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Development of the next generation of synthetic extracellular matrices for 3D cell culture

Development of the next generation of synthetic extracellular matrices for 3D cell culture

Dessert for organ regeneration

Imagine that you are dining at a Japanese restaurant and at the end of the meal, the waiter serves you this beautifully ­laid out and decorated dessert. Besides being brightly colored and simply delicious, some of these desserts might remind you of what is called “pudding” in western cuisine. Believe it or not, these Far Eastern sweet deserts are made of agarose, a polysaccharide extracted from red algae, which has been used as a cooking ingredient for more than 400 years. In Germany however, agarose has only recently been introduced into the vegetarian kitchen as a substitute for gelatine in cakes.

Besides, being found on German bakery racks, walk into a ­biology laboratory and you will find a bottle of agarose neatly tucked away among other laboratory supplies, ready to be dissolved in water and poured into slabs for various biological ­assays. Agarose has become an indispensable tool for the life scientist for analyzing and purifying DNA and proteins. This polysaccharide can form a hydrogel, which is a polymeric water container. This enables it to replicate in vitro the environment found in human tissue and as a result lends itself ideally to the growing field of regenerative medicine, i.e. engineering of tissue or organs, to restore normal function [1]. Since the mechanical properties of agarose recreate the natural surroundings of chon-drocyte cells, its use was confined to only mimicking cartilage tissue. However, a team in our laboratory at the University of Freiburg recently succeeded in extending agarose applications by replicating in vitro the whole range of mechanical properties found in mammalian tissues. This innovation offers completely new perspectives for the development of human tissue replicates in research­ laboratories [2].

The 2D cell culture model and its limitations

Cell cultures in the laboratory are usually performed on gamma-irradiated polystyrene (tissue culture plastic). The properties of the material allow­ the deposition of protein on its surface. Cells de­posited on the protein field attach, spread and multiply. This technique has been in use for decades in every cell biology laboratory around the world because of its convenience, ease of use and affordability. In short: it represents a user-friendly product. Unfortunately, the petri dish technique is not representative of the environment mammalian cells encounter in ­tissues. Indeed, while cells on tissue culture plastic (TCP) are organized in a 2D monolayer, cells in tissues organize around a 3D scaffold called the extracellular matrix (ECM), Figure 1. Made of different components such as macro proteins and polysaccharides, the ECM provides the nutriment to the cells, presents signaling proteins responsible for intercellular communication, and last but not least provides mechanical support. On a 2D monolayer, while the delivery of nutriment and proteins replicates quite accurately what is found in mammalian tissues, the mechanical support provided by the petri dish is completely different from the natural cell surroundings. Indeed, on 2D surfaces cells organize in a different way than in vivo: on TCP, cells that form the inside of blood vessels – endothelial cells – remain single and adopt an elongated shape, Figure 2A, whereas endothelial cells in the kidney collaborate to form a blood vessel, Figure 2D. In addition to the differences in shape, it has been demonstrated that the genetic information of 2D cells diverges from the same cell type in mammalian tissue [3,4].


Fig. 1 In their natural environment, the cells (green) use specific markers (pink) to bind to a mechanical support matrix of polysaccharides (yellow) and fibrous proteins (blue). Dissolved proteins such as growth factors (purple) enable communication between the cells and matrix-degrading enzymes (black), thus remodelling the matrix.


Fig. 2 The substrate on which the cells grow influences their shape and organisation. Fluorescence microscopy image of human umbilical vein ­endothelial cells (HUVECs) on separate substrates. The nucleus is blue, the actin filaments are stained green. A: On a 2D plastic tissue culture, the cells grow separately and have a flat shape. B: On Matrigel®, an animal extra­cellular matrix (ECM), the cells organise themselves into a network of capillaries. C: On the next-generation synthetic ECM, the cells form multicellular structures similar to blood vessels. D: Mouse kidney blood vessel; the cell nuclei are blue and the CAV1 protein specific to endothelial cells is stained green. Bars, 10m.

The tissue engineering paradigm

In order to reduce the gap between in vivo/in vitro experimental results, one of the envisioned solutions is to reproduce the natural tri-dimensional environment found in the different organs. This idea was first put forth by Steinberg in the early 60s [5] and evolved into the tissue-engineering paradigm in the mid-1980’s by Langer and Vacanti [6]. So as to recapitulate the cell environment one has to be able to (1) ­deliver the soluble signals, (2) provide a mechanical support, (3) present the specific and dedicated attachment motifs to the mechanical support and (4) give the possibility for the cell to degrade the support, thus allowing the remodeling and evolution of the tissue [7], Figure 3. Pursuing the goal toward the recapitulation of these features, many systems have been ­developed offering an alternative to the 2D models on TCP.


Fig. 3 The tissue engineering paradigm. A synthetic extracellular matrix (ECM) must copy the essential characteristics of tissue, i.e. it must offer mechanical support to which cells can bind, it must convey dissolved signalling substances to the cell, and it must permit its own degradation and remodelling by cell enzymes. (Aurelien Forget, doctoral dissertation (2013)).

