Review
Biological cardio-micro-pumps for microbioreactors and analytical micro-systems

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Abstract

Bio-hybrid microsystems actuated by living cells, as micro-bio-actuators and micro-bio-pumps have been developed recently. In these devices biological cells may be powered without external energy sources and the movement or the contraction of muscle cells trigger off the flow of fluid (i.e. culture medium or blood) through microchannels in micro-multi-bioreactor systems. Isolated and in vitro cultured cardiomyocytes (cardiac cells) are the most promising bio-material, which can be used to design a micro-bio-pump/actuator. These spontaneously contracting cells are autonomously powered with glucose as an energy source without any external power supply or stimulus, unlike conventional micro-actuators/micro-pumps. Cardio-micro-bio-pumps/actuators are using collective, synchronous contracting forces of cardiac cells or cardiac cell sheets to drive the flow of fluid. The feasibility of building such actuators was demonstrated in a few examples of bio-hybrid microsystems actuated by single or sheeted cardiomyocytes.

Introduction

It has been widely recognized that established substance safety testing of new developed compounds in the chemical, pharmaceutical and cosmetics sectors is poorly predictive to human long-term exposure. Recalls of substances from the market and a significant attrition rate at the level of bioactive compound development are the consequences. Currently available in vitro culture protocols for toxicology testing do not provide a fully acceptable in vitro model of human organ environment for repeated dose testing over long culture periods. Only several concepts nowadays combine a few different organotypic or tissue cultures within one micro-bio-environment system, thus allowing evaluating effects of chemical molecules, chemical active molecules, pharmaceutical drugs and cosmetic compounds on tissues systems [1], [2]. Consequently, engineers and scientists were faced with two main challenges at the beginning of this century [1]:

  • to miniaturize organ/tissue culture space from millilitre to microlitre scale;

  • to provide substantial sources of standardized human tissues.

It was obvious that further miniaturization of organ/tissue culture scale needs innovative realization ideas and radically new fabrication technologies.

It has been well known that every organ consists of multiple, functionally self-reliant, identical, micro- or millimeter scale structural units (from several cell layers up to a few millimeters), so-called sub-organoids [1], [2], [3], [4]. Such organ specific units are the smallest building blocks of each human organ, including several cell layers up to 1 mm, which corresponds to a volume of microliters size. Each very small but sophisticated sub-organoid provides the essential functionality of one of the most prominent organ. Multiplication of micro-sub-organoid structures within a specified organ is a kind of “nature's risk management tool” to prevent total loss of functionality after partial organ damage [1]. Adult stem cell niches were recently found in many relevant tissues and organs of the human body. In vivo, these niches carry the capacity to fully regenerate nearby micro-organoids in case of loss or destruction [4], [5]. In vitro, adult stem cell niches are the basis for the generation of such sub-organoids and form the basis of building artificial multi-micro-organoid systems [2], [6].

The technology which needs to be integrated with culture technologies to overcome the gap in the miniaturization of dynamic bioreactors to micro-scale size is called micro-electro-mechanical systems (MEMS) technology, and is a business that crosses over multiple technologies to provide high performance micro/nano-systems in various applications, combining micro/nano-system research with micro-fluidic technology [1], [6]. MEMS are at an early stage of their development cycle, but are already showing their great application possibilities. Nowadays, a lot of cell-based micro-system research takes place under this “lab-on-a-chip”, “organ-on-a-chip” or a “micro-total-analysis-system” (μTAS) framework that seeks to create micro-systems incorporating several steps of an assay into a single system [6], [7]. Integration of various process devices, cell culture systems and complex operations onto a multi-organ-on-a-chip is currently generating major interest due to the desirable characteristics of such system. “Living” micro-devices exhibit several distinct performance advantages including short diffusion distances, laminar flow regime, high interface-to-volume ratios and low heat capacities [7], [8], [9]. In other words, microchip devices provide several advantages for systems of cellular response analysis because the microfluidics inside the microdevice is appropriate to accommodate cells. New concepts in integrated miniaturized cell cultures systems also cope with the original human counterparts and satisfy the requirements of efficient long-term toxicology testing. Recently, bio-hybrid microsystems actuated by living cells as micro-bio-actuators and micro-bio-pumps have been developed in which biological cells may be powered without external energy sources and the movement or the contraction of muscle cells trigger off the flow of fluid (culture medium or blood) through microchannels in micro-multi-organ-on-a-chip systems [7], [10], [11].

Cardiomyocytes (cardiac cells) are the most promising bio-material, which can be used to design a micro-bio-pump. These spontaneously contracting cells are autonomously actuated just with glucose as the only energy source without any applied electrical power supply or stimulus, unlike conventional microactuators/micropumps. Cardiomyocyte sheets were first produced in the field of tissue engineering to reconstruct or repair a heart without any artificial scaffolds. Sheets of cardiac cells can be harvested intact, and transferred or subcultured to various devices while maintaining their regular and robust contracting phenotype. In cardiomyocyte sheets macroscopic pulsation is visible, therefore they can also be used as an element of micro-bio-pumps [12], [13]. Cardio-micro-bio-pumps (cardio-μ-bio-pump) use collective, synchronous contracting forces of cardiac cell sheets to drive the flow of the fluid. There are examples of hybrid (biotic/abiotic) devices, which consist of synthetic (polymers) and natural living (cardiomyocytes) materials [7], [11]. The feasibility of such microstructures to force the flow of fluids in microchannels will be demonstrated below in a few examples of bio-hybrid microsystems actuated by single or sheeted cardiomyocytes.

