Bridging the gap between in vivo and in vitro testing

Collaborators in The Netherlands have created a 3D well system integrated into biomaterials that can be used either in vitro or in vivo for molecule screening in this HOT Technical Innovation.

The research was led by Gustavo Higuera and Jeanine Hendriks at Screvo Ltd and CellCoTec, with a team at The University of Twente. Avoiding animal experimentation by using 3D systems that mimic the in vivo environment is desirable yet challenging. Optimising animal experiments to be as efficient and effective as possible is a common goal of much research.

This team envisioned the integration of such in vitro 3D cell culture mimicking methods with biocompatible materials into animal models. To do this, they create a 9-well system that can be implanted into four sites under the skin of one mouse for in vivo screening. This enables testing of up to thirty six different conditions at once.

The innovative platform was produced from a wide range of biomaterials chosen for biocompatibility instead of conventional materials like PDMS. The cell culture compatibility of wells made with copolymer PEOT-PBT was tested using human mesenchymal stem cells. They test the systemic effect of the device on surrounding tissue of twenty mice.

This innovative bridge between in vivo and in vitro experimentation has great potential to both increase throughput and minimise animal experimentation by reducing the number of animals needed. Read the article in full now as it’s free to access for the next 4 weeks*:

In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal
Gustavo A. Higuera, Jeanine A. A. Hendriks, Joost van Dalum, Ling Wu, Roka Schotel, Liliana Moreira-Teixeira, Mirella van den Doel, Jeroen C. H. Leijten, Jens Riesle, Marcel Karperien, Clemens A. van Blitterswijk and Lorenzo Moroni 
DOI: 10.1039/C3IB40023A

 *Free access to individuals is provided through an RSC Publishing personal account. Registration is quick, free and simple

 

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HOT article! Contactless dielectrophoresis to assess drug efficacy

Today’s HOT article as recommended by the referees is from Rafael Davalos and colleagues at Virginia Tech, USA. Using contactless dielectrophoresis, the changes in the electrical properties and surface roughness of cancer cells treated with non-toxic doses of spingolipid  metabolites.

One metabolite, S1P, promotes tumour formation and the second, So, has anti-tumour properties. The electrical properties of cancer cells change as they transform from benign to malignant. Contactless electrophoresis means that there is no contact between the cells and the electrodes, ruling out challenges of traditional techniques.

Using this technique, the researchers show that treatment with So partially transforms late stage malignant mouse ovarian surface epithelial cancer cells back to a benign state. S1P as expected does not reverse the transformation.

The exploration of potential non-toxic treatments as an alternative to highly toxic chemotherapy and of drugs whose action is not affected by the presence of variable biomarkers is important. This study shows that So has the potential for such therapy and it also demonstrates contactless dielectrophoresis as a useful tool in drug efficacy studies.

As a HOT article, we’ve made it free to access for the next 4 weeks!

Sphingolipid metabolites modulate dielectric characteristics of cells in a mouse ovarian cancer progression model
Alireza Salmanzadeh, Elizabeth S. Elvington, Paul C. Roberts, Eva M. Schmelz and Rafael V. Davalos
DOI: 10.1039/C3IB00008G

 *Free access to individuals is provided through an RSC Publishing personal account. Registration is quick, free and simple 

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HOT article! Kidney on a chip for drug toxicity tests

HOT research from Donald Ingber and colleagues at the Wyss Institute for Biologically Inspired Engineering at Harvard University, USA, and Seoul National University, Korea, on a more accurate microfluidic mimic of kidney function is described in this article.

The device contains a porous polyester membrane dividing the device into two channels. This provides an apical luminal channel and a basal interstitial channel. They culture primary kidney epithelial cells onto the upper surface of the membrane coated with extracellular matrix.

The microfluidics aspect is in the exposure of the cells to fluid shear stress in the apical channel, mimicking natural kidney tubules and ensuring the device cells phenotype is closer to those in vivo. The researchers compare the cells’ performance with cells cultured in static culture conditions. The flow is vital to maintain many cell functions.

The measurement of cytotoxicity to cisplatin was closer to the in vivo response than conventional culture conditions. Quantitative analysis and visualisation of the biological processes of a kidney tubule are made possible. This more accurate organ-on-a-chip makes innovative steps towards reliable investigation of drug toxicity to the human kidney in an artificial system rather than an animal system.

This HOT article is free to access for 4 weeks*!

Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment
Kyung-Jin Jang, Ali Poyan Mehr, Geraldine A. Hamilton, Lori A. McPartlin, Seyoon Chung, Kahp-Yang Suh and Donald E. Ingber 
DOI: 10.1039/C3IB40049B

 *Free access to individuals is provided through an RSC Publishing personal account. Registration is quick, free and simple 

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Issue 5 now online! 3D cell culture, systems medicine, single-molecule imaging and more high impact Integrative Biology research

The research highlighted by this week’s front cover artwork comes from Hexin Chen and Qian Wang et al. at the University of South Carolina, USA.  This cover article was featured on the blog last week and will now be free to access for the next 6 weeks*. The team are focused on how 3D culture conditions affects cancer cell behaviour. In this cover article, they study the effect of fibrous scaffolds, discovering that the epithelial-to-mesenchymal transition of the MCF-7 cells is enhanced.

Expansion of breast cancer stem cells with fibrous scaffolds
Sheng Feng, Xinrui Duan, Pang-Kuo Lo, Shou Liu, Xinfeng Liu, Hexin Chen and Qian Wang
DOI: 10.1039/C3IB20255K


 

Issue 5 includes a review article from Editorial Board member Philip Day at University of Manchester, UK, and Ehsan Ghayoor Karimiani, Mashhad University of Medical Sciences, Iran. They review the impact of quantitative single cell measurements on future therapies for haematological malignancies. This article was also the subject of an interesting April blog post, which you could read now for a brief idea of what this interesting review covers.

Personalised treatment of haematological malignancies through systems medicine based on single molecules in single cells
Ehsan Ghayoor Karimiani and Philip Day
DOI: 10.1039/C3IB20258E    


 

You will also find a tutorial review on single molecule imaging (featured on the blog here) and of course highly significant primary research and two Technical Innovations in this month’s issue of Integrative Biology:

Single-molecule imaging in vivo: the dancing building blocks of the cell
Miguel Coelho, Nicola Maghelli and Iva M. Tolić-Nørrelykke 
DOI: 10.1039/C3IB40018B

A model of membrane contraction predicting initiation and completion of bacterial cell division
Claire E. Dow, Alison Rodger, David I. Roper and Hugo A. van den Berg
DOI: 10.1039/C3IB20273A

Three-dimensional photolithographic micropatterning: a novel tool to probe the complexities of cell migration
Joseph C. Hoffmann and Jennifer L. West
DOI: 10.1039/C3IB20280A

*Free access to individuals is provided through an RSC Publishing personal account. Registration is quick, free and simple

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Most Read: Using microenvironment activity to predict tumour growth

A cross-departmental team at the University of Wisconsin-Madison led by Elaine Alarid and Integrative Biology Associate Editor David Beebe use a tumour biomarker to study the surrounding microenvironment by applying microfluidics and immunofluorescence techniques in this iBiology article. Using this innovative method, they can predict how the microenvironment will influence the growth of the tumour.

The biomarker used is Estrogen Receptor-α (ERα), which is a therapeutic target in the most common form of breast cancer. There are a number of different factors involved in the ERα signalling pathway; in this article these processes are defined as the microenvironment activity.

The researchers use an array of different microenvironments in a high-throughput microfluidic co-culture model to test the different variables. Quantitative immunofluorescence imaging is used to monitor ERα levels.  Other measures used are gene expression and phosphorylation status.

They discover a previously unrecognised linear correlation between growth of the tumour and decreases in ERα protein levels, indicating activation, under most of the various signalling input conditions. This work enables ERα regulation to be a dynamic biosensor of microenvironment activity for predicting tumour growth. Two of the conditions analysed showed a different correlation with tumour growth, with the growth appearing to be independent of ERα. The group will be looking into this further as it may have implications for the effectiveness of therapy.

They envision co-culturing samples of patient tumour microenvironment with MCF-7 cells acting as a biomarker for ERα activation. Read on for more detail into the assay and the results:

Hormonally responsive breast cancer cells in a microfluidic co-culture model as a sensor of microenvironmental activity
Jessica D. Lang, Scott M. Berry, Ginny L. Powers, David J. Beebe and Elaine T. Alarid
DOI: 10.1039/C3IB20265H

Submit your work to David’s Editorial Office today at http://mc.manuscriptcentral.com/ib!

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HOT article: Testing the mechanisms of touch sensitivity

A team at Stanford University, USA, test how changing the stiffness of the body changes sensitivity of touch of the model worm C. elegans using a microelectomechanical clamp.

