Jens M. Kelm

3D cell culture systems modeling tumor growth determinants in cancer target discovery

Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.

SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus α-amylase

Abstract The Bacillus stearothermophilus α-amylase (amyS) is a heat-stable monomeric exoenzyme which catalyses random hydrolysis of 1,4-α-glucosidic linkages in polyglucosans. The Bacillus α-amylase was engineered for use as an intracellular (AmySΔS) as well as a secreted reporter protein (SAMY; s ecreted α- amy lase) in mammalian cells. The 5′ end of amyS containing the prokaryotic secretion signal was either deleted (amySΔS) or replaced by a murine immunoglobulin secretion signal. SAMY was cloned under control of the cytomegalovirus promoter (PCMV) in a mammalian expression vector or the promoter of the human elongation factor 1α (PEF1α) in a lentiviral expression context. A variety of mammalian and human cell lines growing as monolayers, in suspension or as three-dimensional spheroids were transfected/transduced with SAMY- or amySΔS-encoding expression/lentiviral vectors and α-amylase activity was measured in cell lysates and culture supernatants. These experiments showed that SAMY and AmySΔS were either secreted or remained intracellular as highly sensitive reporter enzymes. SAMY expression and detection was fully compatible with established SEAP (human secreted alkaline phosphatase) and u-PALMW (low molecular weight urokinase-type plasminogen activator) reporter systems and could be used to quantify expression of up to three independent genes in one culture supernatant.

A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.

Current scientific attempts to generate in vitro tissue-engineered living blood vessels (TEBVs) show substantial limitations, thereby preventing routine clinical use. In the present report, we describe a novel biotechnology concept to create living small diameter TEBV based exclusively on microtissue self-assembly (living cellular re-aggregates). A novel bioreactor was designed to assemble microtissues in a vascular shape and apply pulsatile flow and circumferential mechanical stimulation. Microtissues composed of human artery-derived fibroblasts (HAFs) and endothelial cells (HUVECs) were accumulated and cultured for 7 and 14 days under pulsatile flow/mechanical stimulation or static culture conditions with a diameter of 3mm and a wall thickness of 1mm. The resulting vessels were analyzed by immunohistochemistry for extracellular matrix (ECM) and cell phenotype (von Willebrand factor, alpha-SMA, Ki67, VEGF). Self-assembled microtissues composed of fibroblasts displayed significantly accelerated ECM formation compared to monolayer cell sheets. Accumulation of vessel-like tissue occurred within 14 days under both, static and flow/mechanical stimulation conditions. A layered tissue formation was observed only in the dynamic group, as indicated by luminal aligned alpha-SMA positive fibroblasts. We could demonstrate that self-assembled cell-based microtissues can be used to generate small diameter TEBV. The significant enhancement of ECM expression and maturation, together with the pre-vascularization capacity makes this approach highly attractive in terms of generating functional small diameter TEBV devoid of any foreign material.

Design of artificial myocardial microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.

Scaffold-free cell delivery for use in regenerative medicine.

The development of cell-based therapies for diseased tissues is one of the most promising research directions in regenerative medicine. Cell-delivery methods are an essential part of cell therapy concepts. Therapies with the potential to become clinical routine will only be possible if these methods ensure efficient engraftment and therapeutically-relevant number of cells survive. Here we provide an overview of three different scaffold-free cell-delivery concepts: (i) single cell delivery, (ii) cell sheet engineering and (iii) microtissue technology.

Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells

Introduction We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Methods Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. Results Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. Conclusion We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

3D spherical microtissues and microfluidic technology for multi-tissue experiments and analysis

Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue-tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.

Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.