The study of biological organs and tissues in clinical and biomedical research is often based on the use of isolated cell cultures plated in Petri dishes or multi-wells. Scientists have begun to appreciate that this representation of living processes is overly simplistic and much attention is now being paid to the development of so-called “physiologically-relevant” in vitro models. Such models have the potential to better approximate in vivo responses, offering new advanced systems with greater translational and predictive value. Key improvements to traditional models are focused on conferring three-dimensionality and the presence of multiple cell types and mechanical stimuli such as flow and stress.
Environmental parameters are also essential for the development of more physiologically-relevant in vitro models, as several studies have demonstrated the crucial role of the microenvironment on cell growth and function [1
]. Indeed, as in the body, cells are extremely sensitive to chemical, as well as physical, stimuli, like oxygen concentration, pressure, temperature, and pH. However, traditional, as well as advanced (i.e., in bioreactors, on scaffolds, or in organoids), in vitro models are still maintained in cell culture incubators, with a physiological temperature of 37 °C and a controlled partial pressure of CO2
(5%). Most commercial incubators have very large volumes compared to the culture media and the temperature and CO2
gas sensors are placed a large distance away from the cells. Thus, the temperature and CO2
concentration measured by the instrument is often not that actually perceived by the cells. Moreover, even the opening/closing of the incubator door can briefly modify the gas and temperature levels, as well as the pressure in the whole chamber. An additional parameter, which is often ignored by cell biologists, is the variation in oxygen concentration with media height and the cell density. In both monolayer cultures and scaffolds the oxygen level at the cell membrane depends on the distance of the cell from other cells (i.e., the cell density), the distance of the cell from the gas-medium interface, and its oxygen consumption rate [3
In the case of highly-sensitive cells, like stem cells or undifferentiated cell-lines, these variations may compromise experimental results leading to false negatives or positives. In fact, it is well known that even small and brief changes of the physical and chemical conditions of the cell environment can activate (or inhibit) signal processing for cell differentiation [4
]. For instance, it has been observed that transient fluctuations in oxygen partial pressure can change gene transcription, thus influencing the fate of stem cells in culture [5
The importance of chemical and physical measurements is reported in [6
], where the authors also outlined the difference between on-line, in-line, and at-line measurements. In particular, on-line data are defined as signals that are available continuously, while sequential data can be defined as quasi-on-line measurements. Finally, when the sensors are placed inside the bioreactor, we can refer to in-line measurements, or to at-line measurements when the sensors are not in the culture chamber and sampling is necessary. This distinction can be important for the correct design of culture parameter measuring systems, where the measured variables can be physical (pressure, temperature, flow, and stirrer speed), chemical (pH, pO2
, nutrients, metabolites), or biological variables (cell metabolism, etc.). For each type of measurement, different sensing methods can be used and the best sensor for a specific application should be sterilizable and should not interfere with the medium. In addition to monitoring, a further step is the control of environmental conditions depending on the measured variables. This implies the necessity of actuators able to operate on the culture environment in order to maintain variables of interest within a desired range.
An example of integration of in-line measurements in bioreactors can be found in Abu-Absi et al. [7
], where Raman spectroscopy was implemented for monitoring culture parameters, such as glutamine, glutamate, glucose, lactate, ammonium, viable cell density, and total cell density. In particular, large, 500-L bioreactors operated in fed-batch mode were used, and immersion probes were constructed of stainless steel and connected to a RamanRXN3 (Kaiser Optical Systems Inc., Écully, France). Moreover, the pH and dissolved oxygen tension (DOT) were maintained around 7.0 and 50% of air saturation. The pH was controlled using CO2
gas to decrease pH and 3N NaOH to increase pH.
Several studies in the literature are implemented using commercial bioreactors with environmental controls. For example, the Sixfors multireactor system (Infors AG, Bottmingen, Switzerland) was used by Trummer et al. [8
] to investigate the effect of variation in DOT, pH, and temperature (T) on cell growth, metabolism, and cell cycle distribution. The authors observed that the reduction of T and pH exert the most significant effects on process performance mainly by reducing cell growth and metabolism. A parallel bioreactor system commercialised by DASGIP AG (Jülich, Germany), was used in [9
] for the culture of human pluripotent stem cells. This system is composed of four stem cell culture vessels, and has several monitoring and control opportunities (i.e., temperature, pH, DOT) [9
Different studies with custom control systems can also be found for micro-sized bioreactors. For instance, a microbioreactor platform that allows for independent control of culture parameters in each well of an array has been reported [10
]. To control the physical-chemical mechanisms related to cell activity in microfluidic devices, dissolved oxygen concentrations were monitored in real-time using fluorescence intensity and lifetime imaging of an oxygen sensitive dye by Metha et al. [11
]. In particular, if used at slow flow rates and high cell density, this microfluidic device creates oxygen-limited physiology (hypoxia conditions) that can be useful in several situations, such as maintaining the pluripotency of embryonic stem cells without the need of hypoxic chambers. The authors conclude that the system provides several advantages thanks to the dynamic control of oxygen, achieved by programming temporal changes in the perfusion rates. However, it is known that microfluidic systems are not really representative of physiological conditions and that they present several issues related to edge effects, high shear and nutrient depletion [2
]. On the other hand, the use of very large volume bioreactors can be useful for industrial purposes, but in the case of in vitro studies can lead to an unnecessary expenditure of materials.
