Putting Science into Standards workshop on standards for organ-on-chip.

The European Commission Joint Research Centre and the European Standardization Organizations CEN and CENELEC organized the "Putting Science into Standards" workshop, focusing on organ-on-chip technologies. The workshop, held online on 28-29 April, 2021, aimed at identifying needs and priorities for standards development and suggesting possible ways forward.

Monitoring the microbial contamination of donor cornea during all preservation phases: A prospective study in the Eye Bank of Rome.

BACKGROUND: In most European eye banks, human donor corneas are microbiologically tested after storage in organ culture conditions, and the tissues that are free of contamination are distributed for transplantation. In this prospective study, 100 donor corneas were tested for microbial contamination after cold storage, corneal culture and corneal deswelling at the Eye Bank of Rome. METHODS: Samples of cold storage medium (EUSOL-C), corneal culture medium (TISSUE-C) and deswelling medium (CARRY-C) were tested after three, seven and one days of corneal storage, respectively. The CARRY-C medium, used to transport the cornea to the operation theatre, was retested 1 day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. RESULTS: We found 67% of the EUSOL-C samples were contaminated mainly by Staphylococcus spp, 14% of TISSUE-C media were contaminated by bacteria and fungi and 3% of CARRY-C media by Staphylococcus spp The analysis performed after removing the antimicrobial and antifungal agents showed growth in three additional TISSUE-C samples (S viridans, S haemolyticus and E faecalis) and one CARRY-C (S cerevisiae and P acnes). CONCLUSION: Tissue contamination was unexpectedly high on arrival to the eye bank, indicating the need to review and update decontamination procedures during tissue recovery, and renew training for the recovery teams. Storing donor corneas in organ culture conditions significantly reduced the microorganism burden. Using devices to remove antimicrobial and antifungal agents from samples before testing can increase the sensitivity of the standard microbiological method, and thus help further reduce the risk of microbial transmission.

[Establishment and application of gastric and gastric cancer organoids].

Gastric organoid is the organotypic cultures of gastric stem cells or pluripotent stem cells. Gastric organoid is comprised of all major types of gastric epithelial cells and represent the architecture and function remarkably similar to those of the gastric epithelium, faithfully recapitulating the functional gastric epithelium ex vivo. As ideal basic experimental model, gastric organoid has advantages over animal models and conventional cell model in many aspects. Gastric organoid derived from human gastric tissue, in particular, allows the investigation of the function of human stomach in the ex vivo setting. It has now been applied in the field of formation and physiology of the stomach, Helicobacter pylori infection-associated diseases, research of the pathogenic gene, screening and development of drugs, and regenerative medicine. What is more, as an innovative pre-clinical cancer model, gastric cancer organoid has provided important insights in the development of gastric cancer and screening of antitumor drugs, such as simulating the occurrence and development of gastric cancer, screening and development of antitumor drugs, personalized medication and targeted therapy for gastric cancer, and combined application with patient-derived xenograft. In this review, we summarize the establishment and application of gastric and gastric cancer organoids, especially in modeling gastric cancer, basic research and drug development.

The rise of three-dimensional human brain cultures.

Pluripotent stem cells show a remarkable ability to self-organize and differentiate in vitro in three-dimensional aggregates, known as organoids or organ spheroids, and to recapitulate aspects of human brain development and function. Region-specific 3D brain cultures can be derived from any individual and assembled to model complex cell-cell interactions and to generate circuits in human brain assembloids. Here I discuss how this approach can be used to understand unique features of the human brain and to gain insights into neuropsychiatric disorders. In addition, I consider the challenges faced by researchers in further improving and developing methods to probe and manipulate patient-derived 3D brain cultures.

Methodological standards for in vitro models of epilepsy and epileptic seizures. A TASK1-WG4 report of the AES/ILAE Translational Task Force of the ILAE.

