Keywords

7.1 Research Issue—Heart Diseases

Biomedical studies play a significant role in both science and daily life. Deep study in this research field, development of medical products and fabrication of medical devices, repeatedly contribute to save human lives. To diagnose and cure a particular illness, it is important to have a specific method, which enables scientists to detect and defeat a given ailment. However, the available methods for diagnosis and treatment of diseases are often not enough to recognize and diagnose all mechanisms responsible for an illness (Fryburg et al. 2014). Additionally, selecting the best treatment in clinical cases requires earlier investigation of the disease mechanisms. In vivo or in vivo-like assays are the best ways to test new drugs and treatment methods (Salyers 2009; Zhang et al. 2012). The existing in vitro testing models have many disadvantages (Katt et al. 2016). Consequently, it is necessary to conduct research for the development of new testing models, tools and methods, which could improve disease treatments. The developed solutions could be used by doctors not only to test new drugs but also to optimize treatment parameters. The cardiovascular system is one of such a research area, in which such solutions are needed.

Statistical analysis shows that cardiovascular diseases (CVDs), next to cancers, are the most common cause of death all over the world (World Health Organization 2014). CVDs increase the loss of cardiomyocytes (CMs), which simultaneously deprive cells of their ability to regenerate (Laflamme and Murry 2011). Treatment of heart diseases may include the usage of: various medicines, medical and surgical procedures as well as cardiomyocyte (CM) regeneration (Mampuya 2012; Sheng et al. 2013). The goals of the above methods are, among others, to relieve symptoms and reduce risk factors, which can cause heart attack as well as prevent CVD complications (Hobbs 2004). Lifestyle changes (healthy diet, no smoking, physical activity) are also crucial in treating and preventing CVDs. There are various kinds of drugs with different mechanism actions, which are used in cardiac therapy (Lundin et al. 2016). The common cardiac medications are divided into such groups as: anticoagulants, beta blockers, calcium channel blockers, digitalis preparations, cholesterol-lowering medications, angiotensin-converting enzyme (ACE) inhibitors, diuretics, and vasodilators. The effects of these drugs include, among others: the regulation of blood clotting (coagulating), decreasing blood pressure, decreasing low-density lipoprotein (LDL) cholesterol, reducing swelling from excess buildup of fluid in the body, and mitigation of chest pain (angina) (Lundin et al. 2016). The nonsurgical procedure—percutaneous coronary intervention (PCI, angioplasty) that opens blocked or narrowed coronary arteries is also used for the treatment (Hoyt et al. 2013). Another method is coronary artery bypass grafting (CABG). CABG involves removing arteries or veins from other areas of the body and using them to bypass narrowed or blocked coronary arteries (Iqbal et al. 2013). There are also advanced methods for treating heart failure such as heart transplants, artificial hearts, and mechanical devices supporting left ventricular function (Kozar-Kaminska 2012). However, these treatments are not widely available, and their usage is limited to a small group of patients. Because heart failure is becoming increasingly common, great emphasis is placed on the development of new methods, which improve and restore heart functions. Regenerative medicine has been playing an important role in cardiology. At the end of the previous century, it was stipulated that with the use of acquired knowledge and experience in tissue engineering , it should be possible to grow a fully functioning heart. However, engineered heart tissue (EHT) is still being developed. Stem cells (SCs) are more commonly used as an alternative therapy for heart diseases (Beeres et al. 2007; Zhang et al. 2015). SCs have the ability to regenerate and differentiate into other types of cells. Appropriate growth factors and external stimuli (electrical, mechanical, optical, or magnetic pulses) can differentiate SCs to the cardiac cells (Batalov and Feinberg 2015; Nadal-Ginard et al. 2014). Therefore, SCs constitute the material filling the destroyed CMs, which lack the ability to regenerate (Cambria et al. 2016). Additionally, scaffolds and multilayer cell cultures are used to improve in vivo conditions. EHT provide an in vitro model reproducing heart tissue, in which therapies for patients with CVDs can be investigated (Doppler et al. 2013; Schaaf et al. 2011; Sondergaard et al. 2012). Research conducted so far has shown that heart regeneration is a long and complex process. It could result from the fact that conventional in vitro methods have some obstacles restricting their application (Bernstein and Srivastava 2012; Lovell and Mathur 2010). Despite long and advanced research based on EHT development and improvement in regenerative medicine there are many aspects, which should be solved and improved. First of all, a large amount of SCs is required. The creation of a heart model with an appropriate system of vascularisation (angiogenesis) responsible for supplying nutritive substances is also difficult. Therefore, scientists have developed new methods for cardiac cell analysis and propose new in vivo-like models to understand processes present in a fully functioning heart.

