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Chapter 1
1.1 Introduction: in-vitro models evolution
“Can Nature be Imitated?” [1].
This question represents the key point that drives tissue engineering. There are two main approaches to study physiology and try to simulate tissues functions. The first, that in this thesis will be called “tissue engineering method”, is a replication of organ functions in order to substitute parts of an injured tissue with a new one developed in-vitro. This field followed a rapid evolution in past few years, because of the increase in requests. Here the focus is the replication of large parts of tissue. Generally the volume of devices that are involved in the development of tissues following this approach is relevant. It is not important to understand the cross talk between tissues. The second approach, called “model approach”, followed in particular in this thesis, is the simulation of a physiological environment in order to replicate suitable conditions for cell growth and development. The focus of the analysis is the study of organ regulation, metabolism, cross talk between different tissues. The end point is the replication of a physiological system, simulating physiological stimuli and pathological conditions. This second approach is deeply involved in pharmacokinetic studies. Once a tissue is replicated biologists can use it as a useful method to study a particular feature of the tissue and its interaction with particular stimuli. An example is represented by drug interaction studies with in-vitro organs, in order to develop new rapid and non invasive delivery systems. Generally devices in this study are characterized by small volumes, in order to increase cells product concentrations, facilitating their detection.
Although 2D monocultures seeded in a multiwells and characterized by static conditions are still the standard in biology, in past few years new 3D models developed in dynamic conditions. The reason is that scaffolds and 3D models are more representative of the physiologic environment than a classical cells monolayer. Moreover it has to be underlined that for a deeper analysis of physiological features by in-vitro models, devices where it is
2 possible to mimic cross talk between several cell types are required. In fact the most sophisticated in-vitro organ models culminate in the use of multiple cell types arranged in a 3D structure in dynamic conditions. Despite of the optimum represented by 3D models, in most cases cell or tissue crosstalk is simulated using growth factors and hormones or conditioned media, for example to induce differentiation or to maintain cell phenotype. Cocultures are an alternative option, but they are difficult to characterize particularly if the different cell types are not spatially separated. For this reason, non-contact or connected cultures can often be used to reproduce aspects of cellular crosstalk. In-vitro models underwent an evolution in past few decades. Static conditions are in most cases substituted by dynamic architecture, characterized by cross talk models that simulate multi organs systems. The evolution in model complexity is a phenomena strictly correlated to design of new devices. The evolution of technology is often an answer to requests of new device to establish in-vitro models closer to physiology.
1.2 Evolution of devices
In the past decade the development of bioreactors allows biologists to study new tissues mimicking both physiological and pathological conditions. A bioreactor is a system that supports a biological active environment. In biology a bioreactor is a device where cells can grow in order to develop their function in sterile conditions. These devices have several shapes and dimensions based on their task and their functionalities. In general they permit the development of cell cultures in sterile conditions and in a dynamic environment. “Dynamic environment” means the presence of time dependent concentrations of nutrients or drugs and velocity gradients. Both characteristics are very important. Exchange between fresh media and old permits the use of low nutrient concentration, simulating what happens in the physiologic environment and driving away toxic compounds that could be created by cells or could be products of drug degradation. These points can be classified as chemical stimuli. It is important to decide the suitable flow on cells because in parallel to the advantages described previously the action of flow action on the cell surface (mechanical stimuli) has to be considered. Each cell type has a mechanical stimulus range. As described by fluid dynamic studies a flow creates a mechanical action on a surface that can be described by a longitudinal vector to flow lines but characterized by an opposite
3 direction. This quantity is called shear stress. It should be clear that in order to simulate the physiological environment, shear stress should not pass a threshold limit to avoid cell damage. So a bioreactor chamber has to be designed in order to impose a suitable shear stress on cell in a certain range of flow. Generally in-vitro models require monitoring of cells products. It is important that a bioreactor allows biologists to take samples of culture medium in order to analyze solutes.