In this paper, we describe a “milli-scaled” platform, SUITE (Supervising Unit for In Vitro Testing), for cell culture under controlled environmental conditions. The platform can be used to carry out experiments in well-defined flow, temperature, pressure, and pH, and enables the study of specific physiological and pathological scenarios in vitro. In previous studies interconnected modular bioreactors with liver, adipose tissue, and endothelial cells were used to generate an advanced in vitro model of the metabolic system [12
]. Following this approach, a feasibility study was conducted to implement a model of portal hypertension using an environmental control system embedded in SUITE.
Portal hypertension syndrome is characterized by a pathologic increase in portal venous pressure which leads to an increase in the pressure gradient between the portal vein and the hepatic veins (i.e., inferior vena cava). The study of this pathology poses a variety of difficulties both in in vitro and in clinical studies, and the knowledge of portal hypertension pathophysiology is mainly based on animal models [15
]. The few examples of portal hypertension in vitro studies in the literature are based on rat hepatectomy [16
] or are focused on understanding the correlations between endothelial dysfunction and liver diseases [17
]. However, in many instances animal models are not sufficiently predictive of human pathophysiology [18
]. Moreover, as far as in vitro methods are concerned, traditional incubators cannot be used to provide pressure variations typical of those observed in portal hypertension. The SUITE platform was hence employed to determine the influence of hydrostatic pressure generated by the system on the two cell types primarily involved in portal hypertension: hepatocytes (represented by the human hepatic cell line C3A) and endothelial cells (human umbilical vein endothelial cells or HUVEC). The study demonstrates the potential of the platform to control and maintain different pathophysiologic pressure conditions and paves the way towards the establishment of more complex hypertension models, thanks to the possibility of connecting different bioreactors to reproduce the interaction between different cell types (e.g., endothelial and hepatic cells).
The SUITE platform is a free-standing cell culture system able to measure and regulate “on-line” cell culture parameters, such as temperature, pH, and pressure in a fluidic cell culture system without the support of an incubator. The system consists of a pump, modular bioreactors, and a mixing chamber, along with sensing, actuation, and control components. Measurements are performed directly in the mixing chamber connected to the bioreactors, without the need for sampling. Therefore they can be referred to as “in-line”. An ensemble of actuators, such as electro-valves, pressure regulators, and heating elements allow precise control of cell culture parameters. Moreover, the use of the LB1 bioreactor as a module in SUITE presents several advantages: (i) pressure and shear stress values close to the physiologic ones in the liver; and (ii) the possibility of optical real-time monitoring.
SUITE is also able to reproduce in vitro conditions related to complex pathologies which are related with an alteration of physico-chemical parameters. Our results show that thanks to a real-time algorithm which adjusts gases injection time considering the actual pressure and temperature, it is possible to maintain pre-determined values of pressure and pH. In particular, the system was able to regulate the pH at around 7.27 ± 0.06, both at different hydrostatic pressures and in the case of external perturbations (i.e., with solutions at different pH). The response time of the system is about 50 s. This type of regulation is not available in traditional incubators, where the constant imposed partial pressure of CO2
combined with cellular metabolic activity leads to a progressively acidic medium over time [20
Portal hypertension, like other diseases characterized by high vascular pressure, are rarely modelled in vitro because of the technical difficulties associated with the generation of a constant overpressure and fixed pH concomitantly. For this reason, human hypertension is modelled in animals, where high blood pressure in different regions is induced, either through the use of drugs or by ligation. To test the feasibility of SUITE as a platform for the design of in vitro experiments of hypertension, a portal hypertension model was implemented with endothelial and hepatic cells (i.e., HUVEC and C3A). HUVECs, as they are endothelial cells, strongly support pressure values up to 50 mmHg, but with a marked increase in vWF expression; while hepatic C3A cell viability begins to decrease at 20 mmHg and their function is severely affected at 40 mmHg. While several biological aspects still need to be addressed to validate the model the technology, nevertheless, paves the way for further investigations into non-animal models of human pathologies associated with high pressures such as stenosis, glaucoma, and intracranial hypertension.
The study presented in this paper demonstrates the importance of cell culture parameter control both for in vitro modelling of complex diseases and for maintaining optimal culture conditions. In particular, this last aspect is fundamental both for the reliability of cell experiments and for the culture of sensitive cells, such as stem cells.
In conclusion, “physiologically relevant” in vitro models are not just about recreating a 3D dynamic environment, but also require the control of environmental parameters to ensure that the in vivo physico-chemical milieu is also recapitulated. Since traditional incubators cannot accurately control these parameters, the SUITE platform represents a valid option for experiments that require fine regulation of pressure, temperature, and pH, and can provide a cell culture environment capable of recreating pathophysiological conditions for the study of disease models.