Analysis and comparison of oxygen consumption of HepG2 cells in a monolayer and three-dimensional high density cell culture by use of a matrigrid®.

By the use of a MatriGrid® we have established a three-dimensional high density cell culture. The MatriGrid® is a culture medium permeable, polymeric scaffold with 187 microcavities. In these cavities (300 µm diameter and 207 µm deep) the cells can growth three-dimensionally. For these experiments we measured the oxygen consumption of HepG2 cell cultures in order to optimize cultivation conditions. We measured and compared the oxygen consumption, growth rate and vitality under three different cultivation conditions: monolayer, three-dimensional static and three-dimensional actively perfused. The results show that the cells in a three-dimensional cell culture consume less oxygen as in a monolayer cell culture and that the actively perfused three-dimensional cell culture in the MatriGrid® has a similar growth rate and vitality as the monolayer culture.

Automated Analysis of Acetaminophen Toxicity on 3D HepaRG Cell Culture in Microbioreactor.

Real-time monitoring of bioanalytes in organotypic cell cultivation devices is a major research challenge in establishing stand-alone diagnostic systems. Presently, no general technical facility is available that offers a plug-in system for bioanalytics in diversely available organotypic culture models. Therefore, each analytical device has to be tuned according to the microfluidic and interface environment of the 3D in vitro system. Herein, we report the design and function of a 3D automated culture and analysis device (3D-ACAD) which actively perfuses a custom-made 3D microbioreactor, samples the culture medium and simultaneously performs capillary-based flow ELISA. A microstructured MatriGrid® has been explored as a 3D scaffold for culturing HepaRG cells, with albumin investigated as a bioanalytical marker using flow ELISA. We investigated the effect of acetaminophen (APAP) on the albumin secretion of HepaRG cells over 96 h and compared this with the albumin secretion of 2D monolayer HepaRG cultures. Automated on-line monitoring of albumin secretion in the 3D in vitro mode revealed that the application of hepatotoxic drug-like APAP results in decreased albumin secretion. Furthermore, a higher sensitivity of the HepaRG cell culture in the automated 3D-ACAD system to APAP was observed compared to HepaRG cells cultivated as a monolayer. The results support the use of the 3D-ACAD model as a stand-alone device, working in real time and capable of analyzing the condition of the cell culture by measuring a functional analyte. Information obtained from our system is compared with conventional cell culture and plate ELISA, the results of which are presented herein.

Functionalized Thick Film Impedance Sensors for Use in In Vitro Cell Culture.

Multi-electrode arrays find application in electrophysiological recordings. The quality of the captured signals depends on the interfacial contact between electrogenic cells and the electronic system. Therefore, it requires reliable low-impedance electrodes. Low-temperature cofired ceramic technology offers a suitable platform for rapid prototyping of biological reactors and can provide both stable fluid supply and integrated bio-hardware interfaces for recordings in electrogenic cell cultures. The 3D assembly of thick film gold electrodes in in vitro bio-reactors has been demonstrated for neuronal recordings. However, especially when dimensions become small, their performance varies strongly. This work investigates the influence of different coatings on thick film gold electrodes with regard to their influence on impedance behavior. PEDOT: PSS layer, titanium oxynitride and laminin coatings are deposited on LTCC gold electrodes using different 2D and 3D MEA chip designs. Their impedance characteristics are compared and discussed. Titanium oxynitride layers emerged as suitable functionalization. Small 86-µm-electrodes have a serial resistance Rs of 32 kOhm and serial capacitance Cs of 4.1 pF at 1 kHz. Thick film gold electrodes with such coatings are thus qualified for signal recording in 3-dimensional in vitro cell cultures.

Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance.

Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.

Regulation of leukocyte recruitment to the murine maternal/fetal interface.

Controlled immune cell access to the pregnant uterus may be one of the mechanisms involved in maternal tolerance leading to the presence of a selected population of immune cells at the maternal/fetal interface. The molecular determinants responsible for coordinating recruitment of leukocytes include the cellular adhesion molecules and members of the chemokine superfamily. During the critical period of initial placenta development in the mouse an elegantly orchestrated progression of leukocyte homing events in the decidua basalis has been described. Moreover, the maternal/fetal interface displays an unparalleled compartmentalization of microdomains associated with highly differentiated vessels expressing vascular addressins in nonoverlapping patterns. These expression patterns are functionally correlated with the distinct localization of uterine NK cells, monocyte-like cells and neutrophils. Switches in vascular specificity and the partial loss of microenvironmental specialization during the second half of mouse development have been shown to parallel dramatic changes in the populations of leukocytes recruited to the maternal/fetal interface. Recently, complex expression patterns of chemokines and their receptors were described in the human pregnant uterus suggesting that along with adhesion molecules these determinants are critical for leukocyte trafficking to the pregnant uterus.

Thermoforming techniques for manufacturing porous scaffolds for application in 3D cell cultivation.

Within the scientific community, there is an increasing demand to apply advanced cell cultivation substrates with increased physiological functionalities for studying spatially defined cellular interactions. Porous polymeric scaffolds are utilized for mimicking an organ-like structure or engineering complex tissues and have become a key element for three-dimensional (3D) cell cultivation in the meantime. As a consequence, efficient 3D scaffold fabrication methods play an important role in modern biotechnology. Here, we present a novel thermoforming procedure for manufacturing porous 3D scaffolds from permeable materials. We address the issue of precise thermoforming of porous polymer foils by using multilayer polymer thermoforming technology. This technology offers a new method for structuring porous polymer foils that are otherwise available for non-porous polymers only. We successfully manufactured 3D scaffolds from solvent casted and phase separated polylactic acid (PLA) foils and investigated their biocompatibility and basic cellular performance. The HepG2 cell culture in PLA scaffold has shown enhanced albumin secretion rate in comparison to a previously reported polycarbonate based scaffold with similar geometry.