1.3 Bioreactors and in-vitro models
Bioreactors permit an increase in model complexity. However this evolution is not the same for all organs, as described by figure 1.1 . An explanation of this phenomena could be the high request of new methods to cure pathologies that affect some organs. For example request of skin substitution in case of severe burns forced a rapid evolution in commercial dermal and epidermal components. Recently Sun et al [2] described a closely bioreactor system with air liquid interface (ALI) which can be used for clinical and experimental testing of in-vitro cultured skin. The system is designed to promote the proliferation of skin cells on 3D scaffolds in a dynamic environment, allowing multiple experiments in sterile conditions. However in the evolution of in-vitro skin models, nowadays the replication of features such as in vivo permeability, anisotropic mechanical properties, or vascular perfusion is not possible [3, 4]. As described in the case of skin both model approaches and tissue engineering methods, discussed in the first part of the chapter, are often the driving force that promotes the evolution of in-vitro systems. Another example of an organ studied by both approaches is represented by the lung. Pulmonary epithelium is an excellent example of a physiological barrier. Alveolar tissue is highly selective and allows passage of gas, while blocking the passage of dust and other particles. However drug inhalation represents one of the best methods to administer pharmaceuticals as they rapidly enter in bloodstream once past the pulmonary epithelia. Therefore many lung in-vitro models are focused on the assessment of drug uptake and passage through the epithelial barrier. In parallel in-vitro models are employed in order to investigate the effects of tobacco on alveolar tissue. Probably the most advanced pulmonary in-vitro model is represented by Petersen’s study [5]. His group presented a decellurarized extra cellular matrix (dECM) from the whole rat lung tissue seeded with
4 pulmonary epithelial and vascular endothelial cells in a two flow ALI bioreactor. Although lung in-vitro models are widely diffused it is not possible have a lung tissue transplantable as in the case of skin. Figure 1.1 represents a summary of the state of the art in in-vitro models. As described previously, tissue engineering approaches forced an evolution for lung and skin that is not comparable with other tissues such as intestine or blood brain barrier. Clusters of organs can be recognized basing the analysis on the architecture that each model presents and the milieu that is offered to cell cultures.
Figure 1.1 Schematic representation of state of art in in-vitro models.
Although skin and lung represent tissues that are widely studied, models that are correlated to these organs are not characterized by the highest architectured complexity. An example of an organ that shows so evolved models is the liver. Around year 2000 an integration between a dynamic milieu and a 3D architecture matrix characterized studies of this tissue. Liver is widely used in multi organs models [6, 7, 8]. Around the same period, micro patterned liver cultures and micro scaled bioreactors also paved the way for small liver-on-a-chip devices [6]. Clearly more physiologically relevant liver models must include the
5 whole ensemble of stimuli which promote the contribution to maintaining tissue function and homeostasis.
1.4 In-vitro relevant models: thesis overview
The meaningfulness of in-vitro models that actually is accepted is a point of discussion. For instance is it sufficient to have a well structured 3D model to have a good simulation of physiology? Correlated to this reasoning is the analysis of results that are obtained by a 3D in-vitro model and its corresponding physiological equivalent. An in-vitro model where for example cells number is not fixed in order to correspond to specific ratios that are characteristic of physiological environment cannot give well correlated outputs with the task to mimic physiological features. So the research follows new approaches in order to design bioreactors or establish experiments that increase the meaningfulness of the in-vitro models and their correlation with physiology.
In this context, this PhD thesis is focused on the design, realization and characterization of fluidic devices that could be useful in order to establish new physiologically relevant vitro models. The word “relevant” in the title is referred to the correspondence between in-vitro models and physiology. As said previously, in a multi organs model it is important to maintain physiological ratios between tissues. These ratios can be correlated to cell numbers, or surface areas where cells are deposited or to the volume of fresh blood that reaches each tissue. Allometry is the science that permits to establish ratio between physiological features and in-vitro characteristics, as will be described in chapter 2. Allometry is a mathematical approach that describes the relationship between a particular features of an animal, such as basal metabolic rate (BMR), life span, body surface area and its body mass. The allometric method can be used in order to increase the relevance of an in-vitro model if compared to physiology. In this thesis some in-vitro models are studied in order to analyze cross talk between different cell types. An example is represented by the gastro intestinal model. This model is the task of the European Project, InLiveTox. The aim of the analysis is to simulate cross talk of intestinal epithelial, endothelial cells and hepatocytes in the presence of different nano particles. Recently researchers have focused on newly developed nano-compounds to study interaction between nanoparticles (NPs) and cells, in order to emanate guidelines for NP safety. In order to evaluate suitable
6 parameters to design a relevant in-vitro model a study of physiology was undertaken. The attention was focused on two main aspects: tissues surface area of exchange, in case of physiological barriers, and number of cells. These points represent two different guidelines that can be followed in order to estimate in-vitro model parameters. The first part of the thesis is focused on the analysis of these approaches and discusses the best solutions in order to evaluate useful parameters to evaluate a relevant in-vitro model. However when a physiological barrier has to be simulated, there are other important specifications that have to be met that could contrast the theoretical allometric analysis. The most important is represented by porous membrane dimensions which support where cells are going to be seeded in the model. In particular cells have to be in confluent in order to create a barrier and simulate the physiological features. Allometric studies can formulate guidelines, but geometrical specifications often required a compromise. This represents the discrimination between theoretical studies and in-vitro models. This thesis proposes several in-vitro relevant models, using an allometric approach. In particular the attention is focused on the gastro intestinal duct. The study of cross talk between intestine, vascular endothelium and liver is the goal. The relevant model that is established has to find a compromise between an allometric approach applied to physiological barrier and other allometric evaluations that are valid for organs correlated to basal metabolic rate, such as liver.
