Literature Picks Archive

10/2014 - T4 Workshop Report: Consensus Report on the Future of Animal-Free Systemic Toxicity Testing

Leist M., et al. (2014). "Consensus report on the future of animal-free systemic toxicity testing." ALTEX. 31(3):341-56. doi: http://dx.doi.org/10.14573/altex.1406091.
http://www.ncbi.nlm.nih.gov/pubmed/25061899

The March 2013 European ban on animal use for cosmetics testing has required a re-assment of the role of animal models in toxicity testing, as well as a re-examination of in vitro and in silico models that can coordinately reduce animal use and improve predictivity of DILI and other toxicities in humans.  This transatlantic think tank for toxicology (T4) Workshop Report summarizes the final proposed roadmap for systemic toxicity testing, including recommendations for the five toxicological endpoints that still lack validated replacement methods:  skin sensitization, repeated dose toxicity, toxicokinetics, reproductive toxicity, and carcinogenicity.  

09/2014 - 3D bioprinting of tissues and organs

Murphy SV and Atala A.  Nat Biotechnol. 2014 Aug 5;32(8):773-85.
doi: 10.1038/nbt.2958

The production of tissues and organs using 3D bioprinting holds considerable promise not only for the field of regenerative medicine, but also for drug discovery and toxicology research.  This review covers the technical and biological building blocks required to make 3D bioprinting a reality, and the complex imaging, materials, and engineering needed to bring them all together in stepwise fashion.  Limitations and challenges of the current tissue engineering strategies are discussed.  Perhaps microtissues will be the "ink" consumed by future bioprinters?       

09/2014 - Microfluidic organs-on-chips

Bhatia SN and Ingber DE.  Nat Biotechnol. 2014 Aug 5;32(8):760-72.
doi: 10.1038/nbt.2989.32

This excellent review addresses most everything you wanted to know about organs-on-chips.  What are they?  Can they mimic organ-level functions and disease?  How might they be used for ADMET, PK/PD modeling, and efficacy testing and drug discovery?  What are the promises and pitfalls of such systems, and how close is this technology, in its relative infancy, to reducing the use of animal models in research? 

05/2014 - The application of 3D cell models to support drug safety assessment: Opportunities & challenges.

Adrian Roth and Thomas Singer.  Advanced Drug Delivery Reviews.
http://dx.doi.org/10.1016/j.addr.2013.12.005

This excellently written review covers the pain and needs of the pharmaceutical industry for the assessment of drug safety. Limited predictive power of animal experiments, but also ethical issues, cost and time constraints have spurred the demand for biologically more relevant in vitro systems which also are amenable to standard screening processes.

Despite recent advancements in omics technologies, high content imaging, and corresponding bioinformatic tools, organ toxicity can only be as predictive as the in vitro model from which such data originate. The review highlights current in vitro systems involving the co-culture of organotypic cells representing the multicellularity of organs, as well as emerging 3D culture formats enabling the spatial arrangement typical for a given organ. Finally the authors offer some guidance as to the criteria future in vitro models need to fulfill and the corresponding challenges which need to be tackled, such as cell sourcing, optimized culture condition for multi cell type systems, long term stability, flow control, avoidance of unspecific binding of drugs, the complexity of system validation, and finally the acceptance by regulatory authorities.

05/2014 - Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

Gieseck III RL, Hannan NRF, Bort R, Hanley NA, Drake RAL, et al. PLoS ONE 9(1): e86372.
doi:10.1371/journal.pone.0086372

The lack of primary human hepatocytes and their limited in vitro life span have led to intensive research aimed at the differentiation of iPSCs towards cells with liver specific functions. However current protocols for hepatocyte differentiation fail to generate cells with satisfactory hepatocyte specific features. The presented work describes an alternative approach by performing late stage differentiation in a 3D configuration by a collagen matrix culture. The authors demonstrated that the preservation of cell-cell junctions by transferring small epithelial clumps from 2D to the 3D culture format is fundamental for hepatocyte iPSC differentiation and maturation. When compared to single cell matrix culture, the 3D clump cultures demonstrated enhanced glycogen synthesis, improved phase I/II and III gene expression and progressive loss of fetal markers. Long term functionality assessed by CYP3A4 activity could be demonstrated for up to 75 days of culture. This work convincingly demonstrates the beneficial effect of 3D culture for the differentiation of iPS cells towards a more mature and physiologically relevant hepatocyte phenotype for toxicological studies.

03/2014 - 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

Wenzel C, Riefke B, Gründemann S, Krebs A, Christian S, Prinz F, Osterland M, Golfier S, Räse S, Ansari N, Esner M, Bickle M, Pampaloni F, Mattheyer C, Stelzer EH, Parczyk K, Prechtl S, Steigemann P. Exp Cell Res. 2014 Jan 27. (e-pub ahead of print)

This article describes a very elegant procedure for the identification of compounds targeting dormant cells within a tumor which commonly contribute to the limited effectiveness of cytostatic-based chemotherapy. Tumor spheroids produced from various cancer cell lines (T47D, DLD1, DU145) and primary colon cancer cells were exposed to compounds from two different libraries in a high-throughput setting and analysed by HCA. Nine compounds could be identified which induced cell death predominantly in the core region of the spheroid and which have been identified to inhibit cellular respiration. Effectiveness of those compounds was dependent on extracellular glucose concentrations indicating that the targeted cells represent a subpopulation in the core of the spheroid which still depend on respiration for survival due to low glucose levels. The author could demonstrate that combining the hit compounds with cytostatica was more effective than either substance alone, supporting the notion of a different mode of action of the hit compounds. This work is an excellent example of how spheroid culture can help to better understand tumor biology and to identify new treatment strategies which would not have been possible with standard 2D cultures.