7.2 Lab-on-a-chip Systems—A New Approach for Heart Investigation

Elaboration of new therapeutic methods for heart diseases is a great challenge for scientists and doctors dealing with this type of ailment. Therefore, to avoid problems which appear during research conducted in conventional laboratories, Lab-on-a-chip systems were developed (Mehling and Tay 2014; Ziolkowska et al. 2011). The aim of the fabrication of such systems is to develop an in vivo-like cardiac model, in which the investigation of cardiac cell processes, as well as the elaboration of new therapies for heart failure will be possible. In such microsystems, fully functioning and vascularized heart tissue could be mimicked (Jastrzebska et al. 2016; Lee et al. 2015). These models, called Heart-on-a-chip systems, are specific types of Organ-on-a-chip systems. The usage of the microsystems for cardiac cell engineering has many advantages. First of all, miniaturization allows for a reduction in volumes of reagents used for experiments. The microsystems allow also to shorten the time of assays and to automate the whole process of biological sample studies (Halldorsson et al. 2015; Sackmann et al. 2014; Tehranirokh et al. 2013). Additionally, it is possible to control precisely the spatio-temporal phenomena present in a microsystem. This is particularly important when difficult-to-isolate or expensive cells (e.g., primary and stem cells ) are examined (Visone et al. 2016). However, the most important advantage of Heart-on-a-chip systems is the possibility to mimic in vivo conditions better than in conventional (two-dimensional, 2D) culture methods (Jastrzebska et al. 2016; Lee et al. 2015). A network of microstructures can be designed in such a way that the microenvironment created in Lab-on-a-chip systems is similar to natural cell growth. Additionally, microstructure dimensions are similar to the dimensions of the cells (Bhatia and Ingber 2014; Mehling and Tay 2014). The in vivo cardiovascular system is characterized by blood circulation, which allows necessary nutrients, oxygen, and hormones taken from components of the metabolism to be supplied to the cells (Pittman 2011). Because the microsystems provide dynamic conditions, culture models obtained in microscale brings even more to in vivo. The flow environment is an important property especially for cardiac cell cultures (Kobuszewska et al. 2017). The next feature is that the cells in tissue are surrounded by an extracellular matrix (ECM), the mixture secreted by the cells and filling the space between them (Alberts et al. 2002). ECM plays a significant role in cell behavior. It regulates intercellular communication and a dosage of various cellular growth factors absorbed in this matrix. Moreover, ECM maintains a spatial (three-dimensional, 3D) arrangement of the cells. Scaffolds, hydrogels, and multicellular spheroids are used to culture cells in such conditions (Bray et al. 2015; Nath and Devi 2016; Tomecka et al. 2018; Yan et al. 2015). However, conventional techniques (macroscale) have still not been optimized.

Cell culture methods used so far in conventional biological laboratories (e.g., using 96-well plates) do not ensure the conditions described above (Yamada and Cukierman 2007; Ziolkowska et al. 2011). A scheme of cell environments provided in vivo and in vitro monolayer cultures is shown in Fig. 7.1. Monolayer cell culture does not correspond well to natural tissue conditions. However, the usage of the microsystems for spatial heart culture can allow to mimic in vivo environment and to control culture conditions precisely.