The second part of the thesis is dedicated to the development of a new type of bioreactor, realized and characterized to mimic the GI tract. The presence of intestinal epithelia requires the introduction of a membrane in a fluidic circuit, in order to evaluate the filtration of the physiological equivalent. In literature there are lots of bioreactors that are characterized by a membrane interposed in a fluidic system, such as for example the multi-core bioreactor system patented by El-Sabban M. & Pedersen S. [9]. The device permits the placement of one or more membranes between two chambers that are independent from a fluidic point of view. Co-cultures of different cells types can be cultured in this bioreactor. As said previously, it is important to minimize chamber volume in order to facilitate solute detection. A new system has to be developed in order to simulate tissues of interest, characterized by modularity, easy case of use, sterilizing by autoclave or gas plasma and easy to be produced by custom devices that represent the equipment of a
7 standard chemical laboratory. A bioreactor called MCmB or, for the purpose of the project, ILT0, was developed by Mazzei et al. (figure 1.2) [10].
Figure 1.2 Picture represents ILT0 system realized by Ahluwalia’s group
This device is a good starting point because it meets the specifications that were previously cited. However an evolution of the culture chamber is required in order to permit membrane insertion and the placement of sensors as described in chapter 3. In this thesis essentially two bioreactors were designed and realized: one represents the evolution of the other. Once specifications of physiological environment were analyzed, the architectures of the in-vitro models were decided. Both new models are composed essentially of two different fluidic circuits where medium flows with different velocities. The communication way between circuits is represented by a porous membrane where intestinal epithelial cells are seeded. This architecture permits to identify an upper circuit above the membrane and a bottom one. In the second one endothelial and hepatic cells have to be cultivated. A bioreactor composed of an upper and a bottom chamber separated by a membrane in order to create the communication between upper and bottom fluidic circuit is designed. In this device intestinal epithelia are seeded on upper membrane surface and endothelial cells are cultivated on a glass disc and placed into the bottom chamber (known as ILT1 and its evolution as ILT2).Hepatocytes are cultivated in the ILT0 [11].The new systems are
8 characterized as described in chapter 3 in order to evaluate the percentage of solute that passes through membrane in the filter cell.
Despite the improvement in technology, nowadays protocols to analyze a particular solute are characterized for a multiwell device that represents the gold standard of static cultures. This consideration is not only referred to chemical analysis but is also related to optical analysis based on fluorescence or absorbance that are involved in these kind of experiments. If the multiwell dimensions are the gold standard, it is hard to introduce a new device that could substitute the old approaches. A good compromise between innovations and commercial specifications has to be found if the aim is to develop a device that could be used in different laboratories.
The last part of the thesis is dedicated to the design and realization of a system which is a compromise between the technical evolution that is represented by bioreactors and the classical static devices such as multiwells common in cell culture laboratories. It is hard to propose and introduce innovative systems when technology that permits rapid analysis in real time during an experiment, avoiding any cell damages, is based on devices for static cultures specifications and as consequence it is not compatible with systems of different shapes or dimensions. This has slowed down the progress of new tools for cell culture. In parallel with the growth of devices that permit dynamic cultures this thesis tries to find a meeting point between paradigms influenced by old technologies and new evolutions in technology. A key role is represented by geometrical dimensions of proposed devices. A hybrid solution that unifies bioreactors and multiwell plates topology, keeping the advantages of each component was designed and patented in the form of a plate similar in shape and dimensions to a multiwell plate, provided by slots where bioreactors can be housed has been designed and patented [12]. Moreover a system of channels was studied and integrated as communication ways between bioreactors. Dynamic conditions are assured by a peristaltic pump that applies a flow of culture medium to the device. A multiwell where channels permit communications between slots is a project patented in 1983 by Mr. John Ernest Foster Holley [13]. In this prototype dynamic conditions were assured by the slope imposed on mutliwell plate. Liquid flowed from one slot to the next one through channels realized directly in the bulk. In the past 20 years many systems have been studied, in order to solve fluid dynamic problems and permit integration of new
9 technologies and strategies to take real time measurements, in order to monitor what it happens inside bioreactor devices [14, 15]. A step forward is represented by the possibility to change the topology of fluid dynamic circuit. Devices where On / Off valves placed on channels between slots permit to set opened or closed connection have been developed [16]. As it can be argued an On / Off valve permits few circuit topology combinations. A strategy in the development of a new apparatus is to find solutions that permit changes in flow direction, in order to vary connections between slots. In this way the topology of the fluid dynamic circuit can be switched between a lot of alternatives and it is possible to set a new combination in real time during an experiment, as described in chapter 5. Moreover pumps, sensors and modules that permit to monitor what happens inside the plate can be integrated in order to create a device that can be used stand alone, as described by figure 1.3.
Figure 1.3 Schematic view of multiple sensors, peristaltic pump and valves integration in fluidic device dimensioned on a multiwell plate