03/2014 - Self-organization and the self-assembling process in tissue engineering

Athanasiou KA, Eswaramoorthy R, Hadidi P, Hu JC. Annu Rev Biomed Eng. 2013; 15:115-36

This excellently written review focuses on scaffoldless tissue engineering, particularly on self-organisation and self-assembling of cells. While the use of scaffolds provides some advantages for traditional tissue engineering (serve as predefined structure for anchor-dependent cells, controlled release of growth factors, long shelf lives) certain disadvantages let to alternative methods in tissue engineering, such as scaffoldless tissue generation which does not depend on seeding or adherence within an exogenous, three dimensional material. Self-organization refers to a process which generates scaffoldless tissue under the influence of external forces (bioprinting, cell-sheet engineering). Self-assembly on the other hand describes the transition of cells to a higher order arrangement in a spontaneous manner and driven by differential adhesion, recapitulating the native tissue formation that generally occurs spontaneously in vivo. The review highlights some aspects of self-organization and self-assembling, their use for the production of replacement tissues and clinical applications thereof.

Dec 2013 - Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Xia Y, Nivet E, Sancho-Martinez I, Gallegos T, Suzuki K, Okamura D, Wu MZ, Dubova I, Esteban CR, Montserrat N, Campistol JM, Belmonte JC. Nat Cell Biol. 2013 Nov 17. (e-pub ahead of print)

The kidney is one of the most complex organs to model in vitro and, consequently, the pharmaceutical industry still lacks a suitable platform to study kidney disease for the development of drug–based therapeutics. The authors exposed human iPS or ES cells to a chemically defined medium differentiating these cells towards a ureteric bud progenitor-like phenotype which, after 3D co-culture with murine cells of the developing kidney, matured into ureteric bud structures. The murine embryonic kidney cells provide the appropriate developmental cues for the progenitor cells to acquire a uretic bud phenotype, ultimately making regular nephrogenesis possible. The authors also demonstrated the feasibility of disease modelling by utilising iPS cells from a patient suffering from polycystic kidney disease. This approach opens new possibilities to coax human renal lineages into the complex 3D structures indispensable for mimicking kidney development and function and the modelling of renal disease.

Nov 2013 - Generation of co-culture spheroids as vascularisation units for bone tissue engineering

Walser R, Metzger W, Görg A, Pohlemann T, Menger MD, Laschke MW. Eur Cell Mater. 2013; 26:222-33.

This publication highlights the use spheroids as building units for tissue engineered constructs in regenerative medicine. In this study, co-culture spheroids were produced from human primary osteoblasts and dermal microvascular endothelial cells as building blocks to generate scaffolds for the reconstruction of large bone defects. The objective of the co-culture approach using microvascular endothelial cells was to overcome the limitation of insufficient blood supply in current bone tissue engineering models. Co-culture spheroids had a dense network of tubular-like vessel structures Fourteen days following transplantation into the dorsal skinfold chambers of mice, intravital microscopy revealed that the spheroids attached to the host's vascular system and increased in size over time, suggesting long-term viability and angiogenic activity of the transplanted human endothelial cells. This study demonstrates that spheroid cultures are functional tissue engineered constructs that are suitable for use in reconstructive medicine.

Nov 2012 - Multicellular Tumor Spheroids as an In Vivo-Like Tumor Model for Three-Dimensional Imaging of Chemotherapeutic and Nano Material Cellular Penetration

Ma Hl, Jiang Q,Han S, Wu Y, Tomshine JC, Wang D, Gan Y, Zou G,and Liang XJ. Mol Imaging. 2012; 11(6):487-98.

The value of 3D cell culture models in mimicking early stage tumor biology has long been recognized, but has not been widely used in conjunction with imaging technologies due to the lack of standardized protocols. This article describes the use of different imaging modalities to characterize tumor spheroids, but also to investigate the penetration kinetics and properties of novel anti-cancer therapeutics. HeLa spheroids were morphologically characterized by scanning and transmission electron microscopy as well as multi-photon microscopy to obtain high resolution 3D images. By conventional histology in conjunction with flow cytometry the authors could clearly identify three distinct cell populations (proliferating-quiescent-necrotic) commonly observed in native solid tumors. Multi-Photon microscopy revealed reduced kinetics for cellular doxorubicin uptake in spheroids compared to monolayer cultures, indicative for the frequently observed in vivo chemotherapeutic resistance. Being able to identify quantifiable differences for nanoparticle delivery (positively and negatively charged quantum dots) in 2D versus spheroid cultures suggests altered biochemical properties of cellular membranes and extracellular matrix which warrants the use of advanced 3D cell culture formats to study chemotherapeutic drug delivery.

Nov 2012 - Novel Technologies and an Overall Strategy to Allow Hazard Assessment and Risk Prediction of Chemicals, Cosmetics, and Drugs with Animal-Free Methods

Leist M, Lidbury BA, Yang C, Hayden PJ, Kelm JM, Ringeissen S, Detroyer A, Meunier JR, Rathman JF, Jackson GR, Stolper G, and Hasiwa N. ALTEX. 2012; 29(4):373-88

In the past, several programs with the goal to implement emerging technologies into new concepts for animal-free, in vitro based risk assessments have been started, such as the Tox21/ToxCast or the European SEURAT-1 initiative. Leist et al. present an overall strategy for the valuation of compound toxicity, which incorporates a profound assessment of physicochemical properties of parent compounds and metabolites, and a “hazard assessment” on a set of in vitro models. Compound characteristics, in vitro properties and omics data will be fed into databases and simulation programs, corresponding to toxicity pathways and which will serve to provide an in vitro-in vivo extrapolation to predict relevant human doses. In vitro modelling is an integral part of the entire risk assessment process and will become increasingly more important for ethical reasons and the avoidance of cross species interpolation. This review exemplifies representative models, covering advanced 2D cell cultures (LUHMES cells, conditionally immortalized human neurons), InSphero’s scaffold-free 3D organotypic microtissues, MatTek’s human epithelial tissues with engineered toxicological reporter functions and Altamira’s in silico modelling approach to predict skin irritancy based on various pre-existing data. This review convincingly demonstrates on how new in vitro models in conjunction with in silico methods could represent the cornerstones of future animal-free risk assessment strategies.>