Section snippets

Cardiomyocyte sheets

Cardiomyocyte based sheets were first produced for applications in tissue engineering and regenerative medicine [14]. The basic idea of cardiac cell sheets was to propose an alternative therapy to cardiac transplantation and to create a kind of smart bio-material to reconstruct or repair a human heart without any artificial scaffolds [15]. The contraction and viability of grafted myoblasts were confirmed in the early 2000s. Multipotent bone marrow cells or embryonic stem cells have been

Flexible hydrogel micropillar micro-bio-actuators

The concept of bio-micro-actuators using cultured cardiomyocytes coupled to polymer-based microstructures is to convert chemical energy of culture medium into mechanical energy of micropillars’ movement. Two kinds of polymers are used as a material of micropillars: acrylic acid/N,N′-dimethylacrylamide (polyacrylamide gel, PAM) based hydrogel [27] and poly(dimethylsiloxane) (PDMS) [28], both studied by the Kitamori and co-workers. Fig. 2 presents the outline of polymer-based micropillar

Micro-bio-pump based on a pulsating cardiomyocyte sheet

Cardiomyocyte sheets establish electrical communications between neighbouring cells, which result in their strong and collective synchronous macroscopic pulsation. More practical and stronger actuating micro-bio-pumps/actuators could be created by exploiting long surviving and simultaneous beating sheets of cardiac myocytes [15]. A bio-actuated pump on a microchip powered by a cultured cardiomyocyte sheet and cell-coupled fluid mechanical motion was first demonstrated by Tanaka et al. [35]. A

Other cardiomyocyte-based non-fluidic micro- and millimeter-scale devices

Single or sheeted myocytes of cardiac muscle were also used for assembling bio-powered hybrid micromechanical devices, i.e. a coiled strip oscillator, a helical linear actuator, or motile robotic walking/swimming actuators [38], [39]. Some of them are schematically presented in Fig. 8. These milli- and centimeter-scale constructs were not considered as real micro-pumps but they also could be classified as bio-hybrid cardio-micro-actuators despite the fact that they perform functions as diverse

Conclusions

So far only few prototypes, which can be named as cardio-micro-bio-pumps/actuators have been described in literature. Basic properties of those devices have been compared in Table 1. They are interesting actuators working only with chemical energy input and mechanical force generation output. Cardiac myocytes, the most heavily working muscle cells in the living bodies, were used as generator of the force, which triggers the flow of fluid in microchannels. Such trials are of great interest for

Maciej Pilarek was born in Inowrocław, Poland in 1975. He is an assistant professor in the Department of Biotechnology and Bioprocess Engineering, Warsaw University of Technology, Poland. He received his M.Sc. degree in industrial biotechnology and Ph.D. degree in biochemical engineering from the Faculty of Chemical and Process Engineering at Warsaw University of Technology and performed a postdoc in the laboratory of Professor Peter Neubauer at the TU-Berlin, Germany. His scientific interest

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    Maciej Pilarek was born in Inowrocław, Poland in 1975. He is an assistant professor in the Department of Biotechnology and Bioprocess Engineering, Warsaw University of Technology, Poland. He received his M.Sc. degree in industrial biotechnology and Ph.D. degree in biochemical engineering from the Faculty of Chemical and Process Engineering at Warsaw University of Technology and performed a postdoc in the laboratory of Professor Peter Neubauer at the TU-Berlin, Germany. His scientific interest concerns bioreactors for plant and animal culture, particularly applications of perfluorinated liquid oxygen carriers in cell cultures and in micro-bioreactor systems. He is an author of over 15 peer-reviewed papers, chapters in one book, and one European patent declaration.

    Peter Neubauer obtained Ph.D. in microbiology from the Ernst-Moritz-Arndt University of Greifswald, Germany. After performing postdoctoral studies at KTH Stockholm, Sweden in the group of S.O. Enfors and being a group leader at the Department of Biotechnology, Martin-Luther University of Halle-Wittenberg (group of R. Rudolph) he obtained a professorship in bioprocess engineering from the University of Oulu, Finland, where he started collaborations in the field of microsystems, mainly directed to diagnostic applications. In 2008 he accepted a professorship at the Technische Universität Berlin in bioprocess engineering. His current interests are bioprocess scale up/down, miniaturized controlled culture systems and their application for the production of heterologous biocatalysts.

    Uwe Marx received his doctorate degree in immunology from the Humboldt University in Berlin after finishing his medical training. In 1995, Dr. Marx joined the University of Leipzig as head of the Department of Medical Biotechnology. His research projects focused on various aspects of tissue engineering, e.g. umbilical cord blood stem cell expansion and in vitro blood vessel formation. Between 2000 and 2010, Uwe Marx worked as the Chief Scientific Officer at ProBioGen – a biotech company he founded in 1994. In 2010, Dr. Marx joined the supervisory board of ProBioGen and became the head of a research group at the Department of Biotechnology of the Technische Universität Berlin concentrating now on the establishment of a new multi-organ chip technology platform.

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