Biocompatible micro-sized cell culture chamber for the detection of nanoparticle-induced IL8 promoter activity on a small cell population

In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds

Neuro-Muscular Differentiation of Adult Porcine Skin Derived Stem Cell-Like Cells

BACKGROUND: Due to the genetic relationship to humans, porcine stem cells are a very important model system to investigate cell differentiation, associated cell signaling pathways, and cell fate. Porcine skin derived stem cells have been isolated from mid-gestation porcine fetus recently. To our knowledge, stem cells from the skin of the adult porcine organism have not been isolated until now. Hence, to our knowledge, we here describe the isolation, expansion, characterization and differentiation of multipotent porcine skin derived stem cell-like cells (pSSCs) from the adult porcine organism for the first time. METHODOLOGY/PRINCIPAL FINDINGS: pSSCs had a spindle shaped morphology similar to mesenchymal stem cells (MSCs). They could be maintained proliferatively active in vitro for more than 120 days and were able to form colonies from single cells. pSSCs expressed Sox2 and Oct3/4, both transcription factors essential to the pluripotent and self-renewing phenotypes of embryonic stem cells, which recently gained attention due to their function in inducing pluripotent stem cells. Furthermore, the expression of the progenitor marker nestin, the somatic stem cell markers Bcrp1/ABCG2, Bmi1, and Stat3 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in undifferentiated pSSCs. Flow cytometry revealed the expression of the MSC related proteins CD9, CD29, CD44 and CD105, but not CD90. After neuronal differentiation cells with a characteristic morphology of neuronal and smooth muscle-like cells were present in the cultures. Subsequent immunochemistry and flow cytometry revealed the down-regulation of nestin and the up-regulation of the neuron specific protein beta-III-tubulin and the astrocyte marker GFAP. Also, alpha-SMA expressing cells increased during differentiation suggesting the neuro-muscular differentiation of these skin derived cells. pSSCs could also be induced to differentiate into adipocyte-like cells when cultured under specific conditions. CONCLUSIONS/SIGNIFICANCE: Adult porcine skin harbors a population of stem cell-like cells (pSSCs) that can be isolated via enzymatic digestion. These pSSCs show characteristic features of MSCs originated in other tissues and express the embryonic stem cell marker Oct3/4, Sox2, and Stat3. Furthermore, pSSCs have the potential to differentiate into cells from two different germ lines, the ectoderm (neurons, astrocytes) and the mesoderm (smooth muscle cells, adipocytes)

Synthesis and in vitro evaluation of cyclodextrin hyaluronic acid conjugates as a new candidate for intestinal drug carrier for steroid hormones

Steroid hormones became increasingly interesting as active pharmaceutical ingredients for the treatment of endocrine disorders. However, medical applications of many steroidal drugs are inhibited by their very low aqueous solubilities giving rise to low bioavailabilities. Therefore, the prioritized oral administration of steroidal drugs remains problematic. Cyclodextrins are promising candidates for the development of drug delivery systems for oral route applications, since they solubilize hydrophobic steroids and increase their rate of transport in aqueous environments. In this study, the synthesis and characterization of polymeric β-cyclodextrin derivates is described, which result from the attachment of a hydrophilic β-CD-thioether to hyaluronic acid. Host-guest complexes of the synthesized β-cyclodextrin hyaluronic acid conjugates were formed with two poorly soluble model steroids (β-estradiol, dexamethasone) and compared to monomeric β-cyclodextrin derivates regarding solubilization and complexation efficiency. The β-cyclodextrin-drug (host-guest) complexes were evaluated in vitro for their suitability (cytotoxicity and transport rate) as intestinal drug carriers for steroid hormones. In case of β-estradiol, higher solubilities could be achieved by complexation with both synthesized β-cyclodextrin derivates, leading to significantly higher intestinal transport rates in vitro. However, this success could not be shown for dexamethasone, which namely solubilized better, but could not enhance the transport rate significantly. Thus, this study demonstrates the biocompatibility of the synthesized and characterized β-cyclodextrin derivates and shows their potential as new candidate for intestinal drug carrier for steroid hormones like β-estradiol

Nanoparticulate Transport of Oximes over an In Vitro Blood-Brain Barrier Model

Background: Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB. Methodology/Principal Findings: In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes. Conclusions/Significance: With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulation

Influence of Physicochemical Characteristics and Stability of Gold and Silver Nanoparticles on Biological Effects and Translocation across an Intestinal Barrier—A Case Study from In Vitro to In Silico