Oct 2012 - Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality

Ramasamy TS, Yu JS, Selden C, Hodgson H, Cui W. Tissue Eng Part A. 2012: Sep 24 (e-pub ahead of print)

In order to generate hepatocytes from induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs) well defined treatment steps in a sequential order need to be applied to coerce the cells into the definitive endoderm (DE) and further into hepatocytes. In an attempt to mimic the microenvironment of a developing liver, the authors performed the last differentiation steps with hESCs with definitive endoderm features in Algimatrix culture plates to foster spheroid formation and to enhance cell-cell contacts. This approach let to hepatocytes cultures with increased expression of liver specific genes, augmented P450 activity and urea production as compared to a differentiation procedure carried out solely in 2D. Interestingly the cell density in Algimatrix cultures influenced the spheroid size and hence also liver specific features and functions. The highest degree of differentiation was observed in spheroids at a size of 50-100 µm. This work nicely demonstrates the impact of 3D-culture on the ability to improve differentiation of pluripotent stem cells into a desired lineage.

Oct 2012 - Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3D spheroid cell culture system

Karlsson H, Fryknäs M, Larsson R, Nygren P. Exp Cell Res. 2012: 318(13):1577-85

This article investigates compound activity with respect to survival of a colon cancer HCT-116 spheroid model. Spheroids were formed by means of a micropatterned culture format. The tumor spheroids showed distinct features from regular monolayer cultures (i.e. increased expression of cell adhesion proteins and p21), but also traits which depended on spheroid size as a function of culture time (proliferation rate, hypoxia). Spheroid viability was assessed either by a fluorescein-diacetate based read out or of fluorescence measurement in spheroids composed of GFP expressing HCT-116 cells. When compared to monolayer cultures the spheroids were less sensitive to the cytotoxic effect of all compounds applied which was even more pronounced with older and larger spheroids becoming nearly resistant to the compound's activities. Further investigations are needed to verify whether this frequently observed resistance towards cytostatic compounds in 3D models compared to monolayer cultures is related to altered tumor biology or whether this, at least partially, may also be related to limited compound penetration into the tissue.

Oct 2013 - Cerebral organoids model human brain development and microcephaly

Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA. Nature 2013. 19;501:373-9

This excellent work describes the powerful capabilities of 3D cell culture to study human brain development and relating disorders in vitro. The authors relied on the self-organization capacity of pluripotent stem cells to form complex tissues by providing the proper environment necessary for the generation of intrinsic signals and cues which orchestrate brain development. This was achieved by culturing neuroectodermal cells obtained from embryoid bodies in Matrigel droplets which after transfer to a spinning bioreactor formed cerebral organoids with defined brain regions. Organoid differentiation was demonstrated by increasing expression and regionalization of neuronal markers at the expense of stem cell markers, which indicated the development of a variety of brain regions organized into discrete but interdependent domains. This in vitro model bears the great potential to study neurodevelopmental disorders in vitro as the authors were able to recapitulate hypoplasia of cerebral organoids derived from induced pluripotent stem cells of a patient suffering from microcephaly. This is even more striking as mouse models failed to demonstrate the same defect due to species specific differences in brain development. This pioneering work is an important contribution to improve in vitro models for the study of brain development and neurological diseases and for the assessment of developmental and neurological toxicity in the field of drug discovery

Oct 2013 - Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells

Subramanian K, Owens DJ, Raju R, Firpo M, O'Brien TD, Verfaillie C, Hu WS. Stem Cells Dev. 2013; (e-pub ahead of print)

Despite recent advances in stem cell research, current differentiation protocols fail to achieve full tissue specific functionality when starting from either induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). For example, hepatic differentiation of human embryonic stem cells is a process that lasts 20 days and is performed by 4-stages with the addition of various cocktails of growth factors and cytokines and yet results in a heterogeneous population of cells with hepatocyte-like features and embryonic phenotype. In this report Subramanian et al. describe a new approach by applying a 3D cell culture phase subsequent to the conventional 4-stage differentiation protocol. Spheroid formation by spontaneous self-assembly helped to increase the fraction of differentiated cells and liver specific gene expression which could be maintained by at least 15 days. This report nicely demonstrates that cell-cell interactions are crucial to foster phenotypic maturation of embryonic stem cells and maintaining specific structure and function over extended culture periods.

Jun 2013 - Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering

Haraguchi Y, Matsuura K, Shimizu T, Yamato M, Okano T. J Tissue Eng Regen Med. 2013; (e-pub ahead of print)

Human derived induced pluripotent stem cells have become a valuable and important source for therapeutic strategies in regenerative medicine. Classical expansion protocols rely on large culture dishes and the use of feeder cells (e.g. irradiated fibroblasts). This is quite a laborious procedure considering that for the treatment of a myocardial infarction approximately 500 densely grown 100 mm culture dishes would be required to provide the necessary mount of 1-2x 109 cardiac cells. The authors describe an alternative cell expansion method by applying rotary cell culture in spinner flasks, yielding up to 1x108 iPCSs, corresponding to 24 100 mm dishes. The cells grown under constant rotation form aggregates and stay undifferentiated with sustained pluripotency. Subjecting them afterwards to standard differentiation procedures yields electroactive cardiomyocytes with a conversion rate of 50-60%. Such new culture protocols will be essential in the field of regenerative medicine which will eventually help to provide the large amounts of cells required to achieve therapeutic success.

