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Chapter 6
6.1 Conclusions: overview
It is useful to repeat the question that was used to open the introduction: “Can nature be imitated?”
The answer to this question is not a simple “yes” or “no”, but requires careful elaboration and contextualization. It has to be underlined that the answer is strictly related to technological evolution of the devices that are used to establish in-vitro models. The advance in technology allows biologists to expand new studies, focusing the attention on new aspects that in the past were avoided because of the lack in suitable devices. An example of the opportunities that derive from the technological evolution is represented by physiological barriers. Tissues such as intestine or lung require an interface between two phases, such as air-liquid or liquid-liquid. Simulating these kind of tissues with a cell monolayer seeded on a glass disc or on the bottom of a multiwell plate makes it impossible to evaluate any physiological organ functions. The growth up of transwells permitted the establishment of in-vitro models to study barriers in static conditions. The introduction of porous membranes inside bioreactors permitted the study of functions in a dynamic environment. The inverse is also true: device evolution is often driven by specifications for the evolution of in-vitro models. Therefore a collaboration between biologists and engineers is highly recommended. Advantages that derive from this partnership are described in this thesis. In fact in this work the reader can find solutions to meet biologist’s specifications or technological improvements to push new approaches and methods in the establishment of in-vitro models. An example of the first situation is represented by the ILT family of bioreactors.
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6.1.1 ILT bioreactors
The development of the culture chambers called ILT1 and ILT2 was driven by the necessity to have suitable devices to establish an in-vitro model to study cross talk in the gastro-intestinal duct. As described in chapter 3, a European project called InLiveTox is dedicated to development of a GI tract/metabolism model. The analysed tissues are intestine epithelium, vascular endothelium, and hepatic tissue. A first bioreactor called ILT1 was developed as an evolution of ILT0, an existing chamber realized by Ahluwalia’s group years ago. Various specifications led the development of the new prototype. An example is represented by the necessity to have two fluidic circuits where media flows at different velocities, in order to simulate the physiological environment in the GI tract. The communication way between circuits is represented by a porous micro fabricated membrane where intestinal epithelial cells are seeded. A slot to insert the membrane in a fixed position is realized. The new device allows biologists to take TEER measures using 4 gold electrodes directly inserted in the bioreactor and connected to a machine called EVOM that is able to evaluate the impedance imposed by a cell layer and then correlate the value to the integrity of the barrier. In order to meet specifications of the biologists the first prototype was used and analysed by project partners. Feedback specifications forced the development of a new bioreactor, in order to solve some of the problems of ILT1. The new technology is called ILT2. In this device a holder system permits the protection of the membrane and cells seeded on porous surface during the growth and differentiation phases. Chambers are completely redesigned in order to offer an easy way to join a membrane holder system to PDMS parts. A problem solving phase follows the realization of ILT2, in order to avoid for example the passage of the upper circuit media to the bottom level. Several models are evaluated in order to characterize the fluid dynamics inside the bioreactors, solutes passage through the membrane layer and nanoparticles deposition onto circuit surfaces. Finally all theoretical data are verified by in-vitro experiments. In these cases TEER measurements are taken in order to evaluate the efficacy of the implemented solutions. Once all the aspects are evaluated ILT2 is used by biologists in order to cultivate cells. As an alternative to the use of micro fabricated membranes a holder system in PDMS is designed and realized in order to house commercial porous membranes. In chapter 3 experiments performed to characterize the new improvement are described. This represents
106 an attractive alternative to ILT2 system, but improvements are required in order to monitor barrier integrity.
