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O2-sensitive Microcavity Arrays for 3D Cell Culture Monitoring
Oxygen Imaging in Spheroids with VisiSens TD Technology
C. Grün1, G. Liebsch2 und E. Gottwald1
1Institute of Functional Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany
2PreSens Precision Sensing GmbH, Regensburg, Germany
Microthermoforming is applied to create an array of microcavities in oxygen sensor foils. 3D cell aggregates can be cultured in these cavities and the oxygen content can be quantified via imaging. With the help of this new platform technology it is now possible for the first time to monitor oxygen in the direct microenvironment of spheroids and organoids and by this, create near in vivo conditions in vitro.
Cell culture technologies have been a valuable tool in basic research and drug development for decades. In vitro 3D cultures have shown many advantages compared to 2-dimensonal cultivation approaches, as key parameters like e. g. cellular behavior, metabolism, and morphology better mimic the cell status in vivo, and are therefore better suited as an alternative for animal testing. However, it is often neglected to set appropriate culture conditions to create near in vivo conditions for the cells. For example, cell cultures are still often performed under standard incubator conditions, at atmospheric oxygen levels supplemented with 5 % CO2, which is not necessarily the typical oxygen concentration cells are exposed to in live tissue. This can lead to a state of hyperoxia or in some cases to hypoxia, which will cause cell stress. To provide the cultured cells with oxygen concentrations that better reflect in vivo conditions, appropriate measurement techniques are necessary. Many oxygen measurement methods, like e. g. Clark-type electrodes consume oxygen during the measurement process and are therefore not applicable in small-scale culture. Optical oxygen sensors on the other hand do not consume oxygen, can be miniaturized and manufactured in different designs for different applications, which makes these sensors more and more popular for cell culture monitoring. Optical sensors are based on fluorescent dyes, that react with oxygen and can be incorporated for example in hydrogels or nanoparticles. The fluorescent dye can also be immobilized in a polymer film and oxygen-sensitive sensor foils can be created. This allows even the 2-dimensional recording of oxygen gradients over a whole area with an appropriate imaging camera. This method is already applied in many microfluidic and lab-on-a-chip applications. Here we describe the manufacturing of a microcavity array with oxygen sensing capabilities based on an optical sensor foil. Spheroids can be cultivated in these microcavities and oxygen can diffuse freely from the medium above into the 3D cell construct creating near physioxic conditions. The oxygen content in the microenvironment of multiple spheroids can be recorded simultaneously with the VisiSens imaging technology, which allows for high throughput and label-free oxygen measurements without disturbing the 3D cell aggregates in any way.
Sensor Arrays with Oxygen-Sensing Capabilities
For the construction of the microcavity sensor array a polycarbonate film of 50 µm thickness is coated with oxygen-sensitive fluorescent dye incorporated in a polymer (PreSens GmbH). This sensor foil is placed on a brass molding tool and the microcavity arrays are created by a microthermoforming process (Karlsruhe Institute of Technology). In this way accurate arrays can be produced, with an inner microcavity diameter of 300 µm, and towards the upper end a bevel with 500 µm outer diameter (Fig. 2 A,B). The microstructure geometry is not restricted to round cavities or the here mentioned dimensions and can be tailored to customer needs. Fitted into CellCrown™ 12 NX (Scaffdex) cell culture inserts the microcavity sensor foils are available in 12-well format (Fig. 2 C,D). In our study the sensor foils proved to be non-toxic for the applied cell type. The cells grow directly on the oxygen-sensitive layer; plasma-treated and collagen-coated sensor foils even show enhanced adherent cell growth. BIOFLOAT™ (FaCellitate) treatment on the other hand prevents adherent cell growth allowing spheroids to be formed by pipetting the cells directly into the cavities (self-assembly). This offers a convenient and direct way of obtaining 3D cell aggregates.
Spatio-temporal Oxygen Measurements in 3D Culture
The plates with sensor array inserts are placed in a chamber above the camera and the VisiSens system takes oxygen images from below (Fig. 4A). This way, real-time oxygen measurements over whole insert areas and a multitude of spheroids can be taken. Unlike other currently available 3D cell culture monitoring systems, in this set-up the microcavities remain open during spheroid cultivation and measurements, so there is no risk of hypoxia (Fig. 6). An oxygen gradient develops corresponding to O2 diffusion from the culture medium above the micocavity to the cell aggregate equivalent to the native vascular system in vivo. Diffusion is only possible from the above, as the sensor microcavity forms a diffusion barrier from below (Fig. 5). The special shape of the microcavities allows to monitor the oxygen content in the direct microenvironment of the spheroid in the center of the cavity so physioxia can be verified. At the same time the diffusion zone in the bevel, the area towards the edge of the microcavity, can be evaluated (Fig. 5). Figure 7 shows an example for an oxygen image taken of one sensor array with 120 microcavities in total. Regions of interest can be selected in the center of a microcavity, the longest oxygen diffusion distance through the 3D cell culture, and in the inner and outer bevel areas, the diffusion region, and can be compared. In a microcavity array, the individual measurements are independent of each other, as the gradient is directed into each cavity individually. The respective boundary layers are permanently supplied with O2 by the overlying medium, identical to the in vivo situation.
New Perspective for Many Applications
This culture platform with oxygen sensing capabilities gives the possibility to test the effects of pharmaceutical active compounds on cell metabolism. As the microcavities ease the process of creating 3D cell aggregates in high numbers, up to 1400 spheroids in one 12-well plate, this method is not only timesaving for assays but also enables to create a large amount of data for comparison. As the cells are not altered during the measurement process, they can even be used for downstream processing / analysis. In initial proof-of-concept experiments this set-up was used for mitochondrial stress tests, and after adding the respective test substances, expected oxygen concentrations could be detected in the 3D cell cultures (Fig. 8). Ongoing research is currently on the way to establish protocols for cardiotoxicity tests of pharmaceutical agents with the microcavity sensor array.