 

Jun 2013 - Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system

Arai K, Sakamoto R, Kubota D, Kondo T. Proteomics. 2013; (e-pub ahead of print)

Spheroid culture models have been known to mimic chemoresistance frequently observed in clinical cancer therapies. Here the authors convincingly demonstrate that proteome analysis of spheroid cultures can identify proteins which correlate to the degree of resistance towards a chemotherapeutic agent. 9 out of 11 investigated osteosarcoma cell lines were less sensitive towards the cytotoxic effect of doxorubicin when cultured as spheroids compared to monolayer culture conditions. The degree of chemoresistance was neither correlated to spheroid size nor to the prevalence of hypoxia in the centre of the spheroids. However, proteome analysis by 2-dimensional gel electrophoresis identified 17 proteins which were differentially expressed in spheroid cultures and monolayer cultures. Expression levels of 4 proteins, including Cathepsin D, were positively correlated with increased IC50 values for doxorubicin. Cathepsin D has been associated with malignant types of various cancers and attenuation of apoptosis. This work underscores the potency of tumor cell spheroid cultures for the identification of cellular components conferring chemoresistance and the detection of potential new therapeutic targets for cancer treatment.

Sep 2012 - An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids

Anada T, Fukuda J, Sai Y, Suzukia O. Biomaterials 2012: 33 (33); 8430-8441

This article highlights an important aspect in 3D cell culture, namely the provision of oxygen in spheroid cultures that exceed a certain size that is considered to be critical to provoke hypoxia, depending on the cell's metabolic activity. The authors developed a PDMS based oxygen-permeable chip which included microcavities for the formation of similar-sized spheroids. HepG2 spheroids showed a remarkable enhancement in growth, reduced anaerobic metabolism and increased albumin secretion as compared to spheroids cultured in a non-oxygen-permeable chip. The improved metabolic activity was reflected by a drastic reduction of hypoxia and diminished central necrosis suggesting that oxygen rather than glucose is the rate limiting supply for spheroid cultures in conventional formats. These findings are of significant importance for future developments in 3D cell culture formats, which need to take the limitations in oxygen supply into account.

Sep 2012 - Human embryonic stem cell differentiation into insulin secreting beta cells for diabetes

Bose B, Shenoy SP, Konda S, Wangikar P. Cell Biol Int. 2012 Aug 17 (e-pub ahead of print)

Generating insulin producing cells from embryonic stem cells has been followed for almost ten years with promising results but has never reached clinical relevance, primarily due to immunogenicity and risk for tumorgenesis. Recent protocols applied a five-step differentiation procedure mimicking beta-cell organogenesis, which finally led to approx. 25% of insulin producing cells. Bose et al. applied a 3D culture step for beta-cell maturation in presence of a stable GLP-1 analogue, known to induce beta-cell mass in vivo. With this improved protocol the fraction of insulin producing cells could be increased to nearly 70%, ameliorating streptozotocin-induced diabetes in mice for up to 96 days post transplantation. This work underlines the importance of 3D cell culture for the formation of morphogenic gradients that are necessary in order for stem cells to differentiate into a desired cell type.

May 2013 - Next-Generation Regenerative Medicine: Organogenesis from Stem Cells in 3D Culture

Sasai Y. Cell Stem Cell. 2013; 2(5):520-30

This excellently written review provides a comprehensive overview on the current understanding of organogenesis as observed and influenced using 3D stem cell cultures. The author presents nature’s concept of tissue formation by cellular self-organization and spontaneous formation of highly organized structures, guided by morphogenic gradients in a spatiotemporal or “4-dimensional” context. Based on the cells own internal program, 4D stem cell biology bears an enormous potential to generate self-organized complex tissues for future therapeutic applications in regenerative medicine.

Apr 2013 - Multicellular self-assembled spheroidal model of the blood brain barrier

Urich E, Patsch C, Aigner S, Graf M, Iacone R, Freskgård PO. Sci Rep. 2013, 3:1500

This intriguing study demonstrates the power of scaffold-free spheroid culture formats to produce highly organotypic structures, recapitulating the complex architecture of the blood brain barrier (BBB), simply by gravitational force and the cell’s inherent ability of self-arrangement. Human primary brain endothelial cells, human primary pericytes and human primary astrocytes were seeded into hanging drops at a uniform ratio which spontaneously formed a spheroid consisting of an astrocyte core, an endothelial cell mantle and pericytes residing in between the parenchyma and the endothelial outer layer - a structure highly reminiscent of the native cell organization of the BBB. The addition of endothelial cells stopped the growth of otherwise continuously growing astrocyte/pericyte co-cultures while the presence of pericytes within this model system had a profound effect on the expression levels of endothelial cell surface receptors, which could not be observed in a standard transwell co-culture set up. The authors present a convincing and ground-breaking new in vitro model for the study of BBB transport and CNS drug delivery.

Apr 2013 - A Novel 3 Dimensional Stromal-based Model for In Vitro Chemotherapy Sensitivity Testing of Leukemia Cells

Aljitawi OS, Li D, Xiao Y, Zhang D, Ramachandran K, Stehno-Bittel L, Van Veldhuizen P, Lin TL, Kambhampati S, Garimella R. Leuk Lymphoma. 2013 (e-pub ahead of print)

Lately it has become widely accepted that suspension cell culture models have a poor predictivity when testing treatment strategies against leukemia, mainly due to the self-protective niche formed by interacting stromal cells from the bone marrow. To overcome this limitation, the authors established a 3D co-culture system of leukemia cells (HL-60, Kasumi-1, and MV411) with expanded human bone-marrow-derived mesenchymal stem cells (hu-BM-MSC) in a synthetic polyglycolic acid/ poly L-lactic acid copolymer scaffold. All leukemia cells showed similar or higher proliferative activity in response to doxorubicin in 3D co-culture when compared to 2D co-culture or 2D mono-culture which according to the authors could be at least partially explained by an increased expression of N-cadherin in 3D co-cultures. Observations of reduced sensitivity in scaffold based culture systems are often questioned as this might be related to limited compound penetration or unspecific adsorption by the scaffold material – both of which concerns have been addressed by the authors. Even though this model needs further clarification and validation the presented results point out that such a co-culture system may reflect the tumor microenvironment more closely than simple suspension cell cultures and thus provide a better prediction of future treatment options.

