Homunculi instead of guinea pigs
Artificial man on a chip will change pharmaceuticals
Maria Sotskova, Vitaly Kavtaradze, "Popular Mechanics" No. 1-2015
Published on the website "Elements"
A homunculus is a test tube creature, an artificial person, a dream and an impossible goal of medieval alchemists. But if the scientists of the past had rather selfish goals – to get closer to the Creator and learn some "truth" of life, now the approach is more than humanistic.
Have you ever thought about pharmaceutical manufacturing technology? From the development to the introduction of drugs go a long way – from a chemical formula to a successful marketing project. But the most difficult and ethically controversial point is animal testing and further human testing. Mice are usually used for tests, because their genome is very close to human, but still some specific reactions cannot be traced.
There are more than 200 factors of compatibility of blood groups, about the same number of target substances for drugs responsible for immunity, and many other unique features. What happens if the manufacturer fails to take into account at least one factor, not to mention the individual features of the body? Such involuntary mistakes lead to a lot of lost lives (1,000 testers died in ten years) and to huge financial costs for pharmaceutical companies to withdraw the drug from production, as well as lawsuits and restoration of reputation. Of course, all these costs fall on the shoulders of consumers.
As an alternative to animal testing, the technology of testing on individual cell cultures has been actively used in recent years. However, although this technique removes the ethical problem, it does not provide a systematic approach to research. After all, a drug designed, for example, to treat the liver, can have a detrimental effect on the stomach and kidneys, or even cause a deadly reaction of the immune system. Scientists are looking for a way out of the stalemate at the junction of biology and high technology.
The Road to Russia"Popular Mechanics" visited the laboratory of the Moscow Scientific and Technical Center "BioClinicum", where a unique project of creating an "artificial human" Homunculus has been conducted since 2008 under the leadership of Corresponding member of the Russian Academy of Sciences Alexander Tonevitsky.
The idea is simple and this makes it even more ingenious: to place human cells on a plate the size of a credit card and combine them with a system of "vessels" in the likeness of a living organism.
The golden mean. In terms of accessibility and relevance of the results of Homunculus research, they seem to be the optimal compromise between human trials and tests on 3D cultures and animals. Although, of course, bioreactor tests are not intended to completely replace other types of tests.
The idea of such a bioreactor originated relatively recently: in 2007, the first works of German scientists led by Uwe Marx appeared, in which the idea was expressed to arrange several types of cells side by side in order to simulate their interaction. Since then, many laboratories around the world have begun to solve this problem in their own way, but most specialists have managed to create only highly specialized systems. In the laboratory of Kae Sato from the Faculty of Applied Biochemistry of the University of Tokyo (Japan), the interaction of cancer cells of tumors with other tissues is studied, Donald Imber (Weiss Institute, USA) creates a filigree model of a "lung on a chip" capable of contractions and natural gas exchange.
The idea of creating individual models quickly develops into the concept of a full-fledged living human model, and Uwe Marx and a team of scientists from the TissUse laboratory turn to his former scientific supervisor Alexander Tonevitsky, head of the Scientific Research Center "BioClinicum". Thus began the history of the Russian project "Man on a Chip", which currently has the only working samples of the system in the world.
The laboratory has everything you need for prototyping,
and for the full assembly of all mechanical and electronic components of the system
The technology combines the achievements of a variety of sciences, microbiologists, chemists, physicists, programmers and engineers work at the STC. "Here we have everything we need: sterile PCR boxes, a low-pressure plasma furnace, a workshop equipped with the latest tools, including a 3D printer, a laser engraver and computer-controlled machines," says our guide Dmitry Sakharov, director of the Homunculus project, not without pride. In such conditions, a group of talented young scientists is creating the future of medicine – little "men" on glass and plastic, which are designed to save many lives.
Flesh and bloodThe Homunculus platform consists of a cellular chip and a control unit that monitors the progress of the experiment and supports the life of a little man.
Cell cultures are placed on the chip, primarily those through which the test substance will enter the body, as well as those on which it should affect.
