Joose Kreutzer

Pneumatic cell stretching system for cardiac differentiation and culture

This paper introduces a compact mechanical stimulation device suitable for applications to study cellular mechanobiology. The pneumatically controlled device provides equiaxial strain for cells on a coated polydimethylsiloxane (PDMS) membrane and enables real time observation of cells with an inverted microscope. This study presents the implementation and operation principles of the device and characterizes membrane stretching. Different coating materials are also analyzed on an unstretched membrane to optimize the cell attachment on PDMS. As a result, gelatin coating was selected for further experiments to demonstrate the function of the device and evaluate the effect of long-term cyclic equiaxial stretching on human pluripotent stem cells (hPSCs). Cardiac differentiation was induced with mouse visceral endoderm-like (END-2) cells, either on an unstretched membrane or with mechanical stretching. In conclusion, hPSCs grew well on the stretching platform and cardiac differentiation was induced. Thus, the platform provides a new possibility to study the effect of stretching on cellular properties including differentiation and stress induced cardiac diseases.

Structured PDMS Chambers for Enhanced Human Neuronal Cell Activity on MEA Platforms

Structured poly(dimethylsiloxane) (PDMS) chambers were designed and fabricated to enhance the signaling of human Embryonic Stem Cell (hESC) - derived neuronal networks on Microelectrode Array (MEA) platforms. The structured PDMS chambers enable cell seeding on restricted areas and thus, reduce the amount of needed coating materials and cells. In addition, the neuronal cells formed spontaneously active networks faster in the structured PDMS chambers than that in control chambers. In the PDMS chambers, the neuronal networks were more active and able to develop their signaling into organized signal trains faster than control cultures. The PDMS chamber design enables much more repeatable analysis and rapid growth of functional neuronal network in vitro. Moreover, due to its easy and cheap fabrication process, new configurations can be easily fabricated based on investigator requirements.

Mechanical Analysis of a Pneumatically Actuated Concentric Double-Shell Structure for Cell Stretching

An available novel system for studying the cellular mechanobiology applies an equiaxial strain field to cells cultured on a PolyDiMethylSiloxane (PDMS) substrate membrane, which is stretched over the deformation of a cylindrical shell. In its application of in vitro cell culture, the in-plane strain of the substrate membrane provides mechanical stimulation to cells, and out-of-plane displacement plays an important role in monitoring the cells by a microscope. However, no analysis of the parameters has been reported yet. Therefore, in this paper, we employ analytical and computational models to investigate the mechanical behavior of the device, in terms of in-plane strain and out-of-plane displacement of the substrate membrane. As a result, mathematical descriptions are given, which are not only for quantitatively determining the applied load, but also provide the theoretical basis for the researchers to carry out structural modification, according to their needs in specific cell culture experiments. Furthermore, by computational study, the elastic modulus of PDMS is determined to allow the mechanical behavior analysis of a fabricated device. Finally, compared to the experimental results of characterizing a fabricated device, good agreement is obtained between the predicted and experimental results.

Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array

BACKGROUND Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO2 supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential. NEW METHOD We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber. RESULTS We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments. COMPARISON WITH EXISTING METHODS Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO2 gas. Utilizing dry gas supply makes the proposed chamber simple to use. CONCLUSION Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture.

Indirect Temperature Measurement and Control Method for Cell Culture Devices

Microfluidic devices are promising tools with which to create an environment that mimics a cell’s natural microenvironment more closely than traditional macroscopic cell culture approaches. In these devices, temperature is one of the most important environmental factors to monitor and control. However, direct temperature measurement at the cell area can disturb cell growth and potentially prevent optical monitoring, and is typically difficult to implement. On the other hand, indirect measurement could overcome these challenges. Therefore, using the system identification method, we have developed models to estimate the cell area temperature from external measurements without interfering cells. In order to validate the proposed models, we performed large sets of experiments. The results show that the models are able to catch the dynamics of temperature in a desired area with a high level of accuracy, which means that indirect temperature measurement using the model can be implemented in the future cell culture studies. The usefulness of the model is also demonstrated by simulations that use estimated temperature as a feedback signal in a closed-loop system. We also present tuning of a model-based controller and a noise study, which shows that the tuned controller is robust for typical ambient room temperature variations.Note to Practitioners—In this paper, we tackle the problem related to temperature measurement in microfluidic devices, especially but not only concerning cell culture environments. Even though it would be desirable to place a temperature sensor as close as possible to the location of interest, practical limits usually prevent this; for instance, limited space and requirements for optical monitoring. To overcome these problems in microfluidic devices, we present a novel indirect temperature measurement approach using the system identification method. The idea is to create a model that estimates temperature on the area of interest using measured outside temperature. Because it is required to measure both model input and output signals for the model development, we first fabricated a temperature sensor plate, combined it with our heating system, and measured required temperatures on several experiments. Then, we developed third-order discrete state-space models using measured temperatures and System Identification Toolbox in MATLAB. Model performances were examined and compared with measurements. Furthermore, we created a closed-loop Simulink (from MATLAB) model, and showed how desired temperature could be controlled using only measured outside temperature and the developed model. In the future research, we will implement the designed closed-loop system to our cell culture system to precisely control temperature in the cell area.