Mar 2013 - 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing

Longati P, Jia X, Eimer J, Wagman A, Witt MR, Rehnmark S, Verbeke C, Toftgård R, Löhr M, Heuchel RL. BMC Cancer. 2013;13(1):95. (e-pub ahead of print)

Chemoresistance is the leading cause for the lack of efficacy of compounds developed against pancreatic cancer and which is often not detected in preclinical testing. In an approach to identify the mechanisms leading to chemoresistance and to improve the predictivity of compound screening the authors applied 3D spheroid cultures with cell lines derived from pancreatic ductal adenocarcinoma. Compared to 2D cultures various matrix proteins (e.g. Lumican, SNED1), signal transduction molecules controlling critical pathways (e.g. DARPP32) or micro RNA known to regulate pancreatic metastasis formation (miRNA-146a) or involved in enhanced chemoresistance (miRNA-21 and miRNA-335) were upregulated in spheroid cultures. With the exception of three compounds all drugs tested were less effective than in 2D cultures. The authors proposed the 3D spheroid culture as a more relevant in vitro system for predicting drug efficacy, as it reflects the chemoresistant state of pancreatic cancer mediated by stroma formation, cell adhesion and metabolic shift towards glycolysis.

Mar 2013 - 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies

Gunness P, Mueller D, Shevchenko V, Heinzle E, Ingelman-Sundberg M, Noor F. Toxicol. Sci. 2013. (e-pub ahead of print)

HepaRG cells have been heavily investigated as a suitable replacement for primary human hepatocytes to study drug induced liver toxicity in vitro. To study the impact of 3-dimensional culture on morphology and hepatocyte specific function, HepaRG spheroids were produced by InSphero’s GravityPLUS™ hanging drop culture format. In comparison to 2D HepaRG grown in monolayers, spheroids showed an improved metabolic activity with a higher albumin and urea production rate throughout the entire culture time up to three weeks. Interestingly, glucose production was consistently increased over time as did lactate release and this might be explained by a tight compartmentalization of gluconeogenesis and glycolysis. Compared to 2D cultures CYP2E1 enzyme activity was significantly higher which was also reflected by an increased sensitivity towards acetaminophen induced toxicity, with an IC50 value comparable to in vivo data. In contrast to 2D cultures, HepaRG spheroids were not sensitive to troglitazone, suggesting that enhanced CYP3A4 expression let to complete detoxification. This excellent article convincingly demonstrates that in the 3D microtissue format even advanced cell models like HepaRG will be dramatically improved in terms of functionality and predictive power.

Jan 2013 - 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells

Wang W, Itaka K, Ohba S , Nishiyama N, Chung U, Yamasaki Y, Kataoka K. Biomaterials. 2009; 30: 2705-15

Mesenchymal stem cells (MSCs) are believed to offer great therapeutic potential as they are capable of differentiating into multiple cell lineages, such as chondrocytes, osteocytes or adipocites. However current monolayer cell culture techniques prevent high efficiency rates for differentiating MSCs into desired cell lineages, as conventional 2D culture limits cell-cell interaction which is fundamental to the differentiation process. 3D culture formats relying on biomimetic scaffolds are difficult to standardize and the fate of MSCs is largely dependent on the seeding density within such culture formats. In this work a comparative gene expression study was performed with respect to the differentiation capacities of MSCs cultured either in conventional 2D formats or as spheroids grown on micropatterned substrates. The authors could not only demonstrate a higher expression of marker genes representing either adipocytes or osteoblasts in spheroid cultures but also observed a significant down regulation of genes that confer stemness to MSCs when compared to monolayer cultures. These data nicely substantiate the notion that differentiation of stem cells into specific cell lineages is strongly influenced by their microenvironment and that 3D spheroid culture can mimic this environment better than any other culture format.

Jan 2013 - Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition

Smith SJ, Wilson M, Ward JH, Rahman CV, Peet AC, Macarthur DC, Rose FRAJ, Grundy RG, Rahman R. PLOS ONE. 2012: 7 (12): e52335

This article is a comprehensive work investigating the differences of glioma cell lines (U87 and KNS42) that were grown in regular 2D cultures or in a 3D Rotary Cell Culture System (RCCS) where a heterogeneous population of differently sized aggregates are formed. The morphology of the aggregates was similar to pediatric gliomas, featuring proliferating and necrotic areas, while the gene expression profile of the rotary cultures was intermediate between cells cultured in 2D and primary brain tumors. Metabolic analysis of aggregates revealed a profile distinct to 2D cultures with elevated levels of lipids and decreased phosphocholine as commonly observed in tumors. The sensitivity towards the histone deacetylase inhibitor Vorinostat was markedly decreased in 3D versus 2D cultures. Such an increased drug resistance is often observed in 3D tumor models and may simply reflect the inability of the compound to completely penetrate the tissue but may also be conferred by the formation of a cell subpopulation with altered sensitivity to xenobiotic substances. This article underlines the advantages of the RCCS culture format to obtain spheroids that mimic the heterogeneity of a solid brain tumor, but which is achieved at the expense of standardization and throughput.