In vitro preparations are a powerful tool to explore the mechanisms and processes underlying epileptogenesis and ictogenesis. In this review, we critically review the numerous in vitro methodologies utilized in epilepsy research. We provide support for the inclusion of detailed descriptions of techniques, including often ignored parameters with unpredictable yet significant effects on study reproducibility and outcomes. In addition, we explore how recent developments in brain slice preparation relate to their use as models of epileptic activity.

Better science with human cell-based organ and tissue models.

At present, animal-based models are the major test systems for assessing the tolerability and safety of chemical substances for regulatory purposes, and also for pivotal efficacy testing in pharmaceutical development. In spite of the high genetic similarity between many laboratory animals and humans, animal models are very poor predictors of human health effects and pathophysiological processes. Thus, models and testing strategies that are more relevant to human biology, are needed for these purposes. The best predictability is achieved with human organotypic models that mimic the microenvironment of human tissues. During their development, such models have to be characterised at the structural, genetic and functional levels, and compared to the respective human tissues. Their predictivity should be confirmed by using known reference chemicals with corresponding human data. The use of these methods in safety assessment and biomedical research, combined with the knowledge gained of the underlying biological processes on gene and protein expression, as well as on cellular signalling, will ultimately lead to better human science and animal welfare.

Efficacy of decontamination protocol by antimicrobial treatment in Iranian Tissue Bank (ITB).

Iranian Tissue Bank established in 1994 provides soft tissues for implantation in Iran. This study was designed to evaluate the efficacy of decontamination process of cardiac and soft tissues in Iranian Tissue Bank. In this bank after initial assessments, the tissues were incubated in a 5-antibiotic cocktail at room temperature for 24 h and then at 4 °C for 14 days. Contamination status was compared before and after antibiotic cocktail incubation. Of 3,315 assessed tissues, 1,057 were pericardia, 1,051 were fascia and 1,207 were other soft tissues including tibialis and aorta. The initial contamination rate was 36.86%. Pericardia showed the highest contamination rate. Klebsiella species was the most prevalent organism causing contamination. Decontamination rate after antibiotic incubation was 86.91% with the highest successful decontamination rate for fascia tissue. Klebsiella species was the major source of contamination in tissues that remained contaminated after antibiotic incubation. This may be due to resistance of this organism to applied antibiotics in the decontamination cocktail possibly due to a negative drug interaction between aminoglycoside and penicillin derivatives in this antibiotic cocktail. In conclusion collected data shows comparable efficacy of the decontamination process that is used in ITB compared with homograft banks of other countries.

LUMOR: an app for standardized control and monitoring of a porcine lung and its nutrient cycle.

The outcome of the EU-funded project ElBik has been the lung simulator 'iLung', which imitates an actively breathing human lung with a porcine lung. In order to keep the explanted lung in a nearly physiological state during transportation from the slaughterhouse to the ventilation laboratory the tissue needs to be nourished and temperature controlled. The Project AlveoPic designs a mobile transport vehicle implementing an ISO/IEEE 11073-20601 compliant communication interface for the exchange of the physical parameters, alert messages and setpoint-values. An appropriate 11073 domain information model is designed and limitations of the defined services and attributes are identified. For monitoring purposes the Android App LUMOR is implemented providing a user with an easy-to-handle GUI. It was found, that alert capabilities and remote set features are not well supported in ISO/IEEE 11073-20601 at the moment and possible workarounds are discussed.

A high-throughput template for optimizing Drosophila organ culture with response-surface methods.

The Drosophila wing imaginal disc is a key model organ for molecular developmental genetics. Wing disc studies are generally restricted to end-point analyses of fixed tissues. Recently several studies have relied on limited data from discs cultured in uncharacterized conditions. Systematic efforts towards developing Drosophila organ culture techniques are becoming crucial for further progress. Here, we have designed a multi-tiered, high-throughput pipeline that employs design-of-experiment methods to design a culture medium for wing discs. The resulting formula sustains high levels of proliferation for more than 12 hours. This approach results in a statistical model of proliferation as a function of extrinsic growth supplements and identifies synergies that improve insulin-stimulated growth. A more dynamic view of organogenesis emerges from the optimized culture system that highlights important facets of growth: spatiotemporal clustering of cell divisions and cell junction rearrangements. The same approach could be used to improve culture conditions for other organ systems.