SANDWICH WITH TECHNOLOGYDesign of the latest generation PDMS substrate
From the point of view of the design, the Homunculus chip looks simple, but this simplicity is apparent. Almost every component of it is manufactured using the most modern technologies, such as injection molding and plasma activation.
1. Depending on the tasks, the cells may contain cells of the kidney, heart, brain, skin, lungs, or optical or electrochemical sensors may be installed in them. 2. Intestinal cells. 3. Liver cells. 4. Expansion chamber. 5. Channel for carbon dioxide enrichment. 6. Valves of the micro-pump and valves of tanks for changing the medium. The "blood" of an artificial human is a nutrient solution that supplies cells with all the compounds necessary for life.
The test substance is also injected through it. The solution contains a set of salts that maintain a constant acidity of the medium, since the cells secrete their alkaline metabolites into the solution. It also contains a nutritious broth, and the "diet" is clearly observed – there is the necessary amount of proteins, fats and available sugars. The composition of the solution is very close to the composition of blood plasma, but there are no analogues of erythrocytes with hemoglobin – oxygen enters the system dissolved in liquid.
Cell cultures are located in transwells – special cells with a semi-permeable membrane from below, through which cells exchange substances with the nutrient medium and with each other. Perhaps someday scientists will be able to place on the platform and bring into interaction all (or almost all) types of cells contained in the human body. However, while the number of cell cells on the chip does not exceed six, the developers are trying to focus on the organs that are directly in contact with the tested drug, participate in its transmission and isolation.
For example, if we are talking about tablets, the active substance is placed in a cell with intestinal cells, absorbed by them and enters the nutrient medium through the membrane. External agents are injected through the skin cells, and intravenous – directly into the solution. The liver and kidneys involved in the metabolism and excretion of the drug are certainly tested. The heart and brain, especially sensitive to toxic drugs, are not deprived of attention. The list of available cells can be expanded constantly – the main thing is that within the test cell cultures function and interact in the most plausible way.
The test lasts about 28 days, after which specialists begin processing the results. The most obvious indicator is the number of living cells by the end of the experiment, but the most accurate results are obtained by microbiological and genetic studies of RNA and DNA. This makes it possible to determine the delayed toxicity of the drug in the case when it does not kill the cell instantly, but causes mutations in the genome and metabolic disorders. The consequences of such poisoning can be noticeable only after a few years, and standard laboratory testing methods do not allow them to be fixed.
By the time the work began, the question of which cells to use for cultivation was particularly important for researchers. Embryonic? Cells of living people? Or something else? The answer was found unexpected – scientists use cancer cell lines obtained from the world's cell banks. What does oncology have to do with it? Cancer cells live longer, grow better, and most importantly, they are standard, widely available and described in detail in numerous publications, while their functions are exactly the same as in healthy ones.
Cyborg EvolutionDespite the seeming completeness and independence, the miniature man cannot breathe by himself or drive nutrient fluid through his "veins".
This requires a control unit – the electronic brain, heart and lungs of the "homunculus". The device contains micro pumps that ensure the circulation of the nutrient medium and vacuum control valves installed in the silicone layer of the chip. In addition, it supplies carbon dioxide and oxygen to the system, and also maintains a constant temperature of the chip.
The control unit. The external control unit is the heart, lungs and autonomic nervous system of the "homunculus". It feeds oxygen and carbon dioxide into the chip, monitors its temperature and informs researchers of any changes in the chip's parameters, wherever they are at that moment. The compact device takes up no more space than the system unit of a desktop computer, and has a friendly interface that can be used not only by employees of the Bioclinicum, but also by any other specialists.
All parameters can be adjusted according to the objectives of the experiment. The device has a built-in touchscreen display and is connected to a computer via USB or LAN, and the software interface is designed so that doctors and researchers from other laboratories can easily and quickly master all the functions. In general, this is not a device created for a specific task, but a multifunctional platform ready for release to the market.
The control unit, like the chip, is the brainchild of the center. "All the components used have long been known to engineers all over the world, but their correct combination and calibration make the development unique and innovative," says Dmitry Sakharov. At first, all the parts of the device were made by hand, including the printing of boards and the assembly of cases, but when sales began, the main components began to be ordered to an assembly company in St. Petersburg, from where the device comes in the form of a designer.
