Enhancing pre-clinical research with simplified intestinal cell line models.

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.

Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease.

BACKGROUND: The function of the blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the associated molecular mechanisms, particularly with respect to the high-risk APOE4/4 genotype, are not well understood. For this purpose, we developed a multicellular isogenic model of the neurovascular unit (NVU) based on human induced pluripotent stem cells. METHODS: The human NVU was modeled in vitro using isogenic co-cultures of astrocytes, brain capillary endothelial-like cells (BCECs), microglia-like cells, neural stem cells (NSCs), and pericytes. Physiological and pathophysiological properties were investigated as well as the influence of each single cell type on the characteristics and function of BCECs. The barriers established by BCECs were analyzed for specific gene transcription using high-throughput quantitative PCR. RESULTS: Co-cultures were found to tighten the barrier of BCECs and alter its transcriptomic profile under both healthy and disease conditions. In vitro differentiation of brain cell types that constitute the NVU was not affected by the LOAD background. The supportive effect of NSCs on the barrier established by BCECs was diminished under LOAD conditions. Transcriptomes of LOAD BCECs were modulated by different brain cell types. NSCs were found to have the strongest effect on BCEC gene regulation and maintenance of the BBB. Co-cultures showed cell type-specific functional contributions to BBB integrity under healthy and LOAD conditions. CONCLUSIONS: Cell type-dependent transcriptional effects on LOAD BCECs were identified. Our study suggests that different brain cell types of the NVU have unique roles in maintaining barrier integrity that vary under healthy and LOAD conditions. .

Challenges in Permeability Assessment for Oral Drug Product Development.

Drug permeation across the intestinal epithelium is a prerequisite for successful oral drug delivery. The increased interest in oral administration of peptides, as well as poorly soluble and poorly permeable compounds such as drugs for targeted protein degradation, have made permeability a key parameter in oral drug product development. This review describes the various in vitro, in silico and in vivo methodologies that are applied to determine drug permeability in the human gastrointestinal tract and identifies how they are applied in the different stages of drug development. The various methods used to predict, estimate or measure permeability values, ranging from in silico and in vitro methods all the way to studies in animals and humans, are discussed with regard to their advantages, limitations and applications. A special focus is put on novel techniques such as computational approaches, gut-on-chip models and human tissue-based models, where significant progress has been made in the last few years. In addition, the impact of permeability estimations on PK predictions in PBPK modeling, the degree to which excipients can affect drug permeability in clinical studies and the requirements for colonic drug absorption are addressed.

Biomimetic Connection of Transcutaneous Implants with Skin.

Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.

Locked Out: Phoenixin-14 Does Not Cross a Stem-Cell-Derived Blood-Brain Barrier Model.

Phoenixin-14 is a recently discovered peptide regulating appetite. Interestingly, it is expressed in the gastrointestinal tract; however, its supposed receptor, GPR173, is predominantly found in hypothalamic areas. To date, it is unknown how peripherally secreted phoenixin-14 is able to reach its centrally located receptor. To investigate whether phoenixin is able to pass the blood-brain barrier, we used an in vitro mono-culture blood-brain barrier (BBB) model consisting of brain capillary-like endothelial cells derived from human induced-pluripotent stem cells (hiPSC-BCECs). The passage of 1 nMol and 10 nMol of phoenixin-14 via the mono-culture was measured after 30, 60, 90, 120, 150, 180, 210, and 240 min using a commercial ELISA kit. The permeability coefficients (PC) of 1 nMol and 10 nMol phoenixin-14 were 0.021 ± 0.003 and 0.044 ± 0.013 µm/min, respectively. In comparison with the PC of solutes known to cross the BBB in vivo, those of phoenixin-14 in both concentrations are very low. Here, we show that phoenixin-14 alone is not able to cross the BBB, suggesting that the effects of peripherally secreted phoenixin-14 depend on a co-transport mechanism at the BBB in vivo. The mechanisms responsible for phoenixin-14's orexigenic property along the gut-brain axis warrant further research.

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models.

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)-derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection.

Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.

Correction: A forty-year review of Rocky Mountain spotted fever cases in California shows clinical and epidemiologic changes.

[This corrects the article DOI: 10.1371/journal.pntd.0010738.].

