Exploring Porcine Precision-Cut Kidney Slices as a Model for Transplant-Related Ischemia-Reperfusion Injury

Marginal donor kidneys are more likely to develop ischemia-reperfusion injury (IRI), resulting in inferior long-term outcomes. Perfusion techniques are used to attenuate IRI and improve graft quality. However, machine perfusion is still in its infancy, and more research is required for optimal conditions and potential repairing therapies. Experimental machine perfusion using porcine kidneys is a great way to investigate transplant-related IRI, but these experiments are costly and time-consuming. Therefore, an intermediate model to study IRI would be of great value. We developed a precision-cut kidney slice (PCKS) model that resembles ischemia-reperfusion and provides opportunities for studying multiple interventions simultaneously. Porcine kidneys were procured from a local slaughterhouse, exposed to 30 min of warm ischemia, and cold preserved. Subsequently, PCKS were prepared and incubated under various conditions. Adenosine triphosphate (ATP) levels and histological tissue integrity were assessed for renal viability and injury. Slicing did not influence tissue viability, and PCKS remained viable up to 72 h incubation with significantly increased ATP levels. Hypothermic and normothermic incubation led to significantly higher ATP levels than baseline. William’s medium E supplemented with Ciprofloxacin (and Amphotericin-B) provided the most beneficial condition for incubation of porcine PCKS. The porcine PCKS model can be used for studying transplant IR

Growth factors of stem cell niche extend the life-span of precision-cut intestinal slices in culture:A proof-of-concept study

Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96 h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24 h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others

Rifampicin Induces Gene, Protein, and Activity of P-Glycoprotein (ABCB1) in Human Precision-Cut Intestinal Slices

P-glycoprotein (ABCB1), an ATP-binding cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug–drug interactions. Drug-mediated induction of intestinal ABCB1 is a clinically relevant phenomenon associated with significantly decreased drug bioavailability. Currently, there are no well-established human models for evaluating its induction, so drug regulatory authorities provide no recommendations for in vitro/ex vivo testing drugs’ ABCB1-inducing activity. Human precision-cut intestinal slices (hPCISs) contain cells in their natural environment and express physiological levels of nuclear factors required for ABCB1 induction. We found that hPCISs incubated in William’s Medium E for 48 h maintained intact morphology, ATP content, and ABCB1 efflux activity. Here, we asked whether rifampicin (a model ligand of pregnane X receptor, PXR), at 30 μM, induces functional expression of ABCB1 in hPCISs over 24- and 48-h incubation (the time to allow complete induction to occur). Rifampicin significantly increased gene expression, protein levels, and efflux activity of ABCB1. Moreover, we described dynamic changes in ABCB1 transcript levels in hPCISs over 48 h incubation. We also observed that peaks of induction are achieved among donors at different times, and the extent of ABCB1 gene induction is proportional to PXR mRNA levels in the intestine. In conclusion, we showed that hPCISs incubated in conditions comparable to those used for inhibition studies can be used to evaluate drugs’ ABCB1-inducing potency in the human intestine. Thus, hPCISs may be valuable experimental tools that can be prospectively used in complex experimental evaluation of drug–drug interactions

Rat precision-cut liver slices predict drug-induced cholestatic injury

Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 μM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC

Prenatal smoke exposure induces persistent Cyp2a5 methylation and increases nicotine metabolism in the liver of neonatal and adult male offspring

Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life

Structural Validity of the Short Musculoskeletal Function Assessment in Patients With Injuries

Background. The Short Musculoskeletal Function Assessment (SMFA) is a widely used patient-reported outcome measure, originally having 2 elements of outcome: the function index and the bother index. In multiple studies, it has been argued that the SMFA should be scored using 3, 4, or 6 subscales instead. Therefore, there is inconsistency with the number of underlying dimensions of the SMFA. Objective. The aim of this study was to evaluate the structural validity of the various proposed subscale configurations of the SMFA in a broad range of Dutch patients with injuries. Design. This study used a prospective cohort design. Methods. Participants with injuries were asked to complete the Dutch SMFA (SMFA-NL) at 5 to 8 weeks postinjury. The structural validity of the 6 different factor structures that have been proposed in other studies was evaluated using confirmatory factor analyses. Internal consistency was analyzed using Cronbach alpha. Results. A total of 491 patients participated (response rate = 74%). A 4-factor structure showed an acceptable fit (root-mean-square error of approximation [RMSEA] = 0.070, comparative fit index = 0.973, Tucker-Lewis index = 0.971). Other models, including the original 2-index structure, showed insufficient structural validity in Dutch patients with injuries. The 4-factor structure showed sufficient discriminant validity and good internal consistency (Cronbach alpha = 0.83). Limitations. It is unclear whether conclusions are generalizable across different countries, people who are elderly, and people without injuries. Conclusion. In a broad range of patients with injuries, the SMFA-NL may be best scored and interpreted using a 4-factor structure. Other factor structures showed insufficient structural validity