Intestinal crypts reproducibly expand in culture.

BACKGROUND: In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro. METHODS AND MATERIALS: Crypts were harvested from 5 cm of proximal, middle, and distal small intestine of C57BL/6J mice aged 4 wk, 6-8 wk, 12-14 wk, and 18-20 wk (n = 4-6 animals) and cultured. For each region, we determined the efficiency of crypts forming enterospheres (day 1) and progressing to enteroids (day 7). Subsequently, enteroids were passaged and cryopreserved to determine if growth was changed by these manipulations. RESULTS: Forty-three to 99% of intestinal crypts formed enterospheres, with higher efficiency in proximal small intestine and in younger mice. Twenty-five to 64% of enterospheres progressed to budding enteroids within 7 d. In vitro expansion was greater in proximal enteroids. This expansion continued in a logarithmic fashion, with ≈ 97% replating efficiency of isolated enteroid crypt buds. Following cryopreservation, ≈ 90% of enteroids recovered normal proliferative capacity. CONCLUSIONS: Intestinal crypt culture is efficient and significantly expands intestinal tissue in a reproducible manner. Regional and age growth differences may reflect distinct stem cell characteristics or differences in support cells. The ability to culture and expand intestinal tissue in vitro provides a potential translational approach toward understanding and treating patients with short bowel syndrome.

Method of euthanasia affects amygdala plasticity in horizontal brain slices from mice.

An important consideration in any terminal experiment is the method used for euthanizing animals. Although the prime consideration is that the method is humane, some methods can have a dramatic impact on experimental outcomes. The standard inhalant anesthetic for experiments in brain slices is isoflurane, which replaced the flammable ethers used in the pioneer days of surgery. To our knowledge, there are no data available evaluating the effects of the method of euthanasia on plasticity changes in brain slices. Here, we compare the magnitude of long-term potentiation (LTP) and long-term depression (LTD) in the lateral nucleus of the amygdala (LA) after euthanasia following either ether or isoflurane anesthesia, as well as in mice decapitated without anesthesia. We found no differences in input-output curves using different methods of euthanasia. The LTP magnitude did not differ between ether and normal isoflurane anesthesia. After deep isoflurane anesthesia LTP induced by high frequency stimulation of cortical or intranuclear afferents was significantly reduced compared to ether anesthesia. In contrast to ether anesthesia and decapitation without anesthesia, the low frequency stimulation of cortical afferents induced a reliable LA-LTD after deep isoflurane anesthesia. Low frequency stimulation of intranuclear afferents only caused LTD after pretreatment with ether anesthesia. The results demonstrate that the method of euthanasia can influence brain plasticity for hours at least in the interface chamber. Therefore, the method of euthanasia is an important consideration when brain plasticity will be evaluated.

Effect of experimental temperature on the permeation of model diffusants across porcine buccal mucosa.

The influence of experimental temperature on the permeability of model diffusants across porcine buccal mucosa was investigated in vitro. The permeability increased significantly as the experimental temperature was increased in increments of approximately 7°C. It was observed that the apparent permeability and temperature were related by an exponential relationship that conformed to the Arrhenius equation. Diffusants with higher lipophilicities--buspirone and bupivacaine--had lower activation energies for diffusion when compared to hydrophilic diffusants--antipyrine and caffeine. The activation energy for diffusion of the model diffusants decreased linearly with increasing distribution coefficients across porcine buccal mucosa. The results suggested that the buccal mucosa acts as a stronger barrier to the diffusion of hydrophilic diffusants than the lipophilic ones. The log-linear relationship between permeability and temperature indicates that temperature should be carefully controlled in diffusion experiments. These results also point to the possibility of developing heat-generating buccal delivery devices, especially for hydrophobic diffusants.