Touch is the least understood of the five human senses. It is obvious that force applied to the skin is an important stimulator of neurons and mechanoreceptors below the surface. The mechanics of this process are not understood. In this HOT article, the researchers test the hypothesis that body stiffness influences touch sensitivity.

The microelectromechaical (MEMS) force clamp applies a force to a freely crawling worm and the team then measured the response stimulated by the level of force. The clamp is integrated with a moving stage. The worms are sensitive to under 1 μN of force and to indentations under 1 μm deep.

The team led by Miriam Goodman and Beth Pruitt then genetically alter the wild type worms’ skin proteins, softening the skin. They also modify their ability to control body wall muscles to show the effects of changing body mechanics.  Even small changes resulted in dramatic changes in force sensitivity, but had little effect on indentation depth sensitivity. They looked into whether the worms were directly responding to the force or whether they were reacting to the production of indentations. The conclusion is that the indentation of the skin drives touch sensitivity.

In this HOT article, the researchers also use a theoretical model to understand different kinds of deformation and force transfer involved. The article also discusses how this research relates to other animals and humans.

We’ve made this HOT article free to access for the next 4 weeks*:

MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation
Bryan C. Petzold, Sung-Jin Park, Eileen A. Mazzochette, Miriam B. Goodman and Beth L. Pruitt
DOI: 10.1039/C3IB20293C

You can also now find Integrative Biology HOT articles on Facebook!

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How does 3D culture affect cancer stem cell behaviour?

A team of researchers at the University of South Carolina are investigating how cancer cells respond to the conditions of 3D cell culture and how we can use their behaviour to study anti-cancer drugs and cancer stem cells. In their work they combine biology with mathematics and biomaterials chemistry.

cancer stem cells, 3D cultureIn this just published Integrative Biology paper, the team led by Hexin Chen and Qian Wang, culture MCF-7 cells on 3D polycaprolactone (PCL) fibrous scaffolds. The fibers are randomly orientated as in a cancerous environment collagen fibres surround breast cancer cells randomly. The fibrous scaffolds are made by electrospinning.

They discover that the epithelial-to-mesenchymal transition of the cells, required for cancer cell migration and malignancy, is enhanced when human epithelial breast cancer cells are cultured on this 3D fibrous substrate. For 2D cultures, the population of cancer stem cells remains constant.

Elucidating the exact mechanism of how will be part of the group’s further research. The researchers will also be expanding their previous mathematical models of cancer stem cell growth in tumours to include transformation of non-stem cells to stem cells. This would give an insight into whether the increase in cancer stem cells is down to non-stem cells converting or increasing division of those cancer stem cells already present.

Expansion of breast cancer stem cells with fibrous scaffolds
Sheng Feng, Xinrui Duan, Pang-Kuo Lo, Shou Liu, Xinfeng Liu, Hexin Chen and Qian Wang
DOI: 10.1039/C3IB20255K

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Personalised treatment using systems biology – a review

Diagnosis and monitoring of many diseases usually involves testing for a specific disease-related molecular biomarkers from a small sample of tissue. Within a single tissue, the contents and functions of individual cells vary greatly. The regulation of the genome varies from cell to cell due to different regulatory mechanisms taking control.

If we could quantify the number of biomarker molecules per cell in a diseased sample this will lead to a more personalised approach to disease. For example, getting such a profile of all of the different cell types involved on one particular person’s leukaemia would reveal the different biomarkers and their combinations that are resulting in the disease. Treatment could then be based on this detailed information.

Integrative Biology Editorial Board member Philip Day, University of Manchester, UK, and Ehsan Karimiani at the Mashhad University of Medical Sciences, Iran, present a complex but fascinating review of the steps that are being made towards personalisation of disease monitoring made possible by such a quantitative systems medicine approach to genetic biomarker analysis.

The focus is on quantitative single cell measurements for haematological malignancy monitoring. This review includes: 

  1. Sample preparation
  2. The multidisciplinary future of molecular diagnostics
  3. Nucleic acid profiling for diagnostics
  4. Gene expression profiling of single cells for diagnostics
  5. The role of minaturisation and microfluidics in PCR

The review concludes with a look at what may be possible in the future, with Karimiani and Day emphasising that current practices need to change drastically before this personalised approach can become a reality.