A Human Stem Cell-Derived Brain-Liver Chip for Assessing Blood-Brain-Barrier Permeation of Pharmaceutical Drugs.

Significant advancements in the field of preclinical in vitro blood-brain barrier (BBB) models have been achieved in recent years, by developing monolayer-based culture systems towards complex multi-cellular assays. The coupling of those models with other relevant organoid systems to integrate the investigation of blood-brain barrier permeation in the larger picture of drug distribution and metabolization is still missing. Here, we report for the first time the combination of a human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier model with a cortical brain and a liver spheroid model from the same donor in a closed microfluidic system (MPS). The two model compounds atenolol and propranolol were used to measure permeation at the blood-brain barrier and to assess metabolization. Both substances showed an in vivo-like permeation behavior and were metabolized in vitro. Therefore, the novel multi-organ system enabled not only the measurement of parent compound concentrations but also of metabolite distribution at the blood-brain barrier.

Potential Effects of Environmental Conditions on Prairie Dog Flea Development and Implications for Sylvatic Plague Epizootics.

Fleas are common ectoparasites of vertebrates worldwide and vectors of many pathogens causing disease, such as sylvatic plague in prairie dog colonies. Development of fleas is regulated by environmental conditions, especially temperature and relative humidity. Development rates are typically slower at low temperatures and faster at high temperatures, which are bounded by lower and upper thresholds where development is reduced. Prairie dogs and their associated fleas (mostly Oropsylla spp) live in burrows that moderate outside environmental conditions, remaining cooler in summer and warmer in winter. We found burrow microclimates were characterized by stable daily temperatures and high relative humidity, with temperatures increasing from spring through summer. We previously showed temperature increases corresponded with increasing off-host flea abundance. To evaluate how changes in temperature could affect future prairie dog flea development and abundance, we used development rates of O. montana (a species related to prairie dog fleas), determined how prairie dog burrow microclimates are affected by ambient weather, and combined these results to develop a predictive model. Our model predicts burrow temperatures and flea development rates will increase during the twenty-first century, potentially leading to higher flea abundance and an increased probability of plague epizootics if Y. pestis is present.

A forty-year review of Rocky Mountain spotted fever cases in California shows clinical and epidemiologic changes.

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease documented in North, Central, and South America. In California, RMSF is rare; nonetheless, recent fatal cases highlight ecological cycles of the two genera of ticks, Dermacentor and Rhipicephalus, known to transmit the disease. These ticks occur in completely different habitats (sylvatic and peridomestic, respectively) resulting in different exposure risks for humans. This study summarizes the demographic, exposure, and clinical aspects associated with the last 40 years of reported RMSF cases to the California Department of Public Health (CDPH). Seventy-eight RMSF cases with onsets from 1980 to 2019 were reviewed. The incidence of RMSF has risen in the last 20 years from 0.04 cases per million to 0.07 cases per million (a two-fold increase in reports), though the percentage of cases that were confirmed dropped significantly from 72% to 25% of all reported cases. Notably, Hispanic/Latino populations saw the greatest rise in incidence. Cases of RMSF in California result from autochthonous and out-of-state exposures. During the last 20 years, more cases reported exposure in Southern California or Mexico than in the previous 20 years. The driver of these epidemiologic changes is likely the establishment and expansion of Rhipicephalus sanguineus sensu lato ticks in Southern California and on-going outbreaks of RMSF in northern Mexico. Analysis of available electronically reported clinical data from 2011 to 2019 showed that 57% of reported cases presented with serious illness requiring hospitalization with a 7% mortality. The difficulty in recognizing RMSF is due to a non-specific clinical presentation; however, querying patients on the potential of tick exposure in both sylvatic and peridomestic environments may facilitate appropriate testing and treatment.

Establishment of the SIS scaffold-based 3D model of human peritoneum for studying the dissemination of ovarian cancer.

Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer.

Detection and Establishment of Aedes notoscriptus (Diptera: Culicidae) Mosquitoes in Southern California, United States.