Fig. 7.1
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A scheme of microenvironments provided in vivo and in vitro two-dimensional (2D) culture

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The systems based on Heart-on-a-chip have two main approaches (i) the creation of a beating heart culture model, which mimics heart tissue and (ii) creation of a whole vascular system , which mimics blood flow in vessels (Fig. 7.2) (Lee et al. 2015; Ribas et al. 2016; Simmons et al. 2012). The development of a beating heart model allows to investigate cardiomyocyte contraction under various conditions (static and dynamic). Heart-on-a-chip systems can also be utilized for investigation of an external stimuli influence on cardiomyocyte growth as well as stem cell cardiogenesis. Parameters such as oxygen concentration, pH value, hydrodynamic stress, uptake of intracellular calcium ions, and lactic oxidase level were monitored in cardiac cultures maintained in microscale. Additionally, cytotoxicity of new compounds with cardiac therapeutic activity can be investigated. The microsystems mimicking either heart tissue or the whole vascular system can be good models for the understanding of heart phenomena as well as to verify drug usefulness in heart disease treatment (Jastrzebska et al. 2016; Lee et al. 2015; Simmons et al. 2012; Zhang et al. 2016a).

Fig. 7.2
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Types of Heart-on-a-chip systems: model of beating heart culture, which mimic heart tissue (left) and model of a whole vascular system , which mimic blood flow in vessels (right)

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7.3 Heart-on-a-chip Systems—What Is Specific?

Organ-on-a-chip systems are in vivo-like models, which mimic functioning organs as well as which are used for investigation of both tissue disorders and drug cytotoxicity (An et al. 2015; Young 2013). Each in vitro organ model has parameters which are specific for that tissue. Therefore, to create a fully working heart model in microscale, it is necessary to know the properties of the specific tissue. Native myocardium is characterized by both parallel cardiac muscle fiber and complex electrochemical dynamics. Therefore, these facts are considered during the development of Heart-on-a-chip systems (Ribas et al. 2016). Electrochemical signals present in heart tissue are important because they regulate heart function and demonstrate spontaneous beating of CMs. A laminar flow and a pulsatile flow in the vasculature system are the next essential feature. The electrical field, stretching, the usage of hydrogels, or nanofibers are the key signaling parameters used to mimic native myocardium. Additionally, heart cell culture in the microsystems is often used to simulate heart diseases and investigate heart regeneration using stem cells (Fig. 7.3) (Ghafar-Zadeh et al. 2011; Visone et al. 2016).

Fig. 7.3
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Key signaling parameters used to mimic native myocardium in Heart-on-a-chip systems

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7.3.1 Vasculature and Dynamic Conditions

The microenvironment occurring in the vasculature system, provided by blood flow, is characterized by dynamic conditions. Mimicking these conditions in vitro is important to study the influence of shear flow and shear stress on cell proliferation, morphology, and viability (Cheng et al. 2003; Dahl et al. 2010; Li et al. 2005; World et al. 2006). However, the imitation of a vascular environment is difficult. It results from the fact that components of such a system (arteries, veins, venules, and capillaries) have varying compositions. Because of the dynamic microenvironment presented in a body, the microfluidic systems are excellent tools for mimicking these conditions (Wong et al. 2012). This results from the fact that laminar flow is provided in the microsystems (Young and Simmons 2009). A low Reynolds number and shear stress profile in the microsystems are similar to these, which are presented in vivo. Another feature of microscale, which helps in developing Heart-on-a-chip systems is the fact that microtechnology allows various flow profiles to be precisely designed. Thanks to this, a network of microstructures can be designed in such a way that in various areas of a microsystem different cell types can be cultured and simultaneously different shear stress values can be generated (Rossi et al. 2009). Endothelial cells cushion the inner heart chamber and blood vessels, therefore these cells are most often used to mimic the vasculature system in microscale (Hasenberg et al. 2015; Morgan et al. 2013). So far several types of the microsystems have been developed to mimic vessel connections and angiogenesis formation. Additionally, the microsystems for chemotaxis study and vascular disease modeling have been fabricated (Ribas et al. 2016).

