Since collagen is one of the major extracellular matrix components in vascular tissues, its use for vascular tissue engineering has several advantages. However, collagen extraction and processing for tissue engineering application alters its structure. As a result, collagen-based vascular constructs show poor mechanical properties compared to native tissues. In this work, multi-layer (single, double, and triple) vascular tissue constructs were engineered from porcine smooth muscle cells (PSMCs) entrapped in collagen gel by concentrically and sequentially layering after compaction of the previous layer(s). The engineered tissues were matured for either 14 or 21 days to allow the collagen gel to remodel before viscoelasticity, compliance, histological, and protein expression studies were conducted. While there was no significant difference upon addition of the different layers on the elastic modulus (p > .05), the viscous modulus of the single layer construct was significantly lower than the double and triple layer constructs (p < .05). Increasing the number of layers of the cellularized collagen construct increased the wall thickness and the viscous modulus of the construct. Furthermore, the cellularized single-layer construct had a relatively high compliance, but the double and triple layer constructs had compliance values comparable to both engineered vessels and native vessels. PSMCs were uniformly distributed throughout the cross-section and expressed the anticipated marker proteins smooth muscle-α actin, calponin, and smooth muscle myosin heavy chain. Taken together, this study demonstrated the viscoelastic responsiveness of multi-layer collagen-gel based vascular tissues.

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Replacing or improving organ functions through tissue engineering techniques requires the development of substitutes with biological and mechanical features as close as possible to the natural ones. However, the structural complexity of biological tissues generates a complex mechanical behavior. Hence, biological tissues express a nonlinear viscoelastic mechanical behavior. Natural protein-based hydrogels, especially collagen-based hydrogels, represent adequate scaffolds for the in vitro tissue regeneration not only because they can provide the required biological cues for cells adhesion, proliferation, and differentiation but also because they exhibit similar nonlinear viscoelastic behavior. After a brief description of the mechanical characteristics of native vascular tissue, this chapter describes the conventional techniques listed in literature for the mechanical characterization of such materials and highlights their limits. Since native tissues and collagen hydrogels are unconventional materials, they require unconventional characterization techniques. In the absence of a normative in the field, this chapter proposes an unconventional methodology to evaluate the viscoelastic behavior of biological tissues. In this chapter, adequate mechanical setups and related protocols are suggested for the accurate and repeatable evaluation of the pertinent viscoelastic parameters. The different viscoelastic models that should be considered to assess the mechanical parameters of interest are presented. The way of analyzing the experimental data and of interpreting them are discussed. Based on the selected viscoelastic model, a graphical simulation tool is used to analyze the results and to predict the viscoelastic behavior of the hydrogels in specific environment. Finally, the poroelastic theory is introduced to take into account the porosity of collagen hydrogels and the viscoelastic contribution of the interstitial fluid.

Event Abstract Back to Event On the potential of static culture for the reinforcement of collagen-based tissue-engineered vascular substitutes through cell remodeling Sébastien Meghezi1, Dawit Gezahegn Seifu1, 2, Nina Bono1, 3, Caroline Loy1 and Diego Mantovani1 1 CHU de Québec Research Center, Laval University, Lab. Biomaterials & Bioengineering, CRC-I, Dept Min-Met-Mater Eng, Canada 2 University of Alberta & National Institute for Nanotechnology National Research Council, Department of Chemical and Materials Engineering, Canada 3 Politecnico di Milano, Department Electronics, Information and Bioengineering, Italy Introduction: Vascular tissue engineering has made significant progress over the past decade towards the creation of functional living tissues that can be used not only for the replacement of damaged arteries but also for the development of in vitro models of vascular tissue[1]. Collagen scaffolds provide biological cues that are essential for cells to produce neo-extracellular