The future of the patient-specific Body-on-a-chip.

As significant advancements in technology focused on Organ-on-a-chip continue, it is feasible to consider the future of Body-on-a-chip technology. With serious work being done to realize functioning artificial livers, kidneys, hearts, and lungs on chips, the next step is not only to interconnect these organs but also to consider the integration of stem cell technology to create interconnected patient-specific organs. Such a patient-specific Body-on-a-chip requires a sophisticated set of tools for micropattering cell cultures in 3D to create interconnected tissue-like organ structures. This review discusses advanced methods of the past two years in on-Chip organs, the complex 3D patterning of cultures and state-of-the-art scaffolding, and discusses some of the most relevant advancements in human-induced pluripotent stem cell (hiPSC) research applied to these organs and scaffolds for the future of a patient-specific Body-on-a-chip. We anticipate that such a technology would have a wide area of application, primarily benefiting drug development, chemical safety testing, and disease modeling.

Selectin, platelet plays a critical role in granulocyte access to the pregnant mouse uterus under physiological and pathological conditions.

Leukocyte recruitment to the pregnant mouse uterus is associated with highly regulated patterns of expression of vascular adhesion receptors. One striking observation is the localized expression of mucosal vascular addressin cell adhesion molecule (MADCAM1) and selectin, platelet (SELP, formerly P-selectin) by maternal vessels in the vascular zone (VZ) during the first half of pregnancy. From midgestation onwards, endothelial cells lining the maternal vessels of the VZ in addition express vascular cell adhesion molecule-1 (VCAM1). The predominant cell population within these vessels is monocyte-like cells. Granulocytes and low numbers of lymphocytes are also present. Murine fetal trophoblast cells are almost devoid of adhesion molecules, including SELP. In contrast, spontaneous abortions of allogeneic pregnancies are characterized by dramatic upregulation of SELP on maternal VZ vessels and on fetal trophoblast cells. Upregulation of SELP is associated with a dramatic influx of highly activated granulocytes, which infiltrate the vessels and tissue of the VZ and the trophoblast. The majority of the activated granulocytes within the trophoblast undergo nuclear fragmentation, which can be detected by TUNEL staining. To demonstrate that SELP is involved in the recruitment of granulocytes to the pregnant uterus, we undertook long-term in vivo inhibition studies using a monoclonal antibody to inhibit the contribution of SELP to leukocyte trafficking to the decidua. In addition, the pregnant uteri of syngeneic Selp(-/-) x Selp(-/-) mice were investigated and compared to the controls. Our results clearly demonstrate the importance of SELP for granulocyte access to the pregnant mouse uterus under physiological and pathological conditions.

Functional involvement of P-selectin and MAdCAM-1 in the recruitment of alpha4beta7-integrin-expressing monocyte-like cells to the pregnant mouse uterus.

Leukocyte recruitment to the pregnant mouse uterus has been suggested to be associated with highly regulated expression of distinct patterns of vascular adhesion receptors. One of the most striking observations is the combined expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and P-selectin by maternal vessels of the vascular zone during the critical period of initial placenta development. The predominant cell population within these vessels is of the monocyte/macrophage lineage and expresses the mucosal integrin alpha4beta7, which represents the ligand for MAdCAM-1; neutrophils and lymphocytes are rare. To directly assess the importance of identified adhesion receptors, we undertook long-term in vivo inhibition studies using monoclonal antibodies to inhibit the contribution of MAdCAM-1 in leukocyte trafficking to the decidua or to deplete alpha4beta7(+) leukocytes. In addition, implantation sites of mouse strains genetically deficient in specific adhesion receptors were investigated. Our results underline the importance of predicted adhesion pathways in the recruitment of monocyte-like cells, especially those expressing alpha4beta7. Interestingly, maternal/fetal units with inhibited recruitment of alpha4beta7(+) leukocytes or the absence of these cells are characterized by reduced size and frequency of uterine NK cells.

Alterations in the expression of homing-associated molecules at the maternal/fetal interface during the course of pregnancy.

One of the most fascinating immunologic questions is how the genetically distinct fetus is able to survive and develop within the mother without provoking an immune rejection response. The pregnant uterus undergoes rapid morphological and functional changes, and these changes may influence the nature of local immune responses at the maternal/fetal interface at different stages of gestation. We hypothesized that specialized mechanisms exist to control access of maternal leukocyte subsets to the decidua and that these mechanisms are modulated during the course of pregnancy. At the critical period of initial placenta development, the maternal/fetal interface displays an unparalleled compartmentalization of microenvironmental domains associated with highly differentiated vessels expressing vascular addressins in nonoverlapping patterns and with recruitment of specialized leukocyte subsets (monocytes, granulated metrial gland cells, and granulocytes) thought to support, modulate, and regulate trophoblast invasion. One of the most striking observations at this time of gestation is the almost complete exclusion of lymphocytes from the maternal/fetal interface. The second half of pregnancy is characterized by a partial loss of microenvironmental specialization and different switches in vascular specificity within the decidua basalis, paralleling dramatic changes in the populations of recruited leukocytes (e.g., a striking influx of lymphocytes, especially T cells). In the term pregnant uterus, the expression of all vascular addressins decreased dramatically; only weakly staining maternal vascular segments remained. These segments may define sites of extremely low residual traffic in the term decidua, which contains remarkably few maternal leukocytes overall. Our results suggest that the maternal/fetal interface represents a situation in which leukocyte trafficking is exquisitely regulated to allow entry of specialized leukocyte subsets that may play a fundamental role in immune regulation during pregnancy.