Monitoring pH, temperature and humidity in long-term stem cell culture in CO 2 incubator

Cell culture in stem cell research is on the rise, not only for basic research but also for its potential medical and therapeutic applications. Monitoring culture process using sensors throughout the culture helps to optimize culture conditions for optimal growth and maximize yield from the cells. Further, the sensor data gives insight into developing better cell culture systems. Typical stem cell culture platforms, e.g. well plates or flasks, cannot be easily equipped with sensors and impose many challenges in periodic process measurements in a CO2 incubator. We present an incubator compatible modular measurement system with three sensors, to monitor pH, temperature and humidity continuously throughout the culture. Sensors are assembled around a flow through cuvette for highly sterile non-contact measurements. No sample preparation or sample extraction from the incubator is needed and the measurements are carried out in a closed flow loop without wasting any medium. The modular assembly is novel, reusable and feasible for humid incubator environments. The system has been tested, validated and used in mesenchymal stem cell expansion and differentiation, for periods ranging from two to three weeks. Once the measurement has commenced at the beginning of culture, continuous measurements without sensor recalibration or special manual attention are carried out till the end of the culture. Measurement data clearly show the interplay between measured parameters, indicating a few stress sources present all through the culture. Additionally, it gives an overall picture of behavior of critical control parameters in an incubator and points out the need for bioprocess systems with automatic process monitoring and smart control for maximum yield, optimal growth and maintenance of the cells.

A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications.

The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells

Although mechanical cues are known to affect stem cell fate and mechanobiology, the significance of such stimuli on the osteogenic differentiation of human adipose stem cells (hASCs) remains unclear. In this study, we investigated the effect of long-term mechanical stimulation on the attachment, osteogenic differentiation and mechanical properties of hASCs. Tailor-made, pneumatic cell stretching devices were used to expose hASCs to cyclic equiaxial stretching in osteogenic medium. Cell attachment and focal adhesions were visualised using immunocytochemical vinculin staining on days 3 and 6, and the proliferation and alkaline phosphatase activity, as a sign of early osteogenic differentiation, were analysed on days 0, 6 and 10. Furthermore, the mechanical properties of hASCs, in terms of apparent Youngs modulus and normalised contractility, were obtained using a combination of atomic force microscopy based indentation and computational approaches. Our results indicated that cyclic equiaxial stretching delayed proliferation and promoted osteogenic differentiation of hASCs. Stretching also reduced cell size and intensified focal adhesions and actin cytoskeleton. Moreover, cell stiffening was observed during osteogenic differentiation and especially under mechanical stimulation. These results suggest that cyclic equiaxial stretching modifies cell morphology, focal adhesion formation and mechanical properties of hASCs. This could be exploited to enhance osteogenic differentiation.

Data Rate Performance of Droplet Microfluidic Communication System

We develop a microfluidic communication system prototype and conduct experiments to show the feasibility of data transmission. We apply using on/off keying and communication by silence modulation schemes for signal transmission. Their performance is evaluated by using data obtained experimentally. The data rate of these modulation schemes is computed as well. We identify the noise sources in microfluidic channels as setup related. The experiments reveal that communication by silence is significantly better than on/off keying. Our results show that both modulation schemes are feasible for signal transmission. Even though CtS outperforms OOK in terms of achievable data rate, from a practical point of view CtS is much harder to control, since there are more parameters to be controlled at the same time.

Computational modeling and structural improvement of a pneumatically actuated concentric double-shell structure for cell stretching

Recent studies have shown that mechanical stimulation by means of mechanical stretching can enhance the cardiac differentiation and proliferation. Thus, several types of devices have been developed for mechanically stimulating the cardiomyocytes (CMs). However, some of the emerging devices need to be improved to satisfy the specific needs in applications. In this study, a computational model is developed for a novel cell stretching device by a finite element (FE) approach, and two significant parameters in terms of in-plane strain and out-of-plane displacement of the cell substrate are improved. This paper describes a method of geometric parametric variation to enhance the design and reports a reduction of the out-of-plane displacement from 315 μm to 4.8μm, while maintaining the maximum in-plain strain of more than 5%.