matrix[2]. However, these models still lack adequate mechanical properties and tissue organization to better mimic the natural tissues[3]. This work focuses on the combined effects of the static culture and cell remodeling on tissue organization and mechanical properties. Additionally, it gives an insight on the intimate relationship between cells and collagen fibrils reorganization and the anisotropic viscoelastic properties. Materials and Methods: Collagen gel-based tubular constructs were prepared by mixing collagen solution (4g/L), with buffer solutions and smooth muscle cells (SMCs) at a final concentration of 106 SMCs and 2g of collagen per mL of gel[4],[5]. After 1h of gelation in a tubular mold, this construct was placed in specifically-designed static bioreactor with fresh medium culture (Figure 1) for further static maturation for 1 or 2 weeks. Immunofluorescence (IF) analyses were performed on these samples to observe the organization of cells and collagen fibrils within the constructs. Briefly, after fixation, embedding and mounting of the samples, cell nuclei and actin filaments were stained with Dapi (1:3000) and Rhodamin Phalloïdin (1:200) respectively and observed with an Olympus BX51 fluorescence microscope. To evaluate the anisotropic mechanical and viscoelastic properties of these statically cultured constructs, stress relaxation tests were assessed in two directions: in the longitudinal one or in the circumferential one, respectively directly on the tubular constructs or on rings cut out of them. Samples were mounted on an Electropuls Microtester (Instron Corporation, Norwood, MA, USA), stretched at 10% (5%/s) and maintained constant for 600s while monitoring the decay of the stress. The same procedure was repeated at 20% and 30%. Results and Discussion: The method described here resulted in a homogenous mixture of SMCs-collagen in tubular shape in a one-step process. IF analyses showed that SMCs were uniformly distributed and longitudinally oriented within the constructs after static culture (Figure 2). This cell-driven reorganization had a great impact on the overall viscoelastic behavior of the constructs. Indeed, important mechanical reinforcement occurred during the 2 weeks of static culture since the elastic modulus in the circumferential direction increased by a factor 35. Moreover, cell remodeling during this period resulted in anisotropic elastic properties since the longitudinal elastic modulus was 3 times higher than the circumferential one (similar trend in the viscous behavior) (Figure 3). Conclusions: Cell-driven remodeling of the constructs during static culture resulted in an important reorganization of cells and collagen matrix and overall increase in the mechanical properties. Hence, through an original and simple method, static culture of engineered tissues appears to be crucial step leading to tubular constructs that can be easily manipulated for further mechanical stimulation. Moreover, this work pointed out the intimate relationship between tissue reorganization and anisotropic mechanical properties. Therefore, this rapid easy-to-process method allows to produce and culture viable or pathological arterial models, with more physiological organization and mechanical properties, in the potential of being used for diagnosis purposes, drug screening or therapeutic. This work was partially funded by NSERC-Canada, CIHR-Canada, FRQ-NT-Quebec, CFI-Canada. CL and DS were awarded of a doctoral scholarship from NSERC Create Program in Regenerative Medicine (www.ncprm.ulaval.ca)References:[1] Catto, V., Farè, S., Freddi, G. & Tanzi, M. C. Vascular Tissue Engineering: Recent Advances in Small Diameter Blood Vessel Regeneration. ISRN Vascular Medicine 2014, 1–27, doi:10.1155/2014/923030 (2014)[2] L’Heureux, N., Germain, L., Labbe, R. & Auger, F. A. In vitro construction of a human blood vessel from cultured vascular cells: A morphologic study. Journal of Vascular Surgery 17 (3), 499–509, doi:10.1067/mva.1993.38251 (1993)[3] Meghezi, S., Couet, F., Chevallier, P. & Mantovani, D. Effects of a pseudophysiological environment on the elastic and viscoelastic properties of collagen gels. International Journal of Biomaterials 2012, 319290, doi:10.1155/2012/319290 (2012)[4] Rajan, N., Habermehl, J., Coté, M.-F., Doillon, C. J. & Mantovani, D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nature protocols 1 (6), 2753–8, doi:10.1038/nprot.2006.430 (2006).[5] Meghezi, S., Seifu, D. G., Bono, N., Unsworth, L., Mequanint, K. & Mantovani, D. Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration. Journal of Visualized Experiments (100), doi:10.3791/52812 (2015) Keywords: Tissue Regeneration, 3D scaffold, mechanical property, matrix-cell interaction Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Biomaterials in constructing tissue substitutes Citation: Meghezi S, Gezahegn Seifu D, Bono N, Loy C and Mantovani D (2016). On the potential of static culture for the reinforcement of collagen-based tissue-engineered vascular substitutes through cell remodeling. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02325 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sébastien Meghezi Dawit Gezahegn Seifu Nina Bono Caroline Loy Diego Mantovani Google Sébastien Meghezi Dawit Gezahegn Seifu Nina Bono Caroline Loy Diego Mantovani Google Scholar Sébastien Meghezi Dawit Gezahegn Seifu Nina Bono Caroline Loy Diego Mantovani PubMed Sébastien Meghezi Dawit Gezahegn Seifu Nina Bono Caroline Loy Diego Mantovani Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Event Abstract Back to Event Cellularized collagen gels for tissue engineered vascular wall: in vitro models alternative to in vivo testing Caroline Loy1, Sébastien Meghezi1, Dawit G. Seifu1, 2, Daniele Pezzoli1, Nina Bono1, 3, Lucie Levesque1, Bernard Drouin1 and Diego Mantovani1 1 CHU de Quebec, Research Center, Laval University, Lab. for Biomaterials and Bioengineering, CRC-1, Dept. Min-Met-Materials Eng, Canada 2 University of Alberta & National Institute for Nanotechnology National Research Council, Department of Chemical and Materials Engineering, Canada 3 Politecnico di Milano, Department Electronics, Information and Bioengineering, Italy Introduction: Type I collagen-gels, as scaffolds for vascular tissue engineering, have a high potential for supporting and guiding vascular cells in the regeneration process[1]. With this in mind, our project was to develop a set of easy-to-prepare collagen-based in vitro vascular wall models and experimental techniques to thoroughly characterize them. Materials and Methods: Cellularized collagen gels were prepared by mixing vascular cells (106cells/ml) with a type I collagen solution (4g/L) as described before[2] and molded both in flat and tubular geometries. ECs were seeded on the surface of tubular constructs at the density of 8x105cells/cm2 using a homemade rotating bioreactor[3],[4]. Mono-culture (SMCs), bi-culture (SMCs and ECs) and tri-culture (FBs, SMCs and ECs) multilayered models were developed. The viscoelastic properties were studied on disk shaped constructs by compression test using MACH-1 (Biomomentum Inc), whereas on tubular constructs by stress-relaxation tests using ElectroPuls E1000 (Instron Corporation) longitudinally and circumferentially[4]. Protein expression by SMCs was investigated by western blot and the blood compatibility of constructs by clotting time assays. Tubular constructs were cultured in Instron Tissue Engineering & Regenerative Medicine (TERM) bioreactors with dynamic stimulations. Results and Discussion: Mono-, bi- and tri-culture models of vascular wall composed of collagen and vascular cells in flat disk and tubular geometries were developed. The structure and cellular distribution of the tri-culture models closely matched those of native vascular wall, reproducing tunica intima, tunica media and tunica adventitia. Altogether, the models allowed to investigate the multiple intimate relationships existing among vascular cells. Importantly, SMCs and FBs remodeled the constructs by contracting the structure. A functional monolayer of ECs (Fig. 1) with anticoagulant properties was achieved. Viscoelastic properties were assessed through stepwise stress relaxation tests by stretching the sample at 10% strain, and maintained constant for 600s while monitoring the stress and the same procedure was repeated until 80% strain was achieved. It showed that the total stress (peak at the beginning of the relaxation, Fig. 2 green line) increased with the number of cellularised collagen layers, while the elastic properties (value at the end of relaxation, Fig. 2 red line) remained the same (Fig. 2). Anisotropic mechanical properties in longitudinal and circumferential directions were observed. Tubular constructs, strong enough to be transferred and cultured into the Instron-TERM bioreactor (Fig. 3) were obtained after 1 week static conditioning . Cyclic mechanical stimulations (0-50 mmHg, 1 Hz) allowed to further improve the mechanical properties of the constructs. Conclusions: Herein, we engineered a platform of in vitro collagen gel-based vascular wall tissue models that can be used for physiological genomics studies. Sets of techniques were also developed to maturation and functional characterization of these structures biologically as well as mechanically. Furthermore, these models can serve as platforms for tightly controlled, high-content screening of drugs and devices in pharmacodynamic analyses. This work was partially funded by NSERC-Canada, CIHR-Canada, FRQ-NT-Quebec, CFI-Canada. CL and DS were awarded of a doctoral scholarship from NSERC Create Program in Regenerative Medicine (www.ncprm.ulaval.ca).References:[1] Seifu D, et al., Nat Cardio. 