Three-dimensional Bioreactor System Research Articles

Reduced Cell Adhesion on LightPLAS-Coated Implant Surfaces in a Three-Dimensional Bioreactor System.

Most implants used in trauma surgery are made of steel and remain inside the body only temporarily. The strong tissue interaction of such implants sometimes creates problems with their explantation. Modified implant surfaces, which decrease tissue attachment, might allow an easier removal and therefore a better outcome. Such a modification must retain the implant function, and needs to be biocompatible and cost-effective. Here, we used a novel VUV-light (Vacuum-Ultraviolett)-based coating technology (LightPLAS) to generate coated stainless-steel plates. The tested LightPLAS coating only had an average thickness of around 335 nm, making it unlikely to interfere with implant function. The coated plates showed good biocompatibility according to ISO 10993-5 and ISO 10993-12, and reduced cell adhesion after four different time points in a 2D cell culture system with osteoblast-like MG-63 cells. Furthermore, we could show decreased cell adhesion in our 3D cell culture system, which mimics the fluid flow above the implant materials as commonly present in the in vivo environment. This new method of surface coating could offer extended options to design implant surfaces for trauma surgery to reduce cell adhesion and implant ingrowth. This may allow for a faster removal time, resulting in shorter overall operation times, thereby reducing costs and complication rates and increasing patient wellbeing.

The impact of flow-induced forces on the morphogenesis of the outflow tract.

One percent of infants are born with congenital heart disease (CHD), which commonly involves outflow tract (OFT) defects. These infants often require complex surgeries, which are associated with long term adverse remodeling effects, and receive replacement valves with limited strength, biocompatibility, and growth capability. To address these problematic issues, researchers have carried out investigations in valve development and valve mechanics. A longstanding hypothesis is that flow-induced forces regulate fibrous valve development, however, the specific mechanisms behind this mechanotransduction remain unclear. The purpose of this study was to implement an in vitro system of outflow tract development to test the response of embryonic OFT tissues to fluid flow. A dynamic, three-dimensional bioreactor system was used to culture embryonic OFT tissue under different levels of flow as well as the absence of flow. In the absence of flow, OFT tissues took on a more primitive phenotype that is characteristic of early OFT cushion development where widely dispersed mesenchymal cells are surrounded by a sparse, disorganized extracellular matrix (ECM). Whereas OFT tissues subjected to physiologically matched flow formed compact mounds of cells, initated, fibrous ECM development, while prolonged supraphysiological flow resulted in abnormal tissue remodeling. This study indicates that both the timing and magnitude of flow alter cellular processes that determine if OFT precursor tissue undergoes normal or pathological development. Specifically, these experiments showed that flow-generated forces regulate the deposition and localization of fibrous ECM proteins, indicating that mechanosensitive signaling pathways are capable of driving pathological OFT development if flows are not ideal.

Cardioprotection by placenta-derived stromal cells in a murine myocardial infarction model

BackgroundAutologous cells for cell therapy of ischemic cardiomyopathy often display age- and disease-related functional impairment, whereas an allogenic immunotolerant cell product would allow off-the-shelf application of uncompromised donor cells. We investigated the cardiac regeneration potential of a novel, clinical-grade placenta-derived human stromal cell product (PLX-PAD). MethodsPLX-PAD cells derived from human donor placentas and expanded in a three-dimensional bioreactor system were tested for surface marker expression, proangiogenic, anti-inflammatory, and immunomodulatory properties in vitro. In BALB/C mice, the left anterior descending artery was ligated and PLX-PAD cells (n = 10) or vehicle (n = 10) were injected in the infarct border zone. Four weeks later, heart function was analyzed by two-dimensional and M-mode echocardiography. Scar size, microvessel density, extracellular matrix composition, myocyte apoptosis, and PLX-PAD cell retention were studied by histology. ResultsIn vitro, PLX-PAD cells displayed both proangiogenesis and anti-inflammatory properties, represented by the secretion of both vascular endothelial growth factor and angiopoietin-1 that was upregulated by hypoxia, as well as by the capacity to suppress T-cell proliferation and augment IL-10 secretion when co-cultured with peripheral blood mononuclear cells. Compared with control mice, PLX-PAD-treated hearts had better contractile function, smaller infarct size, greater regional left ventricular wall thickness, and less apoptosis after 4 wk. PLX-PAD stimulated both angiogenesis and arteriogenesis in the infarct border zone, and periostin expression was upregulated in PLX-PAD-treated hearts. ConclusionsClinical-grade PLX-PAD cells exert beneficial effects on ischemic myocardium that are associated with improved contractile function, and may be suitable for further evaluation aiming at clinical pilot trials of cardiac cell therapy.

Evaluation of Organic Anion-Transporting Polypeptide 1B1 and CYP3A4 Activities in Primary Human Hepatocytes and HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor System

The long-term stability of liver cell functions is a major challenge when studying hepatic drug transport, metabolism, and toxicity in vitro. The aim of the present study was to investigate organic anion-transporting polypeptide (OATP) 1B1 and CYP3A4 activities in fresh primary human hepatocytes and differentiated cryopreserved HepaRG cells when cultured in a three-dimensional (3D) bioreactor system. OATP1B1 activity was determined by loss from media experiments of [(3)H]estradiol-17β-D-glucuronide and atorvastatin acid (ATA) for up to 7 days in culture. ATA metabolite formation was determined at days 3 to 4 to evaluate CYP3A4 activity. Overall, the results showed that freshly isolated human hepatocytes inoculated in the bioreactor retained OATP1B1 activity for at least 7 days, whereas in HepaRG cells no OATP1B1 activity was observed beyond day 2. The activity data were in agreement with immunohistochemical stainings, which showed that OATP1B1 protein expression was preserved for at least 9 days in fresh human hepatocytes, whereas OATP1B1 was expressed markedly lower in HepaRG cells after 9 days in culture. Fresh human hepatocytes and HepaRG cells exhibited similar CYP3A4 activity in bioreactor culture, and immunohistochemical stainings supported these findings. Activity and mRNA expression of OATP1B1 and CYP3A4 in primary human hepatocytes compared with HepaRG cells in fresh suspensions were in agreement with data obtained in bioreactor culture. In conclusion, freshly isolated human hepatocytes cultured in a 3D bioreactor system preserve both OATP1B1 and CYP3A4 activities, allowing long-term in vitro studies on drug disposition and toxicity.