6.1.2 Allometry
Another end point of the thesis is the evaluation of a method in order to increase the meaningfulness of in-vitro models. As described in chapter 2 the perception of a lack in correspondence between in-vitro models and physiology is widely diffused. 2D monolayer cultures are not relevant to study an organ. The evolution to 3D cultures, using scaffold or de-cellularized matrices, permits the introduction of new paradigms to analyse physiology, but it is not sufficient. The misalignment between models and the real environment is evident in case of cross talk experiments that take into consideration more than one tissue. In that case it is clear that the ratio between physiologic parameters such as number of cells, flow rate, nutrient support are key parameter while can be tuned to better approximate the physical reality. In this thesis Allometry is used as an useful method to overcome the problems in experimental set up or chamber design in order to increase the correlation with physiology. In particular two models are proposed in order to establish relevant in-vitro models of cross talk between vascular endothelium and liver. Firstly a model that scales down tissue surface areas and permits the preservation of physiological ratios is useful when physiological barriers are studied. In particular this case represents a typical situation where cell number is strictly correlated by micro fabricated membrane surface area where they are seeded. The confluent state is important when a physiological barrier is simulated, because of the tight junctions that are developed in this condition and which regulate solute passage through the cell layer. As argued in chapter 2, using Allometry, it is possible to scale down the organ surface area of exchange, in order to design a suitable membrane for an in-vitro model. If it is impossible to design a membrane based on physiological scaling, it is important to correlate the dimensions of the other parts of the fluidic circuits with the physiological environment. In this case this thesis focuses on the allometric study of endothelial-liver cross talk. As described in chapter 2 the CNSM is based on a surface area allometric scaling and is compared to SSM where on the contrary the cells number is the key parameter in order to maintain physiological ratios between tissues. In-vitro experiments are performed in order to validate models and decide
107 which is closer with physiological conditions. In-vitro data demonstrated that cell number has the key role when organ metabolism is analysed. Evaluating the number of ILT0 bioreactors that are required in order to respect the allometric specifications, it is possible to have an experimental set up and maintain a meaningful correlation with the physiological environment. Generalizing the procedures, in chapter 2 of this thesis it is demonstrate how CNSM and SSM can be used to simulate organs cross talk, using ILT0 bioreactors, avoiding the need to design new chambers for each model.
6.1.3 Bioreactor support
Despite the evolution of new devices that permit cell cultivation in dynamic conditions, the prevalence of multiwell plates and classical static devices in tissue simulation is still widely diffused. As said in chapter 4 this has slowed down the development of new tools for cell cultures. Currently standard devices are made by a transparent material such as polypropylene that permits the use of a microscope in order to perform qualitative and quantitative measurements on the cultivated cells. This is a characteristic that many bioreactors do not possess. Therefore optical analysis cannot be used with new bioreactor technologies. Even if new dynamic chambers are realized in transparent material, they are not compatible with plate analysers. In order to maintain the standard analytical protocols need in cell culture, but provide biologists with new and more physiologically relevant experimental paradigms, a new patented system based on multiwell plate dimensions is realized in order to house ILT like bioreactors in correspondence to the wells in a multiwell plate. The transparent material that is used to realize the support and the cultivation chambers permits the use of optical imaging methods to monitor cells growth and differentiation. Moreover the possibility to develop cells in dynamic conditions represents an improvement of the static conditions commonly used. Finally the system that is described in chapter 4 is a device that run stand alone: sensors, pumps and actuators are integrated in the multiwell plate. A channel pathway is realized to connect each bioreactor that is housed in slots, allowing media exchange between chambers. Valves to control flow direction represent one of the innovations of the patented support. As said in chapter 4 a new modularity concept is associated to the bioreactor support. 3 way valves allow to modify circuit topology, maintaining a fixed and rigid geometry. The modularity concept
108 is now associated with the possibility to decide which kind of circuit to use during experiments, starting from a fixed structure and reducing problems concerning the connection of subunits. In order to simulate physiological models it is important to have several degrees of freedom (DOFs) in the set up of the fluid circuit. Both parallel and series configurations can be used in the new device, permitting several different choices to implement an in-vitro model. Fluid dynamic models are performed to characterize the system, studying shear stresses and flow lines in the channels. The bioreactor support is an example of technology that anticipates the biologist’s requirements. It is a useful device because it allows the integration of common ideas that are widely diffused and improvements represented by devices that allow dynamic cultures.
6.2 Future improvements
The present thesis summarizes some technological in-vitro improvements that are realized during the PhD. The research in tissue engineering and in-vitro models is in continuous development. This thesis is not an exception. Each part that is described is correlated to future improvements. A list of technological improvements that are necessary is summarized below.
ILT2 development:
1) Evolution of the set up to take TEER measurements: in order to avoid 4 electrodes to take the measurements and the necessity of using expensive micro fabricated membrane, it could be useful to test new methods to evaluate the equivalent electrical impedance of cell layer, such as for example signal spectrum analysis. 2) Improvements in optical quality of chambers, in order to monitor cell morphology,
using a microscope.
MCmB 3.0 development:
1) Evolution of MCmB 3.0 placing the electrodes to perform TEER measurements or implement new modalities to evaluate impedance of the cell layer.
109 2) Placement of sensors to characterize the in-vitro parameters directly in the
chamber wet volume.
Bioreactor support plate development:
1) Realization of a new prototype, using a transparent material.