Mussel-mimetic tissue adhesive for fetal membrane repair: a standardized ex vivo evaluation using elastomeric membranes.

OBJECTIVE: Iatrogenic preterm premature rupture of membranes (iPPROM), the main complication of invasive interventions in the prenatal period, seriously limits the benefit of diagnostic or surgical prenatal procedures. This study aimed to evaluate preventive plugging of punctured fetal membranes in an ex vivo situation using a new mussel-mimetic tissue adhesive (mussel glue) to inhibit leakage. METHODS: A novel biomechanical test device that tests the closure of injured membranes under near-physiological conditions was used. Mussel glue, a poly(ethylene glycol)-based hydrogel, was used to seal membrane defects of up to 3 mm in mechanically well-defined elastomeric membranes with three different degrees of stiffness. RESULTS: Elastomeric test membranes were successfully employed for testing mussel glue under well-defined conditions. Mussel glue plugs were distended by up to 94%, which translated to an improved sealing efficiency on elastomeric membranes with high stiffness. For the stiffest membrane tested, a critical burst pressure of 48 mbar (36 mmHg) was accomplished in this ex vivo setting. CONCLUSIONS: Mussel glue appears to efficiently seal membrane defects under well-standardized ex vivo conditions. As repaired membranes resist pressures measured in amniotic cavities, mussel glue might represent a novel sealing method for iatrogenic membrane defects.

A model of the isolated perfused rat small intestine.

Intestinal edema remains a serious clinical problem, and novel approaches to study its pathophysiology are needed. It was our aim to develop a long-term stable isolated perfused rat small bowel preparation permitting analysis of vascular, luminal, interstitial, and lymphatic compartments and to demonstrate the utility of this model by studying the effects of the proinflammatory mediator platelet-activating factor (PAF). A temperature-controlled chamber with an integrated balance was designed to perfuse isolated intestines through the mesenteric artery and the gut lumen. Steroids or oxygen carriers were not needed. Functional and morphological integrity of the tissue was preserved for several hours as confirmed by oxygen consumption, venous lactate-to-pyruvate ratio, arterial and venous pH, lactose digestion and galactose uptake, intravascular and luminal pressures, maintained fluid homeostasis, gut motility, and quantitative light microscopic analysis. Administration of PAF caused typical effects such as vasoconstriction, gut atony, and loss of galactose uptake. PAF also elicited a transient loss of 20% of the perfusate liquid from the mesenteric vascular bed, two-thirds of which were transferred to the lumen. All these responses were entirely reversible. This new model provides detailed insights into the physiology of the small intestine and will allow to study fundamental processes such as fluid homeostasis, barrier functions, transport mechanisms, and immune responses in this organ. Using this model, here we show a dramatic and yet reversible response of the rat small bowel to PAF, suggesting luminal water clearance as a novel safety factor in the intestine that may be of clinical relevance.

Organ culture preservation for corneal tissue. Technical and quality aspects.