A better understanding of their interaction with cell-based tissue is a fundamental prerequisite towards the safe production and application of engineered nanomaterials. Quantitative experimental data on the correlation between physicochemical characteristics and the interaction and transport of engineered nanomaterials across biological barriers, in particular, is still scarce, thus hampering the development of effective predictive non-testing strategies. Against this background, the presented study investigated the translocation of gold and silver nanoparticles across the gastrointestinal barrier along with related biological effects using an in vitro 3D-triple co-culture cell model. Standardized in vitro assays and quantitative polymerase chain reaction showed no significant influence of the applied nanoparticles on both cell viability and generation of reactive oxygen species. Transmission electron microscopy indicated an intact cell barrier during the translocation study. Single particle ICP-MS revealed a time-dependent increase of translocated nanoparticles independent of their size, shape, surface charge, and stability in cell culture medium. This quantitative data provided the experimental basis for the successful mathematical description of the nanoparticle transport kinetics using a non-linear mixed effects modeling approach. The results of this study may serve as a basis for the development of predictive tools for improved risk assessment of engineered nanomaterials in the future

Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier

Experimental Comparison of Primary and hiPS-Based In Vitro Blood–Brain Barrier Models for Pharmacological Research

In vitro model systems of the blood–brain barrier (BBB) play an essential role in pharmacological research, specifically during the development and preclinical evaluation of new drug candidates. Within the past decade, the trend in research and further development has moved away from models based on primary cells of animal origin towards differentiated models derived from human induced pluripotent stem cells (hiPSs). However, this logical progression towards human model systems from renewable cell sources opens up questions about the transferability of results generated in the primary cell models. In this study, we have evaluated both models with identical experimental parameters and achieved a directly comparable characterisation showing no significant differences in protein expression or permeability even though the achieved transendothelial electrical resistance (TEER) values showed significant differences. In the course of this investigation, we also determined a significant deviation of both model systems from the in vivo BBB circumstances, specifically concerning the presence or absence of serum proteins in the culture media. Thus, we have further evaluated both systems when confronted with an in vivo-like distribution of serum and found a notable improvement in the differential permeability of hydrophilic and lipophilic compounds in the hiPS-derived BBB model. We then transferred this model into a microfluidic setup while maintaining the differential serum distribution and evaluated the permeability coefficients, which showed good comparability with values in the literature. Therefore, we have developed a microfluidic hiPS-based BBB model with characteristics comparable to the established primary cell-based model

A novel microfluidic mucus-chip for studying the permeation of compounds over the mucus barrier

A major challenge of drug discovery is to overcome the mucus barrier, which is lined the surface of the gastrointestinal tract. The ability to overcome this barrier must be tested in preclinical in vitro models, prior to in vivo studies. Therefore, a microfluidic chip was developed to examine the possibility of compounds to overcome a mucus barrier. Native porcine intestinal mucus was used as a mucus model. The mucus was loaded inside the chip comprising of a microporous membrane and a fluidic compartment. To validate the system, caffeine as a hydrophilic positive compound, fluorescein isothiocyanate–dextran (FITC-dextran) as high molecular negative compound and diclofenac sodium as poorly permeating compound were tested. Test samples were applied on the surface of the mucus and samples were collected every 30 min from the fluidic compartment underneath the membrane. The permeation was measured over 3 h and the permeated compound was detected by high-pressure liquid chromatography (HPLC) and a microplate fluorescence reader. The results show that caffeine and diclofenac sodium are permeating over the mucus layer proportionally to the time. FITC-dextran, chosen as non-permeating compound, displays equivalently no permeation. The validation provides the functionality of the developed mucus-chip system

Tyramine-conjugated alginate hydrogels as a platform for bioactive scaffolds

Alginate‐based hydrogels represent promising microenvironments for cell culture and tissue engineering, as their mechanical and porous characteristics are adjustable toward in vivo conditions. However, alginate scaffolds are bioinert and thus inhibit cellular interactions. To overcome this disadvantage, bioactive alginate surfaces were produced by conjugating tyramine molecules to high‐molecular‐weight alginates using the carbodiimide chemistry. Structural elucidation using nuclear magnetic resonance spectroscopy and contact angle measurements revealed a surface chemistry and wettability of tyramine‐alginate hydrogels similar to standard cell culture treated polystyrene. In contrast to stiff cell culture plastic, tyramine‐alginate scaffolds were found to be soft (60–80 kPa), meeting the elastic moduli of human tissues such as liver and heart. We further demonstrated an enhanced protein adsorption with increasing tyramine conjugation, stable for several weeks. Cell culture studies with human mesenchymal stem cells and human pluripotent stem cell‐derived cardiomyocytes qualified tyramine‐alginate hydrogels as bioactive platforms enabling cell adhesion and contraction on (structured) 2‐D layer and spherical matrices. Due to the alginate functionalization with tyramines, stable cell–matrix interactions were observed beneficial for an implementation in biology, biotechnology, and medicine toward efficient cell culture and tissue substitutes