After the chip is assembled, its channels are immediately filled with liquid:
the thinnest capillaries must be constantly wetted, otherwise
subsequently, they will not be able to fill with a nutrient medium
"A man on a chip is only the first step towards the goal, but technologies are developing rapidly, and a more advanced model will be created in the near future," Academician Tonevitsky shares his vision. In the near future, the laboratory plans to create a chip on which ten or more cell cultures can be placed simultaneously, and not six, as it is now. It is also important to make cellular models as close as possible to real structures in the human body. After all, we should not forget that now we are dealing with only a few thousand cells in a cell, and real organs have a complex, differentiated structure with tissues that interact unpredictably with each other. The endocrine system is the most difficult to experiment with, in which hundreds of different hormones are targeted at hundreds of different targets throughout the body.
Bioclinicum scientists are following the path of combining the living with the inanimate: the new version of the chip, which is still in development, has semi–permeable channels through which air circulates - an analogue of human lungs. The intestines of the new little man will have bends similar to the bends of the real prototype, and the cells will be arranged in the right order. The laboratory is confident that the project will be improved until it is possible to simulate all organ systems in all their diversity.
Ethics of the futureDespite the fact that "man on a chip" removes animals and humans from the impact of drug trials, many may be wondering if the test cells themselves can experience pain, especially when it comes to experiments on the nervous system?
Fortunately, the fears are in vain, the cells are cultured separately and do not have innervation. These are just lumps of living tissue combined into a system, and not a full-fledged organism, so there is no need to talk about creating artificial life.
Meanwhile, the microbioreactor can not only reduce the number of animal tests, but also significantly accelerate progress in both pharmaceuticals and medicine. At a certain stage of development, the new technology will make it possible to accurately select hormone therapy and minimize risks when selecting medications.
Homunculus is a completely Russian project that receives grants from the Ministry of Education and Science and the Ministry of Health; permits for testing new drugs have already been issued, and there are international contacts. Now BioClinicum is testing its systems in Russia and Germany, because it is important to prove the correctness of the installation to customers all over the world, and not only in the domestic market. When the device passes all the tests and receives full certification, its implementation will not take long, and perhaps very soon we will receive a lot of life-saving drugs created thanks to the silent "feat" of many "little men" on chips.
HOW PEOPLE MAKE PEOPLEThe heart of the bioreactor – a chip with cells for cells – is a sandwich of polycarbonate, polydimethylsiloxane (PDMS) and glass connected in very non-obvious high-tech ways.
1. The silicone layer serves as the circulatory system of the chip and contains a pattern of tiny channels with a depth of only 100 microns. To make it, a polycarbonate plate is laid in a metal mold with a convex pattern (the same 100 microns). The mold is closed, funnels with PDS are inserted into it, and the entire structure is sent to a heated centrifuge, also developed in the center. There, the silicone heats up to 70 degrees, becomes liquid and easily takes its place between the mold and the plate.2. The resulting "semi-finished product" needs to be closed with a slide, but it is not easy to do this.
The glass should not only tightly cover the vessels, but also not affect the test results. Therefore, glue cannot be used. We solved this problem by processing both halves in a plasma furnace. Plasma activates the surfaces of silicone and glass, makes them hydrophilic, and also increases their ability to stick together. After processing, the glass and silicone are combined, and the sandwich can no longer be separated3. In the "clean room", where, as a rule, outsiders are not allowed, various cell cultures rest in liquid nitrogen.
In order for them to turn from cellular ice cream into a material for research, they are cultured separately for several days. Since different types of cells require different time for differentiation, that is, separation by shape, structure and function, they begin to grow with an interval of up to several days, so that by the beginning of the experiment all were at the same stage of development.4. As soon as the organ models are ready, they are placed in transveles.
The chips are unpacked from sterile bags and filled with cells. Throughout the entire assembly, the chip is kept in sterile conditions so that no foreign cell gets inside. The entire assembly process is carried out in one laboratory by one specialist, which makes the development even more convenient to use.Portal "Eternal youth" http://vechnayamolodost.ru