Tissue mechanics as a biological signal

Concomitant to the engineering of 3D substrates, advances in matrix biology have enabled the identification of specific amino acid sequences recognized by the cells as an anchor to the matrix. The study of the impact of matrix topography on the shape and fate of the cells has revealed that the matrix also carries biological cues [8]. Therefore, projects aiming at understanding the biological signals involved in inter- and intra-cellular communication, e.g. research at the cluster of excellence BIOSS Centre for Biological Signalling Studies from the University of Freiburg, need also to take into account the biological implications of physical cues. In this ­regard, our group at the Institut für Makro­molekulare Chemie investigates the interaction of cells with their environment, and how physical information such as surface topography and sub­strate stiffness can affect the organization and function of cells. This knowledge is expected to aid one day in recreating fully functional tissues in the laboratory.

Challenges in engineering ­a synthetic ECM (SynECM)

3D cell culture systems developed to date unfortunately suffer from several drawbacks, key among them being: cost, need to have a customized material for each cell system, complexity of set up and use, and finally, difficulty with translation to the clinic due to the recourse of complex chemistry or materials originating from animals. These characteristics have slowed down the adaptation of 3D scaffolds over the use of 2D TCP in many laboratories for “everyday” experiments, and have limited their use to specialized laboratories having the “know-how” in both material science and cell biology. In order to compete with the user-friendly TCP 2D model, one has to provide innovative solutions offering a universal platform that can be used by any cell biologist without prior knowledge of material science. To fulfill this user need, we have developed an innovative synECM platform that overcomes the limitations of current 3D models by offering characteristics such as: (1) affordability, (2) translatability, (3) versatility and (4) ease of use, Figure 4.


Fig. 4 Characteristics required to develop the next generation of synthetic extracellular matrices

A new generation of synECM

(1) As a matter of fact, we have based our system on an abundantly naturally-occurring polysaccharide used to prepare Japanese dessert. Apart from being edible, agarose is an inert biocompatible material with a history of use as a matrix for in vitro and in vivo applications. Since agarose does not exhibit any biological information, the biological signals presented in the synECM only originates from the soluble or immobilized protein on the backbone introduced by the user, thus resulting in what is referred to by scientist as a low biological background noise system.

(2) Chemical modification of agarose enables physical hydrogels, which exhibit a sol-gel transition around room temperature and can be formed in a body cavity. This characteristic makes agarose a material with good translational potential as it can be injected as a solution that will gel instantly in the tissue without resorting to chemical crosslinkers.

(3) Current synthetic ECM systems are designed for the recapitulation of a single tissue environment. Therefore, we have chemically modified agarose backbone in such a way that the hydrogel mechanical properties can match the one of many tissue environments ranging from cartilage to brain tissue. This unique and versatile property permits the use of our system for the design of environments for various cells.

(4) Finally, by characterizing the modified agarose at the fundamental level, a material system that can be reconstituted to present precise mechanical properties and cell-interaction peptides has been realized.

The agarose-based synthetic ECM recapitulates the different components that constitute natural tissues. In doing so, the system provides a unique environment in which endothelial cells organize in vitro (Figure 2C), in a similar manner to what is observed in vivo (Figure 2D), and which does not occur on 2D TCP platforms and most commonly used animal extract matrix – Matrigel (Figure 2B).

The future of synthetic ECM

SynECMs are expected to play a vital role on many fronts in the burgeoning arena of biotechnology. In addition to providing a reliable alternative to biologically-derived products such as systems made of decellularized animal tissue as research platforms, synECMs also provide an attractive option in the development of automated high throughput screens that are based on organ and organoid cultures. Such organ culture-based screening platforms can aid in the more rapid discovery and development of drugs candidates, which can not only reduce the lead time for the translation of a potential drug from the lab to phase-I clinical trials but also help in reducing animal testing. In order for synECMs to be integrated in high-throughput technology, the cost and ease of manipulation are vital. Ability to impose highly-defined cellular microenvironments both at the physical and biological level may help in unraveling (deciphering) new clues in disease development, detection and treatment. A synECM essentially adds a long-sought third dimension to what is typically a 2D screening platform. The “one pot” synthesis of highly-defined synECMs can further assist this transition from 2D to 3D. With this goal in mind, our agarose platform provides the first step toward a 3D scaffold system in which the physical attributes and biologically active motifs can be assembled by the final user through simple mixing of the different components. Such technology could be developed by leveraging emerging concepts in peptide-protein interactions.

Under the auspices of the cluster of excellence BIOSS Centre for Biological Signalling Studies, our efforts in engineering a naturally-occurring poly-saccharide to possess diverse physicochemical characteristics has accelerated the transition of routine laboratory cell biology studies from a 2D to 3D system. Our focus on the end user has enabled us to identify and implement key properties such as affordability and ease of use to ensure maximum adoption of the agarose-based synECMs. So, next time you visit a Japanese restaurant please don’t forget to order the dessert. You never know – it may save your life one day.

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Foto: © panthermedia.net| Hiroshi Tanaka

L&M int. 2 / 2014

The articles are publishes in issue L&M int. 2 / 2014.
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