Three dimensional characterization of tissue-engineered constructs by contrast enhanced nanofocus computed tomography.

peer reviewedIn order to successfully implement tissue engineered (TE) constructs as part of a clinical therapy, it is necessary to first develop and validate quality control tools that will ensure accurate and consistent TE construct release specifications. Hence advanced methods to monitor TE construct properties need to be further developed. In this study we showed proof of concept for contrast enhanced nanofocus computed tomography (CE-nanoCT) as a 'whole-construct' imaging technique with non-invasive potential that enables 3D visualization and quantification of in vitro engineered extracellular matrix (ECM) in TE constructs. In particular we performed a 3D quantitative and qualitative structural and spatial assessment of the in vitro engineered ECM, formed during static and perfusion bioreactor cell culture in 3D TE scaffolds, using two contrast agents, namely Hexabrix(R) and phosphotungstic acid (PTA). CE-nanoCT image data were validated by comparison to Live/Dead viability/cytotoxicity and picrosirius red staining data, and to the net dry weight of the TE constructs. When using Hexabrix(R) as contrast agent, ECM volume fitted linearly with net dry ECM weight independent from the flow rate used. When using PTA as contrast agent, CE-nanoCT data showed pronounced distinction between flow conditions when compared to both net dry weight and picrosirius red staining data although linearity was maintained, indicating culture-specific structural ECM differences. This was attributed to the binding specificity of this contrast agent. This novel type of information can contribute to optimize bioreactor culture conditions and potentially critical quality characteristics of TE constructs such as ECM quantity and homogeneity, facilitating the gradual transformation of 'TE constructs' in well characterized 'TE products'.I.P. is funded by the ENDEAVOUR project G.0982.11N of the FWO;M.S. is supported by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT/111457). G.K. and L.G. acknowledge support by the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC grant agreement n°279100

Interactive visualization of cell expansion process performance

As cell-based technologies are rapidly evolving beyond the laboratory scale, the demand for mass production of high quality cells is increasing. Unfortunately, only limited amounts of cells can be sourced from (human) donors. Therefore sequences of cell expansion steps are required to multiply the original number of cells taken from the donor biopsy to the amounts required for clinical application. Currently a large variety of expansion processes are used and described in literature. However, it is extremely difficult to compare them as many mesenchymal stem cell (MSC) subtypes are used in different culture vessels, with different medium compositions, etc. Moreover, adding to this variation in expansion strategies, within one process there can be significant fluctuations in outcome due to process variability and inherent donor-to-donor related variability. The aim of this work was to analyze the performance of a range of expansion processes for large-scale MSC expansion. Therefore a literature-based study was performed, currently resulting in a database of 73 individual cell expansion processes in 5 different types of culture vessels (microcarrier, (layered) flasks, hollow fiber-, multiplate-, and packed bed-bioreactor), 6 different types of MSCs and many different media compositions. The scale of the processes in terms of final cell numbers ranged between 7.5x106 and 1.1x1010 cells. An interactive process map was created where the scale, efficiency, cell type, culture method and load on downstream processing can be explored (see figure below). This interactive visualization tool provides an integrated perspective on the different culture processes and is able to increase the understanding on process comparability, attainable cell yield, scale-up strategies and the effect of certain critical process parameters on the expansion result. Please click Additional Files below to see the full abstract

Phenobarbital, Midazolam Pharmacokinetics, Effectiveness, and Drug-Drug Interaction in Asphyxiated Neonates Undergoing Therapeutic Hypothermia

Item does not contain fulltex

The Significance of Cell-related Challenges in the Clinical Application of Tissue Engineering.

Tissue engineering is increasingly being recognized as a new approach that could alleviate the burden of tissue damage currently managed with transplants or synthetic devices. Making this novel approach available in the future for patients who would potentially benefit is largely dependent on understanding and addressing all those factors that impede the translation of this technology to the clinic. Cell-associated factors in particular raise many challenges, including those related to cell sources, up- and downstream techniques, preservation, and the creation of in vitro microenvironments that enable cells to grow and function as far as possible as they would in vivo. This paper highlights the main confounding issues associated with cells in tissue engineering and how these issues may hinder the advancement of therapeutic tissue engineering. This article is protected by copyright. All rights reserved

Pharmacokinetics of morphine in encephalopathic neonates treated with therapeutic hypothermia

Objective Morphine is a commonly used drug in encephalopathic neonates treated with therapeutic hypothermia after perinatal asphyxia. Pharmacokinetics and optimal dosing of morphine in this population are largely unknown. The objective of this study was to describe pharmacokinetics of morphine and its metabolites morphine-3-glucuronide and morphine-6-glucuronide in encephalopathic neonates treated with therapeutic hypothermia and to develop pharmacokinetics based dosing guidelines for this population. Study design Term and near-term encephalopathic neonates treated with therapeutic hypothermia and receiving morphine were included in two multicenter cohort studies between 2008-2010 (SHIVER) and 2010-2014 (PharmaCool). Data were collected during hypothermia and rewarming, including blood samples for quantification of morphine and its metabolites. Parental informed consent was obtained for all participants. Results 244 patients (GA mean (sd) 39.8 (1.6) weeks, BW mean (sd) 3,428 (613) g, male 61.5%) were included. Morphine clearance was reduced under hypothermia (33.5 degrees C) by 6.89%/degrees C (95% CI 5.37%/degrees C-8.41%/degrees C, p<0.001) and metabolite clearance by 4.91%/degrees C (95% CI 3.53%/degrees C-6.22%/degrees C, p<0.001) compared to normothermia (36.5 degrees C). Simulations showed that a loading dose of 50 mu g/kg followed by continuous infusion of 5 mu g/kg/h resulted in morphine plasma concentrations in the desired range (between 10 and 40 mu g/L) during hypothermia. Conclusions Clearance of morphine and its metabolites in neonates is affected by therapeutic hypothermia. The regimen suggested by the simulations will be sufficient in the majority of patients. However, due to the large interpatient variability a higher dose might be necessary in individual patients to achieve the desired effect