2013[2] Rajan N, et al., Nat Protoc. 2006[3] Boccafoschi F, et al., Macromol Biosci. 2007[4] Meghezi S, et al., JoVE. 2015 Keywords: Tissue Engineering, blood vessel, 3D scaffold, mechanical property Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Loy C, Meghezi S, Seifu DG, Pezzoli D, Bono N, Levesque L, Drouin B and Mantovani D (2016). Cellularized collagen gels for tissue engineered vascular wall: in vitro models alternative to in vivo testing. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00135 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Caroline Loy Sébastien Meghezi Dawit G Seifu Daniele Pezzoli Nina Bono Lucie Levesque Bernard Drouin Diego Mantovani Google Caroline Loy Sébastien Meghezi Dawit G Seifu Daniele Pezzoli Nina Bono Lucie Levesque Bernard Drouin Diego Mantovani Google Scholar Caroline Loy Sébastien Meghezi Dawit G Seifu Daniele Pezzoli Nina Bono Lucie Levesque Bernard Drouin Diego Mantovani PubMed Caroline Loy Sébastien Meghezi Dawit G Seifu Daniele Pezzoli Nina Bono Lucie Levesque Bernard Drouin Diego Mantovani Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The acquisition of new thorough knowledge on the interactions existing between vascular cells would represent a step forward in the engineering of vascular tissues. In this light, herein we designed a physiological-like tri-culture in vitro vascular wall model using a planar cellularized collagen gel as the scaffold. The model can be obtained in 24 h and features multi-layered hierarchical organization composed of a fibroblast-containing adventitia-like layer, a media-like layer populated by smooth muscle cells and an intima-like endothelial cell monolayer. After 7 days of static culture, the compaction of the collagen matrix by the vascular cells was achieved, and the deposition of the vascular extracellular matrix components fibronectin, fibrillin-1 and tropoelastin was observed. The blood-compatible functionality of the endothelial cell monolayer was demonstrated by a blood clotting assay: after 7 days of maturation, clotting was prevented on the endothelialized constructs (more than 80% free hemoglobin maintained after 60 min of blood contact) but not at all on non-endothelialized ones (less than 20% free hemoglobin). In addition, western blotting results suggested that in the tri-culture model the loss of smooth muscle cell phenotype was delayed compared to what was observed in the mono-culture model, finally resulting in a behaviour more similar to the in vivo conditions. Overall, our findings indicate that this in vitro model has the potential to be used as an advanced system to examine vascular cell behavioural interactions, as well as for drug testing and the investigation of physiological and pathological processes.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled. The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The "static bioreactor" provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

Vascular tissue engineering aims to regenerate blood vessels to replace diseased arteries for cardiovascular patients. With the scaffold-based approach, cells are seeded on a scaffold showing specific properties and are expected to proliferate and self-organize into a functional vascular tissue. Bioreactors can significantly contribute to this objective by providing a suitable environment for the maturation of the tissue engineered blood vessel. It is recognized from the mechanotransduction principles that mechanical stimuli can influence the protein synthesis of the extra-cellular matrix thus leading to maturation and organization of the tissues. Up to date, no bioreactor is especially conceived to take advantage of the mechanobiology and optimize the construct maturation through an advanced control strategy. In this review, experimental strategies in the field of vascular tissue engineering are detailed, and a new approach inspired by fetal development, mechanobiology and optimal control paradigms is proposed. In this new approach, the culture conditions (i.e. flow, circumferential strain, pressure frequency, and others) are supposed to dynamically evolve to match the maturity of vascular constructs and maximize the efficiency of the regeneration process. Moreover, this approach allows the investigation of the mechanisms of growth, remodeling and mechanotransduction during the culture.