In Vitro Evaluation of Major In Vivo Drug Metabolic Pathways Using Primary Human Hepatocytes and HepaRG Cells in Suspension and a Dynamic Three-Dimensional Bioreactor System

Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for the detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes, and human in vivo data. Furthermore, the maintenance of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time by using HepaRG cells and human hepatocytes. (14)C-diclofenac and a candidate from AstraZeneca's drug development program, (14)C-AZD6610, which are metabolized by P450 and UGT in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favored in primary human hepatocytes, whereas the glucuronidation route was favored in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over 1 week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs.

BMP2 and mechanical loading cooperatively regulate immediate early signalling events in the BMP pathway

BackgroundEfficient osteogenic differentiation is highly dependent on coordinated signals arising from growth factor signalling and mechanical forces. Bone morphogenetic proteins (BMPs) are secreted proteins that trigger Smad and non-Smad pathways and thereby influence transcriptional and non-transcriptional differentiation cues. Crosstalk at multiple levels allows for promotion or attenuation of signalling intensity and specificity. Similar to BMPs, mechanical stimulation enhances bone formation. However, the molecular mechanism by which mechanical forces crosstalk to biochemical signals is still unclear.ResultsHere, we use a three-dimensional bioreactor system to describe how mechanical forces are integrated into the BMP pathway. Time-dependent phosphorylation of Smad, mitogen-activated protein kinases and Akt in human fetal osteoblasts was investigated under loading and/or BMP2 stimulation conditions. The phosphorylation of R-Smads is increased both in intensity and duration under BMP2 stimulation with concurrent mechanical loading. Interestingly, the synergistic effect of both stimuli on immediate early Smad phosphorylation is reflected in the transcription of only a subset of BMP target genes, while others are differently affected. Together this results in a cooperative regulation of osteogenesis that is guided by both signalling pathways.ConclusionsMechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion. This suggests a direct crosstalk of both mechanotransduction and BMP signalling, most likely at the level of the cell surface receptors. Furthermore, the crosstalk of both pathways over longer time periods might occur on several signalling levels.

Long‐term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor

Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g., T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological, and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three-dimensional bioreactor system for long-term, functional culture of human bone marrow cells on macroporous microspheres in a packed-bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell-enriched areas. Strikingly, these cultures generated a significant number of antibody-producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three-dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo.

Long-term functional culture of human tonsil lymphocytes in a three-dimensional bioreactor system (144.32)

Abstract Peripheral lymphoid organs are the primary sites of adaptive immune responses, and harbor characteristic anatomical and histological features reflecting separation of cellular subsets (e.g. T and B lymphocytes) and functional compartments. Disruption of lymphoid organ structure by genetic mutations, pharmacological treatments or infection can lead to defective immunity, highlighting the role of tissue organization in immune responses. The generation of in vitro culture systems recapitulating salient features of peripheral lymphoid organs for experimental, biotechnological and clinical applications would be highly desirable, but has so far been hampered by the complexity of these organ systems and has encountered only limited success. We have previously developed a 3D bioreactor system capable of supporting long-term, functional human bone marrow cultures. Here we show that the same system can be adapted for culture of human primary peripheral lymphoid tissue (tonsil) cells for periods up to several weeks in the absence of specific external growth factors or activators. Cells in this system display extended survival, maintain population diversity and phenotypes largely comparable to primary cells, and organize in cellular aggregates with identifiable separation of T and B cell clusters analogous to primary organs. Strikingly, we also show that these cultures are capable of supporting the generation of antibody-producing cells in response to a panel of diverse antigens.

Tissue bioreactor system for the creation and maturation of organized functional muscle

As a first step toward the development of clinically applicable functional muscle tissues we investigated whether organized functional muscle tissue could be engineered in a three-dimensional bioreactor system. Human skeletal muscle precursor cells (MPCs) were isolated, grown and expanded in culture, and seeded onto collagen-based muscle scaffolds for two days of static culture. Subsequently, MPC-seeded scaffolds were placed in the bioreactor system and subjected to cyclic strain of ±10% of the initial scaffold length at a frequency of 3 times/minute for the first 5 minutes of every hour, for up to 3 weeks; MPC-seeded scaffolds without cyclic stimulation served as controls. The resulting engineered tissue was assessed for structural and functional parameters using scanning electron microscopy (SEM), histo- and immunohistochemistry, and physiologic studies. SEM of engineered muscle tissue showed a uniform attachment of muscle cells on the scaffold surface with unidirectional orientation within 5 days and continued to mature with time. Multinucleated myofibrils were evident and cells expressed muscle markers and produced extracellular matrices over time. Control scaffolds showed disorganized muscle cells without any directional orientation. Engineered muscle exhibited a reproducible contractile response to KCl-induced depolarization (p<0.05), while control scaffolds displayed no detectable contraction. This study demonstrates that an organized functional muscle tissue can be engineered using a unidirectional tissue bioreactor system.