For more in depth explanation, read the review in full:

Personalised treatment of haematological malignancies through systems medicine based on single molecules in single cells
Ehsan Ghayoor Karimiani and Philip Day
DOI: 10.1039/C3IB20258E

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Single-molecule imaging’s insights into the puzzling components of cells

Iva Tolić-Nørrelykke and colleagues at the Max Planck Institute of Molecular Cell Biology and Genetics, Germany, have written a fascinating tutorial review on how single-molecule imaging is revealing the pieces of cellular jigsaw puzzles.

single-molecule imaging

They see the cell as a dynamic puzzle, with pieces being created, destroyed and changing their interactions all the time. These pieces can now be viewed in vivo using various techniques in single-molecule microscopy with high precision. The measurement of intracellular reactions can also be carried out in order to build models of intricate cellular processes.

As well as a brief chronological look at the developments in single-molecule microscopy and useful short explanations of the different techniques as a glossary, this review includes: 

  1. Appropriate labelling methods
  2. Total Internal Reflection Fluorescence (TIRF) in vivo
  3. Widefield microscopy for a low signal-to-noise ratio and Selective Plane Illumination Microscopy (SPIM)
  4. Fluorescence Speckle Microscopy using tubulin and Photoactivation with a specific wavelength
  5. Mapping lots of individual molecules inside a cell using super-resolution fluorescence methods
  6. Single-molecule FRET for studying conformational change

They emphasise that due to the complications arising inside a cell, the molecules must be resolved spatially in different ways according to the different techniques and they discuss which techniques are best suited to which types of investigation. Single-molecule imaging is compared with ensemble methods and the goal of measuring a large number of molecular events simultaneously in real time is discussed.

Learn more about the potential of single-molecule imaging in the full Tutorial review:

Single-molecule imaging in vivo: the dancing building blocks of the cell
Miguel Coelho, Nicola Maghelli and Iva M. Tolić-Nørrelykke
DOI: 10.1039/C3IB40018B

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Issue 4 online! EGF receptor cluster study, dynamic study of NK cells and large scale adjuvant screening

Issue 4 is online is here with these three fascinating articles featured on the cover. All of our cover articles are free to access for 6 weeks*!

A technical innovation from Björn Önfelt and colleagues at Royal Institute of Technology, Karolinska Institute and Science for Life Laboratory, Sweden, is highlighted on the outside front cover. They enable real-time, dynamic study of immune synapse formation and the cytotoxicity of natural killer cells, as individual NK cells in a population have different cytotoxic responses. The method is an assay using ultrasound-assisted cell–cell aggregation in a multi-well chip. The ultrasound allows the timing and positioning of the aggregation to be controlled and interaction between NK cells can be induced mimicking biological forces.

Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity
Athanasia E. Christakou, Mathias Ohlin, Bruno Vanherberghen, Mohammad Ali Khorshidi, Nadir Kadri, Thomas Frisk, Martin Wiklund and Björn Önfelt
DOI: 10.1039/C3IB20253D


 

Following on from this, an article focused on a nanoimmunoassay on a microfluidic chip for vaccine adjuvant screening is featured as the inside front cover. The work led by Jeffrey Hubbell and Sebastian Maerkl at EPFL, Switzerland, is aimed at large scale, high-throughput biomarker quantification using a microarray robot. The reagents cost 1000 times less than those for traditional ELISA and low sample volumes of only a few nanolitres are required.

A high-throughput nanoimmunoassay chip applied to large-scale vaccine adjuvant screening
Jose L. Garcia-Cordero, Chiara Nembrini, Armando Stano, Jeffrey A. Hubbell and Sebastian J. Maerkl
DOI: 10.1039/C3IB20263A


 

The back cover illustrates the work of Khalid Salaita, Emory University, USA. The paper by Salita et al. looks at the assembly of the epidermal growth factor receptor (EGFR) into clusters after EGF has bound. Despite the wide study of EGF, the role of the clusters was previously unknown. To do this, they quantify the difference in phosphorylation of EGFR when the binding occurs on mobile (nanopatterned supported lipid bilayer) versus immobile (glass slide) surfaces. This is to control the clustering and assess the activation levels of the receptors able to cluster compared to those constrained from clustering normally. They find that cluster size affects receptor phosphorylation. This biological insight, interesting in itself, will be useful for surface-tethering in biomaterials.

Manipulating the lateral diffusion of surface-anchored EGF demonstrates that receptor clustering modulates phosphorylation levels
D. Stabley, S. Retterer, S. Marshall and K. Salaita
DOI: 10.1039/C3IB20239A

*Free access to individuals is provided through an RSC Publishing personal account. Registration is quick, free and simple

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