Vascular tissue engineering focuses on the replacement of diseased small-diameter blood vessels with a diameter less than 6 mm for which adequate substitutes still do not exist. One approach to vascular tissue engineering is to culture vascular cells on a scaffold in a bioreactor. The bioreactor establishes pseudophysiological conditions for culture (medium culture, 37°C, mechanical stimulation). Collagen gels are widely used as scaffolds for tissue regeneration due to their biological properties; however, they exhibit low mechanical properties. Mechanical characterization of these scaffolds requires establishing the conditions of testing in regard to the conditions set in the bioreactor. The effects of different parameters used during mechanical testing on the collagen gels were evaluated in terms of mechanical and viscoelastic properties. Thus, a factorial experiment was adopted, and three relevant factors were considered: temperature (23°C or 37°C), hydration (aqueous saline solution or air), and mechanical preconditioning (with or without). Statistical analyses showed significant effects of these factors on the mechanical properties which were assessed by tensile tests as well as stress relaxation tests. The last tests provide a more consistent understanding of the gels' viscoelastic properties. Therefore, performing mechanical analyses on hydrogels requires setting an adequate environment in terms of temperature and aqueous saline solution as well as choosing the adequate test.

Reconstituted collagen gels are widely used as scaffolds even though their low strength and poor elasticity limit their applications in VTE. Here, two approaches are adopted to modify their mechanical behavior: in the first, gels prepared under physiologic conditions are remodeled by cell-mediated contraction; in the second, gels prepared in non-physiologic conditions are chemically crosslinked. Samples are tested under cyclic loading and their viscoelastic behavior is assessed. The results show that both approaches result in lattices with adequate strength, and crosslinking significantly reduces hysteresis and permanent deformation. SEM shows that SMCs are capable of contracting and remodeling all the lattices, confirming that these are suitable supports for tissue regeneration.

Collagen gels constitute an adequate scaffold for supporting the adhesion, proliferation and tissue regeneration of vascular cells inside a bioreactor. However, their mechanical properties should be enhanced not only for their manipulation but also to resist the mechanical constraints applied in the bioreactor. Actually, assessing the mechanical properties of a hydrogel requires many precautions since they are very sensitive to the environmental conditions (temperature, ionic strength, aqueous environment, etc). Whereas mechanical properties are usually measured directly in the air, the aim of this work was to evaluate the effects of a pseudo-physiological environment (PPE) on the mechanical properties of collagen gels. Furthermore, reinforcement was also tested using UV treatments (λ = 254 nm, 20 J/cm2), known to induce crosslinking. Irradiated samples were more resistant to enzymatic degradation and swelling tests showed that the crosslink density was increased by a factor of 30. This increase was thereafter correlated to the mechanical properties. Results showed that the UV-treated samples were stiffer and more brittle than the non-treated ones when tested in air. However, a 20% decrease and 40% increase were respectively measured on the linear modulus and strain at rupture when the gels were tested in the PPE. In the perspective of vascular tissue regeneration, these results show that the mechanical properties of a hydrogel should be performed in PPE in order to take into account the plasticization phenomenon that will occur in a bioreactor.

Functional vascular tissue engineering aims to produce blood vessels in vitro in a controlled environment named bioreactor. In order to control the growth and remodeling of vascular tissues, suitable measurements should be made on the construct in situ, i.e. during the growth. These measurements will feed the controller with information in order to take efficient control decisions. The non-destructive measurement of compliance or elastic modulus in vitro is a potential indicator of the vascular construct maturity. This work shows that compliance and elastic modulus are related: they can be estimated during the growth of constructs in a bioreactor, and thus provide useful feedback information to the controller.