A P19 and P19CL6 cell-based complementary approach to determine paracrine effects in cardiac tissue engineering.

The negligible self-repair potential of the myocardium has led to cell-based tissue engineering approaches to restore heart function. There is more and more consensus that, in addition to cell development, paracrine effects in particular play a pivotal role in the repair of heart tissue. Here, we present two complementary murine P19 and P19CL6 embryonic carcinoma cell-based in vitro test approaches to study the potential of repair cells and the factors secreted by these cells to induce cardiomyogenesis. P19 cells were 3-dimensionally cultured in hanging drops and P19CL6 cells in a monolayer. Both systems, capable of inducible differentiation towards the cardiomyogenic lineage shown by the appearance of beating cells, the expression of connexin 43 and cardiac troponins T and I, were used to test the cardiomyogenesis-inducing potential of human cardiac-derived adherent proliferating (CardAP) cells, which are candidates for heart repair. CardAP cells in coculture as well as CardAP cell-conditioned medium initiated beating in P19 cells, depending on the cell composition and concentration of the medium. CardAP cell-dependent beating was not observed in P19CL6 cultures, but connexin 43 and cardiac troponin formation as well as expression of GATA-binding protein 4 indicated the dose-dependent stimulatory cardiomyogenic effect of human CardAP cells. In summary, in different ways, P19 and P19CL6 cells have shown their capability to detect paracrine effects of human CardAP cells. In a complementary approach, they could be beneficial for determining the stimulatory cardiomyogenic potential of candidate cardiac-repair cells in vitro.

High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor.

BACKGROUND: Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production. RESULTS: Cultivation of E. coli in the BIOSTAT CultiBag RM system in a conventional batch mode without control yielded an optical density (OD(600)) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD(600) of 60 (20 g L(-1) cell dry weight). By the use of an internal controlled glucose delivery system, EnBase Flo, OD(600) of 30 (10 g L(-1) cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor. CONCLUSIONS: Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

Use of a three-dimensional humanized liver model for the study of viral gene vectors.

Reconstituted three-dimensional (3D) liver models obtained by engrafting hepatic cells into an extracellular matrix (ECM) are valuable tools to study tissue regeneration, drug action and toxicology ex vivo. The aim of the present study was to establish a system for the functional investigation of a viral vector in a 3D liver model composed of human HepG2 cells on a rat ECM. An adeno-associated viral (AAV) vector expressing the Emerald green fluorescent protein (EmGFP) and a short hairpin RNA (shRNA) directed against human cyclophilin b (hCycB) was injected into the portal vein of 3D liver models. Application of the vector did not exert toxic effects, as shown by analysis of metabolic parameters. Six days after transduction, fluorescence microscopy analysis of EmGFP production revealed widespread distribution of the AAV vectors. After optimization of the recellularization and transduction conditions, averages of 55 and 90 internalized vector genomes per cell in two replicates of the liver model were achieved, as determined by quantitative PCR analysis. Functionality of the AAV vector was confirmed by efficient shRNA-mediated knockdown of hCycB by 70-90%. Our study provides a proof-of-concept that a recellularized biological ECM provides a valuable model to study viral vectors ex vivo.

The multi-organ chip--a microfluidic platform for long-term multi-tissue coculture.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing.

Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity, leading to high attrition rates in clinical studies (Paul et al., 2010). The phylogenetic distance between humans and laboratory animals is enormous, this affects the transferability of animal data on the efficacy of neuroprotective drugs. Therefore, many neuroprotective treatments that have shown promise in animals have not been successful when transferred to humans (Dragunow, 2008; Gibbons and Dragunow, 2010). We present a multi-organ chip capable of maintaining 3D tissues derived from various cell sources in a combined media circuit which bridges the gap in systemic and human tests. A steady state co-culture of human artificial liver microtissues and human neurospheres exposed to fluid flow over two weeks in the multi-organ chip has successfully proven its long-term performance. Daily lactate dehydrogenase activity measurements of the medium and immunofluorescence end-point staining proved the viability of the tissues and the maintenance of differentiated cellular phenotypes. Moreover, the lactate production and glucose consumption values of the tissues cultured indicated that a stable steady-state was achieved after 6 days of co-cultivation. The neurospheres remained differentiated neurons over the two-week cultivation in the multi-organ chip, proven by qPCR and immunofluorescence of the neuronal markers ßIII-tubulin and microtubule-associated protein-2. Additionally, a two-week toxicity assay with a repeated substance exposure to the neurotoxic 2,5-hexanedione in two different concentrations induced high apoptosis within the neurospheres and liver microtissues, as shown by a strong increase of lactate dehydrogenase activity in the medium. The principal finding of the exposure of the co-culture to 2,5-hexanedione was that not only toxicity profiles of two different doses could be discriminated, but also that the co-cultures were more sensitive to the substance compared to respective single-tissue cultures in the multi-organ-chip. Thus, we provide here a new in vitro tool which might be utilized to predict the safety and efficacy of substances in clinical studies more accurately in the future.

Chip-based human liver-intestine and liver-skin co-cultures--A first step toward systemic repeated dose substance testing in vitro.

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called "human-on-a-chip" concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air-liquid interface for the skin model during their co-culture with the liver equivalents respectively at (1)/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver-skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance - troglitazone - to the chip-based co-cultures.

Chip-based liver equivalents for toxicity testing--organotypicalness versus cost-efficient high throughput.

Drug-induced liver toxicity dominates the reasons for pharmaceutical product ban, withdrawal or non-approval since the thalidomide disaster in the late-1950s. Hopes to finally solve the liver toxicity test dilemma have recently risen to a historic level based on the latest progress in human microfluidic tissue culture devices. Chip-based human liver equivalents are envisaged to identify liver toxic agents regularly undiscovered by current test procedures at industrial throughput. In this review, we focus on advanced microfluidic microscale liver equivalents, appraising them against the level of architectural and, consequently, functional identity with their human counterpart in vivo. We emphasise the inherent relationship between human liver architecture and its drug-induced injury. Furthermore, we plot the current socio-economic drug development environment against the possible value such systems may add. Finally, we try to sketch a forecast for translational innovations in the field.

A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture.

Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, to accurately predict drug toxicity. In this study, we present a multi-organ-chip capable of maintaining 3D tissues derived from cell lines, primary cells and biopsies of various human organs. We designed a multi-organ-chip with co-cultures of human artificial liver microtissues and skin biopsies, each a (1)/100,000 of the biomass of their original human organ counterparts, and have successfully proven its long-term performance. The system supports two different culture modes: i) tissue exposed to the fluid flow, or ii) tissue shielded from the underlying fluid flow by standard Transwell® cultures. Crosstalk between the two tissues was observed in 14-day co-cultures exposed to fluid flow. Applying the same culture mode, liver microtissues showed sensitivity at different molecular levels to the toxic substance troglitazone during a 6-day exposure. Finally, an astonishingly stable long-term performance of the Transwell®-based co-cultures could be observed over a 28-day period. This mode facilitates exposure of skin at the air-liquid interface. Thus, we provide here a potential new tool for systemic substance testing.