Perfusion is also an important factor in fabricating a model of a beating heart culture. Due to the fact, that the heart is still simulated by a flow rate, assays based on the comparison of static and dynamic conditions are performed in the microsystems . These tests are able to check how a flow rate influences on proliferation and contraction of CMs. Additionally, perfusion can enhance parallel CM arrangement, which is specific for native myocardium (Kobuszewska et al. 2017; Xiao et al. 2014).

7.3.2 Materials Versus Cell Alignment

Mimicking of cardiac tissue microenvironment is also related to the selection of construction materials of Heart-on-a-chip systems. To fabricate such systems, poly(dimethyl siloxane) (PDMS) and glass are often used (Halldorsson et al. 2015; Ren et al. 2013). It results from the beneficial properties of these materials, which have been described in Chap. 3. To obtain a suitable microstructure/surface for cardiac cell culture, PDMS and glass are additionally modified. Native myocardial tissue is characterized by a parallel arrangement of the cells. Lack of such cell orientation is the main distinction between in vivo and in vitro cultures. Therefore, parallel cardiomyocyte alignment should be simulated in 2D and 3D in vitro models performed in microscale. Microcontact printing is a method used for obtaining ECM proteins on PDMS or glass plates and parallel CM alignment (Guillemette et al. 2010). Proteins such as collagen, laminin, and fibronectin have been used for this purpose. A fabrication of microgrooves in the culture surface has also been used to CM align (Motlhagh et al. 2003; Yang and Ma 2012). Scaffolds and hydrogels are other methods used to create a native myocardium environment. This can be achieved by the usage of regular nanofibers. Nanofibers have many advantages, which are useful for cell cultures: they have a high surface-to-volume (SAV) ratio and high porosity. Moreover, their structure and nanofiber organization influences the parallel orientation of CMs (Ashammakhi et al. 2012; Carletti et al. 2011). CM arrangement has been tested on nanofibers made of materials such as: poly(l-lactide-co-ε-caprolactone) [P(LLA-CL)] copolymer, poly(lactide-co-glycolide) (PLGA), poly(ε-caprolactone) (PCL), poly(hydroxybutyrate) (PHB), chitosan-polycaprolactone, polymethylglutarimide (PMGI) (Tomecka et al. 2017). The above mentioned materials can be placed on PDMS or glass surfaces as well as inside of the microsystems. Other method used for the creation of a spatial cardiac cell arrangement in the microsystems is the use of hydrogels. They allow to obtain uniform distribution of nutrients in 3D culture (Annabi et al. 2013). Hydrogels which are gelled under the influence of various external factors (ultraviolet irradiation, temperature, chemical factors) were used for the 3D culture in the microsystems (Hoffman 2012; Zhang et al. 2016b; Zuppinger 2016).

7.3.3 Stretching

Cell stretch, is the next feature, simulated in the microfluidic devices (Simmons et al. 2012). Regulating/moving of a surface, on which the cells are cultured, is used to stretch the cells. This is obtained by changing the pressure. Longitudinal, in-plane and out-of-plane mechanical simulations are used for this purpose. However, pressure microchannels are most often used to stretch a thin membrane made from elastic material (Huh et al. 2010; Moraes et al. 2010). This allows to mimic native conditions of heart cells.