Aug 2012 - 3D HepaRG Model as an attractive tool for toxicity testing

Leite SB, Wilk-Zasadna I, Zaldivar Comenges JM, Airola E, Reis-Fernandes MA, Mennecozzi M, Guguen-Guillouzo C, Chesne C, Gouillou C, Alves PM, Coecke S. Toxicol Sci. 2012 Jul 27 (e-pub ahead of print)

This report describes an alternative culture method for HepaRG, a hepatocarcinoma cell line that shows similar hepatotypic functions as primary human hepatocytes. By culturing these cells in a bioreactor (spinner vessel) the authors could demonstrate long term viability and functionality of HepaRG spheroids. Albumin secretion, phase I and phase II enzyme activity and induction could be maintained up to 7 weeks, while urea production was not detectable. A shortened culture protocol where the differentiation of the HepaRG cells took place in the bioreactor still showed long term functionality, however with slightly reduced biotransformation capabilities. Dose response experiments with APAP revealed lower toxicity in spheroids than compared to 2D cultures, which is most likely attributed to altered diffusion/uptake kinetics related to 3-dimensional cell culture.

Aug 2012 - 3D-Spheroid Defects in NPHP Knockdown Cells Are Rescued By The Somatostatin Agonist Octreotide

Ghosh AK, Hurd TW and Hildebrandt F. Am J Physiol Renal Physiol. 2012 Jul 25. (e-pub ahead of print)

This article is an excellent example of applying a 3D cell culture in vitro model for the study of a genetic disorder without the need to go through the lengthy process of creating transgenic mouse models. The authors were studying the spheroid formation of renal cell culture (mIMCD3) after stable knockdown of members of the NPHP gene, known as a mutation hot spot causing chronic kidney disease by renal ciliopathy. Abnormal spheroid formation was associated with reduced cilia numbers and polarity defects similar to the observed clinical phenotype. Treatment with the somatostatin agonist octreotide could partially reverse the phenotype by reducing elevated cAMP levels which rescued normal spheroid formation. These data tie-in well with recent clinical study applying a long acting octreotide which has been reported to slow down the progression of polycystic kidney and liver disease.

Jul 2012 - Advances in the formation, use and understanding of multicellular spheroids

Achilli TM, Julia Meyer J and Morgan JR. Expert Opin Biol Ther. 2012 Jul 12 (e-pub ahead of print)

This review summarizes recent advances, advantages and limitations of the production of multi-cellular tumor spheroids and their application in biomedical research and drug discovery. It became widely accepted that the biology and biochemistry of tumor spheroids is considerably closer to the natural tissues and organs which they are supposed to represent as opposed to 2D cell cultures on rigid plastic dishes. As a consequence, spheroid production methods have been improved and analysis tools have been developed which help to further implement this culture format in basic science and drug discovery. The article compares various techniques for the production of spheroids and highlights the biology of cellular self-assembly. The relevance of spheroid models in cancer research and drug discovery is discussed as well as their use as building blocks for futures applications in tissue engineering and regenerative medicine.n-relevant data for compound de-risking.

Jul 2012 - Changes in Global Gene Expression Associated with 3D Structure of Tumors: An Ex Vivo Matrox-Free Mesothelioma Spheroid Model

Kim H, Phung Y, Ho M. PLoS One. 2012;7(6):e39556 (e-pub ahead of print)

A global gene expression profile analysis was performed with the mesothelioma cell line NCI-H226 from monolayer or matrix-free 3D spheroid cultures. This allowed gene expression profiles to be investigated independently of the unknown effects from artificial extracellular matrix which is frequently used in 3D cell culture. The H226 cells formed spheroids which have previously been shown to mimic avascular tumors with inherent metabolic and proliferative gradients and which display drug resistance similar to clinical observations. Among 54'675 probe sets, the authors identified 1'808 genes that were differentially regulated between 2D and 3D spheroid cultures of which 142 probe sets exhibited at least a 5-fold change in expression levels. Highly upregulated genes were mainly associated with immune response, wound healing, lymphocyte stimulation and response to cytokine stimulation, whereas downregulated transcripts were primarily related to apoptosis. Further analysis revealed that 17 genes were involved in cellular movement, 13 genes associated with cell to cell signalling and 16 genes which were related to cellular growth. The authors propose the matrix-free spheroid model as a clinically relevant in vitro setting which may lay the ground for the identification of new biomarkers or therapeutic targets.

Jun 2012 - Stromal-epithelial metabolic coupling in cancer: Integrating autophagy and metabolismin the tumor microenvironment

Martinez-Outschoorn UE, Pavlidesa S, Howell, A, Pestell RG, Tanowitzf, HB, Sotgiaa F, Lisanti MP. Int J Biochem Cell Biol. 2011. 43(7):1045-51

This review is highlighting the groundbreaking work of U. Martinez-Outschoorna et al. who first described the parasitic behaviour of cancer cells guiding associated stromal fibroblasts into autophagia and mitophagia. By this stromal cells are forced to undergo gycolysis to produced energy rich catabolites which fuel the cancer cells even in the absence of vascular perfusion. The stromal-epithelial coupling is initiated by oxidative stress towards the cancer associated fibroblasts which finally leads to an augmentation of the mitochondrial mass and enhanced oxidative phosphorylation in cancer cells, suggesting that the Warburg effect (glycolytic shift) commonly observed in cancer cells is an in vitro artefact of cancer cell monocultures. This highly intriguing work emphasizes the paramount need to employ in vitro models that enable the close interplay of stromal cells with cancer cells, which can only be achieved by 3D cell culture systems.

Jun 2012 - Organotypic systems in drug metabolism and toxicity: challenges and opportunities

Dash A, Brett R Blackman BR, Wamhoff BR. Expert Opin Drug Metab Toxicol. 2012 May 26 (e-pub ahead of print)

This article gives an excellent overview of the current needs of the biopharmaceutical industry to employ more relevant organotypic in vitro screening systems to obtain early and meaningful data for predicting drug safety and efficacy. The authors focus on a few but most relevant requirements to be fullfilled by a successful organotypic test system, such as i) the proper choice of cells (immortalized, primary or iPSC-derived), ii) the inclusion of parenchymal and non-parenchymal cells to enhance heterotypic cell to cell communication and compartementalization, and iii) the application of circulatory patterns to closely mimick shear stress, oxygen and nutrient supply. As it seems obvious that system complexity opposes high throughput capabilities, organotypic models are pre-destined to fill the gap between pre-clinical high throughput screening and animal studies to provide meaningful and human-relevant data for compound de-risking.