Aedes notoscriptus (Skuse), the Australian backyard mosquito, is a pestiferous daytime-biting species native to Australia and the surrounding southwestern Pacific region. It is suspected to play a role in the transmission of several arboviruses and is considered a competent vector of dog heartworm, Dirofilaria immitis (Leidy). This highly adaptable mosquito thrives in natural and artificial water-holding containers in both forested and urbanized areas, from tropical to temperate climates, and has benefitted from a close association with humans, increasing in abundance within its native range. It invaded and successfully established in New Zealand as well as in previously unoccupied temperate and arid regions of Australia. Ae. notoscriptus was discovered in Los Angeles County, CA, in 2014, marking the first time this species had been found outside the southwestern Pacific region. By the end of 2019, immature and adult mosquitoes had been collected from 364 unique locations within 44 cities spanning three southern California counties. The discovery, establishment, and rapid spread of this species in urban areas may signal the global movement and advent of a new invasive container-inhabiting species. The biting nuisance, public health, and veterinary health implications associated with the invasion of southern California by this mosquito are discussed.

Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity.

Lipids are the most energy-dense components of the diet, and their overconsumption promotes obesity and diabetes. Dietary fat content has been linked to the lipid processing activity by the intestine and its overall capacity to absorb triglycerides (TG). However, the signaling cascades driving intestinal lipid absorption in response to elevated dietary fat are largely unknown. Here, we describe an unexpected role of the protein kinase D2 (PKD2) in lipid homeostasis. We demonstrate that PKD2 activity promotes chylomicron-mediated TG transfer in enterocytes. PKD2 increases chylomicron size to enhance the TG secretion on the basolateral side of the mouse and human enterocytes, which is associated with decreased abundance of APOA4. PKD2 activation in intestine also correlates positively with circulating TG in obese human patients. Importantly, deletion, inactivation, or inhibition of PKD2 ameliorates high-fat diet-induced obesity and diabetes and improves gut microbiota profile in mice. Taken together, our findings suggest that PKD2 represents a key signaling node promoting dietary fat absorption and may serve as an attractive target for the treatment of obesity.

Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity.

Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.

Enteroids Generated from Patients with Severe Inflammation in Crohn's Disease Maintain Alterations of Junctional Proteins.

BACKGROUND: The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn's disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients' specimens. METHODS: Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. RESULTS: Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. CONCLUSIONS: These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

Bacterial nanocellulose as novel carrier for intestinal epithelial cells in drug delivery studies.

Synthetic cell carriers (A) represent common scaffold structures for the development of cell-based in vitro models of the human intestine but due to their low porosity or unwanted molecular adhesion effects, synthetic carriers can negatively affect cell function. Alternative scaffolds such as natural extracellular matrices (ECMs) (B) were shown to overcome some of the common drawbacks. However, their fabrication is time-consuming, less well standardized and not entirely conform to the 3R principle (replacement, reduction, refinement). Nowadays, biopolymers such as bacterial nanocellulose (BNC) (C) represent interesting scaffold materials for innovative tissue engineering concepts, as they can be generated in a faster and more standardized process workflow without the need for animal material. In this study, we demonstrate the BNC as suitable carrier for the development of Caco-2-based in vitro models of the human intestine. The BNC-based models exhibit organ-specific properties comprising typical cellular morphologies, characteristic protein expression profiles, representative ultrastructural features and the formation of a tight epithelial barrier. The proof of in vivo-like transport activities further validates the high quality of the BNC-based Caco-2 models. In summary, this illustrates the BNC as alternative bioscaffold of non-animal origin to develop functional organ models in vitro.

Matrix decoded - A pancreatic extracellular matrix with organ specific cues guiding human iPSC differentiation.

The extracellular matrix represents a dynamic microenvironment regulating essential cell functions in vivo. Tissue engineering approaches aim to recreate the native niche in vitro using biological scaffolds generated by organ decellularization. So far, the organ specific origin of such scaffolds was less considered and potential consequences for in vitro cell culture remain largely elusive. Here, we show that organ specific cues of biological scaffolds affect cellular behavior. In detail, we report on the generation of a well-preserved pancreatic bioscaffold and introduce a scoring system allowing standardized inter-study quality assessment. Using multiple analysis tools for in-depth-characterization of the biological scaffold, we reveal unique compositional, physico-structural, and biophysical properties. Finally, we prove the functional relevance of the biological origin by demonstrating a regulatory effect of the matrix on multi-lineage differentiation of human induced pluripotent stem cells emphasizing the significance of matrix specificity for cellular behavior in artificial microenvironments.