7.3.4 Electrical Field

Electrical depolarization begins in Purkinje fibers and next propagates between the cardiomyocytes . This phenomenon is responsible for heart beat. Electrical stimulation is one method, which can be applied to cardiomyocyte depolarization and stimulation for contraction (Maidhof et al. 2012). Different types of electrical stimulation can be used for tissue engineering. It can be monophasic or biphasic stimuli, in a form of sinusoidal or square waves. Electrical signals can be delivered in pulses or continuously (Balint et al. 2012). The placement of an anode and cathode in the culture medium is the simplest method of inducing cell depolarization (Ribas et al. 2016; Serena et al. 2009). This allows to obtain a uniform electrical field across the cell culture . Either rods or microelectrodes are used in microscale. The integration of the microfluidic devices with planar electrodes and multi-unit electrode arrays (MEA) is the next method, which allows cell stimulation (Ma et al. 2012). In this case, the stimulation can be performed between two or more electrodes on which cells are cultured. Electrodes made of various types of materials are utilized for cell stimulation: stainless steel, carbon, platinum, gold, indium tin oxide (ITO). The integration of the microsystems with the electrodes allow to obtain controllable conditions, which influences cell contraction, alignment as well as differentiation.

7.3.5 Heart Failure Modeling

Lab-on-a-chip systems are used most often to analyze cell proliferation, migration, and interactions. Cytotoxicity assays of new drugs administered into the cells in a form of either solution or nanoparticles are also performed in the microsystems. Additionally, the mimicking of different organs is performed in microscale (Bhise et al. 2014). However, disease modeling is most often performed in Heart-on-a-chip systems. Arythmia, ischemia, and myocardium infarction were simulated and tested in microscale (Ren et al. 2013; Ribas et al. 2016). Ischemia was simulated by limiting the oxygen level or by the usage of a specific oxygen consumption blocking reagent: cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The microfluidic systems provide many possibilities to investigate the repair of “damaged” cardiac cells. Additionally, processes responsible for heart diseases can be studied in detail using in vivo-like models. Modeling of heart failure could be helpful in better understanding drug development and heart regeneration . The utilization of electrical fields, mechanical stimulation, SCs, or myocytes could be used to test cardiac cell regeneration in microscale.

7.3.6 Co-culture with Stem Cells

The microsystems used especially for heart cell cultures have also been used for co-culture. Because of the high importance of SC usage in regenerative medicine, these cells are most often used in co-culture with cardiac cells. They are utilized as a potential method to regenerate CMs (Garbern and Lee 2013). SCs can be differentiated into cardiac cells using different biochemical, mechanical, and electrical methods. Although, differentiation to various types of the cells is described in the literature there still are not too many reports about SC differentiation into cardiac cells (Ghafar-Zadeh et al. 2011; Jastrzebska et al. 2016). Most often such differentiation is started in macroscale, using conventional cultures and later differentiated cells are introduced and cultured in the microsystems (Mathur et al. 2015). The potential interaction between the cells as well as repair properties has been investigated using SCs (He et al. 2014).

7.4 Summary

Lab-on-a-chip systems for mimicking and studying heart cells are more often presented in the literature. Microtechnology allows in vivo conditions well to be mimicked. Therefore, the microsystems are increasingly used to simulate the vascular system , to culture cardiomyocytes (CMs) and to test their action after stimulation with various external factors. The microsystems for heart cell cultures have properties, which are the same as in another microsystems used especially for cell engineering, e.g., similar microstructure dimensions to cell dimensions, a laminar flow, high surface-to-volume (SAV) ratio, and effective culture volume (ECV). Besides that Heart-on-a-chip systems have additional features, which are specific for heart cells. Developing Heart-on-a-chip systems brings many challenges. It results from the fact that dynamic conditions, stretching, and electrical stimulation should be obtained in such microsystems. In the literature, various approaches and solutions have been presented, e.g., utilization of hydrogels and nanofibers, simulation of heart diseases. The microsystems presented so far have also a good potential to test the heart’s function and regeneration with SCs. However, there are many aspects, which should still be investigated and improved in microscale, e.g., long-term CM culture, SC differentiation.