INTRODUCTION: The technical and quality aspects of organ culture as a storage method for human donor corneas are described. MATERIALS AND METHODS: Data electronically stored since 1989 of > 41,000 corneas, processed in the Cornea Bank Amsterdam, are analysed. The technical information of eye banks collected in the Directory of the European Eye Bank Association (EEBA) is used as comparison. European Union (EU) directive for tissue banking and EEBA technical guidelines are references for the quality aspects. RESULTS: Organ culture allows the storage of donor corneas up to 4-5weeks. The storage phase is followed by a generally much shorter phase of 1-7 days, to reverse the corneal swelling occurring in the first phase and to transport the tissue to the clinic. Selection of the corneas based on inspection of the endothelium after storage as well as microbiological testing of the storage solution after a quarantine period are mandatory for this technique. General agreement exists about the outline of the method, but technical variations are applied to suit local circumstances and preferences of corneal surgeons. Agreement exists about a minimum endothelial cell count as selection criterion in case the donor endothelium is meant to be grafted. The use and cutoff points of other selection parameters for the cornea, e.g. the endothelial cell mosaic, are varying. According to EU regulations, a quality management system should be installed. This way each bank is able to issue a standardized product, while the production process is monitored with quality registrations. With the clinical outcome of the graft, the quality of the selection and storage procedures is verified. With the notification of adverse reactions such as primary graft failure and endophthalmitis, minimum risks will be assessed. CONCLUSION: The organ-cultured cornea is a well-documented product concerning microbiological safety and quality of the tissue. However, variations in performance and materials and no definite cut-off points for selection do not make an organ-cultured cornea a generally standardized product. The corneal surgeons have to ascertain themselves of the safety and quality of the followed procedure. It is up to an organization such as the EEBA to formulate tissue-specific additions to the EU regulations such as training opportunities, technical guidelines and criteria based on science.

Characterization of cultured thymus tissue used for transplantation with emphasis on promiscuous expression of thyroid tissue-specific genes.

Autoimmune thyroid disease occurs in some complete DiGeorge anomaly patients after thymus transplantation. This study was designed to assess the effect of culture of thymus tissue on the expression of genes involved in the development of autoimmunity. The expression of autoimmune regulator (AIRE), thyroglobulin (TG), thyroid peroxidase (TPO), and cytokeratin RNAs was examined in thymocytes and thymus tissue on the day of thymus harvest and after 14 and 21 days of culture. Immunohistochemistry was used to evaluate the cytokeratin expression in the thymus tissue. AIRE, TG, TPO, and cytokeratin mRNAs were found in harvest-day, 14-day and 21-day cultured tissues. Levels of AIRE, TG, and cytokeratin mRNAs were mostly higher after culture compared to expression on the harvest day, likely secondary to thymocyte depletion.

Pancreatic islet isolation variables in non-human primates (rhesus macaques).

BACKGROUND: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. METHODS: Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. RESULTS: In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. CONCLUSIONS: Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

The fundamentals of tissue engineering: new scaffolds.

The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.

Amiodarone inhibits the 3,5,3'-triiodothyronine-dependent increase of sodium/potassium adenosine triphosphatase activity and concentration in human atrial myocardial tissue.

In animal models the function of the sodium pump (sodium/potassium-adenosine triphosphatase [Na+/K(+)-ATPase]) is enhanced by 3,5,3'-triiodothyronine (T3) and inhibited by the antiarrhythmic agent amio. However, it is still unclear whether the effect of the drug on Na+/K(+)-ATPase depends on the interference with thyroid hormone action. We evaluated the interaction of T3 with amiodarone on Na+/K(+)-ATPase activity and site number in human myocardium. Right atrial slices were cultured with (T3+) and without (T3-) 3 nM T3 in presence and absence of amiodarone at therapeutical dose (1.5 microM). When compared to T3+, T3- preparations showed decreased 3H-ouabain binding (p < 0.004) and lower 20-minute and 45-minute 86Rb-uptake (p < or = 0.004). Amiodarone caused an average 49% reduction of the T3-dependent 3H-ouabain binding and decreased the Western blot signal for the Na+/K(+)-ATPase alpha1 subunit. The drug also inhibited T3-dependent increase in 86Rb-influx at 20 and 45 minutes by 66% and 42%, respectively, without affecting the affinity of the pump for K+. No differences were found in the 3H-ouabain binding and 86Rb-uptake of T3-, T3- amio and T3(+)-amio. In conclusion, T3 stimulates the Na+/K(+)-ATPase in human atrial myocardium by increasing the number of ouabain-binding sites, whereas amiodarone decreases the sodium pump function secondarily to the antagonism with thyroid hormone.

The fundamentals of tissue engineering: new scaffolds

The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.