May 2012 - Spherical Bullet Formation via E-cadherin Promotes Therapeutic Potency of Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood for Myocardial Infarction

Lee EJ, Park SJ, Kang SK, Kim GH, Kang HJ, Lee SW, Jeon HB and Kim HS. Mol Ther. 2012 Mar 27 (e-pub ahead of print)

Umbilical cord blood MSCs (hUCB-MSC) were cultured in anchorage-deprived suspension culture to produce spheroids (bullets) for therapeutic intervention in a rat myocardial infarct model. 7 weeks after transplantation of hUCB-MSCs 3D bullets cardiac output (left ventrical systolic and diastolic diameter, left ventrical ejection rate) improved significantly compared to treatment with single cell suspension of adherent cells. Improved cardiac function could be explained by enhanced engraftment hUCB-MSCs 3D bullets compared to single cell transplantation, decreased fibrosis and augmented VEGF secretion resulting in a higher capillary density in the peri-infarct area. The authors demonstrated that VEGF expression was downstream of E-cadherin signalling presumably mediated through phosphorylation of ERK and AKT. This work nicely demonstrates that cell-cell interactions in a 3D arrangement are crucial for promoting cell survival, proliferation and differentiation, all of which are fundamental processes in tissue regeneration.roliferation, migration and cell-to-cell contacts.

May 2012 - Genome-Wide Gene Expression Analysis in Cancer Cells Reveals 3D Growth to Affect ECM and Processes Associated with Cell Adhesion but Not DNA Repair

Oliver Zschenker O, Streicher T, Hehlgans S, Cordes N. 2012 April. PLoS One. e34279 (e-pub)

A comparison of a genome wide expression analysis was performed with two cell lines A549 (lung tumor) and SCC15 (squamous cell carcinoma) cultured either on conventional 2D culture dishes or as 3D cultures embedded in Matrigel. The 3D cell culture format affected mainly the expression levels of genes involved in extracellular matrix formation, cell adhesion and defence response, but not so much genes involved in DNA-repair. This suggests that the resistance to radio and chemotherapy, as often observed in 3D cultures, is rather mediated by alterations in cell architecture affecting cell-cell contacts and interactions with the ECM rather than by modifications of the DNA-repair machinery. This also emphasizes the importance of advanced 3D cell culture models which are capable to mediate and process a multitude of external cues and which can reflect true cancer biology more closely than conventional 2D cell culture.

Apr 2012 - Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line

Fey SJ, 2 and Wrzesinski K. 2012. Toxicol Sci. 2012 Mar 27 (e-pub ahead of print)

This article compares the LC50 values of classical hepatotoxic compounds (APAP, Amiodarone, Diclofenac, Metformin, Phenformin and Valproic acid) retrieved from the literature and based on studies performed with primary hepatocyte's (human, rat or mouse) as well as from HepG2 cultures grown in 2D or 3D spheroids. Interestingly, when converted to LD50 values, i.e. LC50 values normalized to the amount of protein in the corresponding test system (mg compound/mg protein), the values from literature where more consistent than the original LC50 values. Comparison of LD50 values obtained from HepG2 3D spheroid cultures showed better correlation to in vivo lethal blood plasma levels than HepG2 2D cultures and were comparable to data obtained with fresh hepatocytes. Thus the authors claim that HepG2 spheroid cultures are equally useful as primary human hepatocytes for studying liver toxicity.

Apr 2012 - Microtissue size and hypoxia in HTS with 3D cultures

Asthana A and Kisaalita WS. 2012 April. Drug Discov Today (in press)

Cell based high throughput screening for drug discovery largely depends on standardized in vitro models reflecting healthy tissue and cancer biology as closely as possible. 3D culture models can emulate the complex physiological processes of our body much better than standard 2D cultures as has been described in numerous publications. This review emphasizes the need for careful selection of an appropriate 3D culture model, as microtissue size control is critical to the outcome of biological studies. The authors refer to various available 3D cell culture models, most of which do not allow precise control over tissue size, or tissue size is limited by physical barriers. Thus they caution that differential gene expression between 2D and 3D cell culture, as described in numerous studies, may also be attributed to hypoxia associated to larger microtissues. Microtissue size also impacts the fate of adult or embryonic stem cells as hypoxia tends to maintain pluripotency while normal environmental oxygen levels enhance the differentiation capabilities of stem cells. This article nicely points out the advantages of 3D cell culture for providing a more physiological relevant in vitro model but at the same time underscores the necessity to choose a format which allows a homogeneous population of equally-sized microtissues to be obtained.

Of note: Contrary to what was stated by the authors, the InSphero GravityPLUS™ system, which is based on hanging drop cultures, enables the formation of scaffold-free 3D microtissues at precisely defined sizes of less than 10% variation in diameter as a distinct number of cells is confined but physically not limited in a drop to form one single spheroid.

Apr 2012 - In Vitro Tri-Culture Platform to Investigate Effects of Crosstalk between Mesenchymal Stem Cells, Osteoblasts and Adipocytes

Hammoudi TM, Rivet CA, Kemp ML, Lu H, and Temenoff JS. Tissue Eng Part A. 2012 Apr 3 (e-pub ahead of print)

Given the fact that cellular behaviour can be determined largely by the surrounding microenvironment, the authors applied photopatterning techniques to generate and assemble 3D laminated hydrogel modules of mesenchymal stem cells, osteoblasts, and adipocytes to recreate the bone marrow stem cell niche. With this approach effects of multi-directional and dynamic crosstalk between multiple cell types could be studied over time.