Phenobarbital, midazolam pharmacokinetics, effectiveness, and drug-drug interaction in asphyxiated neonates undergoing therapeutic hypothermia

Background: Phenobarbital and midazolam are commonly used drugs in (near-)term neonates treated with therapeutic hypothermia for hypoxic-ischaemic encephalopathy, for sedation, and/or as anti-epileptic drug. Phenobarbital is an inducer of cytochrome P450 (CYP) 3A, while midazolam is a CYP3A substrate. Therefore, co-treatment with phenobarbital might impact midazolam clearance. Objectives: To assess pharmacokinetics and clinical anti-epileptic effectiveness of phenobarbital and midazolam in asphyxiated neonates and to develop dosing guidelines. Methods: Data were collected in the prospective multicentre PharmaCool study. In the present study, neonates treated with therapeutic hypothermia and receiving midazolam and/or phenobarbital were included. Plasma concentrations of phenobarbital and midazolam including its metabolites were determined in blood samples drawn on days 2–5 after birth. Pharmacokinetic analyses were performed using non-linear mixed effects modelling; clinical effectiveness was defined as no use of additional anti-epileptic drugs. Results: Data were available from 113 (phenobarbital) and 118 (midazolam) neonates; 68 were treated with both medications. Only clearance of 1-hydroxy midazolam was influenced by hypothermia. Phenobarbital co-administration increased midazolam clearance by a factor 2.3 (95% CI 1.9–2.9, p < 0.05). Anticonvulsant effectiveness was 65.5% for phenobarbital and 37.1% for add-on midazolam. Conclusions: Therapeutic hypothermia does not influence clearance of phenobarbital or midazolam in (near-)term neonates with hypoxic-ischaemic encephalopathy. A phenobarbital dose of 30 mg/kg is advised to reach therapeutic concentrations. Phenobarbital co-administration significantly increased midazolam clearance. Should phenobarbital be substituted by non-CYP3A inducers as first-line anticonvulsant, a 50% lower midazolam maintenance dose might be appropriate to avoid excessive exposure during the first days after birth. © 2019 The Author(s) Published by S. Karger AG, Base

Pharmacokinetics of morphine in encephalopathic neonates treated with therapeutic hypothermia

Objective Morphine is a commonly used drug in encephalopathic neonates treated with therapeutic hypothermia after perinatal asphyxia. Pharmacokinetics and optimal dosing of morphine in thi

Evaluation of a system-specific function to describe the pharmacokinetics of benzylpenicillin in term neonates undergoing moderate hypothermia

The pharmacokinetic (PK) properties of intravenous (i.v.) benzylpenicillin in term neonates undergoing moderate hypothermia after perinatal asphyxia were evaluated, as they have been unknown until now. A system-specific modeling approach was applied, in which our recently developed covariate model describing developmental and temperature-induced changes in amoxicillin clearance (CL) in the same patient study population was incorporated into a population PK model of benzylpenicillin with a priori birthweight (BW)-based allometric scaling. Pediatric population covariate models describing the developmental changes in drug elimination may constitute system-specific information and may therefore be incorporated into PK models of drugs cleared through the same pathway. The performance of this system-specific model was compared to that of a reference model. Furthermore, Monte-Carlo simulations were performed to evaluate the optimal dose. The systemspecific model performed as well as the reference model. Significant correlations were found between CL and postnatal age (PNA), gestational age (GA), body temperature (TEMP), urine output (UO; system-specific model), and multiorgan failure (reference model). For a typical patient with a GA of 40 weeks, BW of 3, 000 g, PNA of 2 days (TEMP, 33.5°C), and normal UO (2 ml/kg/h), benzylpenicillin CL was 0.48 liter/h (interindividual variability [IIV] of 49%) and the volume of distribution of the central compartment was 0.62 liter/kg (IIV of 53%) in the system-specific model. Based on simulations, we advise a benzylpenicillin i.v. dose regimen of 75, 000 IU/kg/day every 8 h (q8h), 150, 000 IU/kg/day q8h, and 200, 000 IU/kg/day q6h for patients with GAs of 36 to 37 weeks, 38 to 41 weeks, and ≥42 weeks, respectively. Thesystem-specific model may be used for other drugs cleared through the same pathway accelerating model development

Limitations on Concept Formation of Person

status: publishe

The atoms of person: Limitations on concept formation

status: publishe