The authors could demonstrate that lineage specific transcription factors were induced by the co-culture and tri-culture set ups, albeit terminal differentiation was not reached. The trend towards adipocyte or osteoblast differentiation of mesenchymal stem cells was solely dependent on paracrine effects, since the experimental layout limited proliferation, migration and cell-to-cell contacts.

Mar 2012 - Synthetic 3D multicellular systems for drug development

Rimann M, Graf-Hausner U. 2012. Current Opinion in Biotechnology 23:1–7

This review highlights the current demand of the pharmaceutical and cosmetics research for more high-throughput, automation-compatible, but yet biologically highly relevant 3D cell-culture models. The authors list at present commercially available 3D cell culture products categorized either in scaffold-based or scaffold-free systems and novel read out techniques specifically tailored to the particular needs of 3D cell culture. The review also points out the challenges remaining for 3D cell culture and emphasizes the necessity for better standardization, automation and improved analytical methods in order for such 3D cell culture technologies to become widely implemented in routine screening processes.ual importance to mimic organ specific functions.

Mar 2012 - A Heterogeneous In Vitro Three Dimensional Model of Tumour-Stroma Interactions Regulating Sprouting Angiogenesis

Correa de Sampaio P, David Auslaender D, Krubasik D, Failla AV, Skepper JN, et al. 2012. PLoS ONE 7(2): e30753

Correa de Sampaio et al. developed a «mini-tumor» model encompassing endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231embedded in a matrix of type I collagen to study stromal-driven tumor angiogenesis. The authors could observe a cancer-cell dependent initial sprouting and subsequent formation of capillary structures with extensive matrix remodelling. An in-vivo like responsiveness to anti-angiogenic agents underscores the relevance of mini-tumor spheroids as an in-vitro model for early stages of tumor angiogenesis. As suggested by the authors, employing cells with constitutive expression of a fluorophore, such a model could also be used to study the invasive behaviour of cancer cells under the influence of a heterogeneous environment.

Mar 2012 - Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets

Kim K, Ohashi K, Utoh R, Kano K, Okano T. 2012. Biomaterials 33: 1406-1413

This report nicely describes the positive effect of an endothelial-hepatocyte co-culture system on the preservation of hepatocellular morphology and function. Sheets of bovine endothelial cells were stratified on top of a rat collagen/hepatocyte cell layer with the aid of a gelatine coated manipulator. While hepatocytes in the co-culture system at day 12 still showed clearly liver-specific morphological features such as Disse space formation, ECM deposition, and expanding bile canaliculi indicating active bile transport, hepatocyte monocultures were gradually losing their cuboidal shape and subcellular integrity. The co-culture model sustained stable long term functionality of albumin secretion, urea production and hepatocyte specific gene expression for up to 30 days. These observations clearly emphasize the need for more organotypic co-culture systems with parenchymal and non-parenchymal cells of equal importance to mimic organ specific functions.

Feb 2012 - Coherent angular motion in the establishment of multicellular architecture of glandular tissues

Tanner K, Mori H, Mroue R, Bruni-Cardoso A, and Bissell MJ. 2012. PNAS 109(6):1973-78

Applying real time fluorescent imaging of human breast epithelial cells in laminin rich ECM, Mina Bissel and her co-workers nicely demonstrate the formation of epithelial architecture (acini) upon cohesive rotational movements as cells undergo mitosis. Angular motion and sphere formation were tightly linked to cellular polarization and cell adhesion via E-cadherin, features that have been lost in malignant cells with deranged morphogenesis leading only to lose aggregates. These observations underscore the relevance of cell polarity and cell-cell contacts for proper epithelial morphogenesis through angular motility. high-throughput drug screening.

Feb 2012 - Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Drewitz M, Helbling M, Fried N, Bieri M, Moritz W, Lichtenberg J, Kelm JM. 2011. Biotechnol J. 6(12):1488-96

As 3D cell culture represents a more heterogeneous cell culture system by forming spatially defined microenvironments with nutrient and oxygen concentration gradients, zones of proliferation and cell cycle arrest and varying exposures to compounds, production and handling of 3D cell culture requires a high degree of standardization. Drewitz et al. investigated the reproducibility of scaffold-free tumor microtissues production using the hanging drop cell culture method handled either manually or with a robotic liquid handling device. The data convincingly demonstrate a high degree of reproducibility with respect to microtissue size and tumor spheroid growth characteristics. This resulted in highly robust IC50 values when HCT116 microtissues were treated with two different compounds. This report clearly shows how hanging drop technology can be readily implemented to produce microtissues for high-throughput drug screening.

Feb 2012 - The 3D tissue microenvironment modulates DNA methylation and E-cadherin expression in squamous cell carcinoma

Urich E, Patsch C, Aigner S, Graf M, Iacone R, Freskgård PO. Sci Rep. 2013, 3:1500

This intriguing study demonstrates the power of scaffold-free spheroid culture formats to produce highly organotypic structures, recapitulating the complex architecture of the blood brain barrier (BBB), simply by gravitational force and the cell’s inherent ability of self-arrangement. Human primary brain endothelial cells, human primary pericytes and human primary astrocytes were seeded into hanging drops at a uniform ratio which spontaneously formed a spheroid consisting of an astrocyte core, an endothelial cell mantle and pericytes residing in between the parenchyma and the endothelial outer layer - a structure highly reminiscent of the native cell organization of the BBB. The addition of endothelial cells stopped the growth of otherwise continuously growing astrocyte/pericyte co-cultures while the presence of pericytes within this model system had a profound effect on the expression levels of endothelial cell surface receptors, which could not be observed in a standard transwell co-culture set up. The authors present a convincing and ground-breaking new in vitro model for the study of BBB transport and CNS drug delivery.

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