All-round immobilization revolutionizes medicine
31 August 2007 (Volume 2 Issue 8)
‘All-round immobilization’, named by Yoshihiro Ito, Chief Scientist, Director of Nano Medical Engineering Laboratory in Discovery Research Institute, is a groundbreaking technique that enables the immobilization of any organic substance on a single substrate. His laboratory is working to create useful articles by combining biomaterials and artificial materials using all-round immobilization. Described below are various technical innovations expected to emerge from the laboratory, including protein chips capable of simultaneously diagnosing multiple allergies from a small-volume blood sample.
What is all-round immobilization?
Integration of medicine and engineering—this is the key phrase for the Nano Medical Engineering Laboratory.
“Recent years have seen remarkable activity toward the creation of new disciplines by combining different branches of learning, but such projects for the most part aim at fusing medicine and engineering,” says Ito referring to current major trends. Some 30 years ago, however, Ito was already involved in the integration of medicine and engineering.
Ito specializes in polymer chemistry. He has been engaged in research with a strong awareness of the application of his materials in actual clinical settings. “I believe there is no use in creating impractical articles,” says Ito in confidence. “Currently, I have two pillars to my work: diagnosis and treatment.” Both are being studied in joint research projects with the Kanagawa Academy of Science and Technology. First to be described is his research into diagnostic methods.
Ito describes the goal of his research as “quick testing for multiple parameters with a small sample volume.” He adds, “We are developing protein chips as an approach to accomplishing our aim.”
DNA chips are beginning to be used in medical practice to diagnose disease based on gene expression. A DNA chip comprises various DNAs mounted on small sections of a substrate. A sample is added dropwise to the chip. Any DNA in the sample that forms a pair with one of the immobilized DNAs on the chip binds to the partner and produces fluorescence. By identifying the fluorescent spot and reading the intensity of the fluorescence, it is possible to identify and quantify the disease-related gene expressed.
Protein chips have proteins, instead of DNAs, mounted on a substrate. A protein binds to another particular protein to exhibit its function. This specific binding of particular pairs of proteins enables us to identify and quantify the proteins in a sample by the same principles as those for DNA chips. Because different diseases can produce different kinds and amounts of proteins, protein chips are expected to serve for diagnostic purposes.
It has been said, however, that protein chips are difficult to incorporate into practical applications. “Because DNA has quite a simple structure with only four bases, any DNA can be immobilized on a substrate using the same method. In contrast, proteins, unlike DNAs, are structurally complicated and diverse, so that their immobilization has been impossible by a single [all-purpose] method.”
This problem has been beautifully overcome by Ito. His approach is as follows: prepare a substance that responds to light and mix it with a set of proteins to be applied to a substrate; mount this mixture on the substrate and expose it to light; a reaction known as radical crosslinking takes place, resulting in the immobilization of the proteins on the substrate. “A big feature of this method resides in the fact that not only proteins, but also any organic substance, can be immobilized. Hence, I named the method ‘all-round immobilization’ (Fig. 1).”
Figure 1: All-round immobilization methodenlarge image
Simultaneous diagnosis of 20 allergies
Among the various protein chips Ito has prepared using the all-round immobilization method is the allergy diagnostic chip (Fig. 2). It comprises a variety of allergy-causing antigens immobilized on a substrate. A 0.2 ml (about five drops) blood sample is added dropwise to the substrate. Any antibody that is reactive to one of the immobilized antigens binds to that antigen to produce fluorescence. Then, the fluorescence is determined using an automated tester developed by Ito’s laboratory (photograph on page 2 of this issue). Any allergies can easily be diagnosed within 40 minutes. “At present, we have only 10 to 20 kinds of antigens immobilized on a single chip, but this number can, in principle, be increased infinitely. Our method is advantageously capable of testing for multiple allergies with a small blood sample volume.”
There are about 400 kinds of antigens involved in allergies known to date, of which 100 relatively common ones are targeted in the development of the protein chip for simultaneous diagnosis. The chip has been developed through a joint research project with a private company, and is now under preparatory work for launch in the clinical market. According to Ito, inquiries are voiced from an unexpected sector. “We are receiving inquiries about our chip from animal hospitals. It seems that allergies among animal companions have recently become a serious problem. We will expand the coverage of our research to include animal applications.”
In autoimmune diseases, including rheumatism, a broad range of autoantibodies are produced in the body, which attack the body’s own cells. Chips for diagnosing such autoantibodies are also feasible. Identifying the autoantibodies produced in the patient’s body would enable an increase in therapeutic efficacy and help preventative treatments.
“Don’t you think it would be good to have chips for diagnosing, for example, coughs and diarrhea?” continues Ito. “If the physician is able to identify the pathogenic organism or virus contained in the patient’s sputum or feces, optimal treatment can be administered quickly.” He adds that diarrhea can be caused by very uncommon viruses. So far, there has been no diagnostic approach other than examining possibly causative viruses one by one. “It would be wonderful to achieve simultaneous testing in such cases.”
Because of its versatility, which gives all-round immobilization on a substrate, the allergy-diagnostic chip will find an infinite range of applications.
Figure 2: Allergy diagnostic chipenlarge image
Building a bridge between stem-cell technology and regenerative medicine
Ito has also been successful in another pillar of his work, the treatment of disease. As stated in the beginning of this article, Ito was engaged in the development of artificial blood vessels when he was a university student. “In those days, researchers were striving to fabricate various organs, including blood vessels, essentially with artificial materials,” says Ito in retrospect. “But as expected, they encountered limitations.” Ito adds that over the past decade or so, however, the mainstream of research has changed toward fabricating organs using a combination of biomaterial, such as cells, and artificial material. A representative example of the successful application of this approach is artificial skin. Another interesting finding was that the human body has stem cells capable of differentiating into a wide variety of cells. Accordingly, the idea emerged to use stem cells to produce regenerative medicine.
Traditionally, it had been thought that the cells that constitute the developed human body, except liver cells and other special types of cells, have finished their differentiation into cells with particular functions. It has been found, however, that there are hematopoietic stem cells capable of differentiating into various blood cells, such as erythrocytes, leukocytes, and platelets, and nervous stem cells capable of differentiating into various nerve cells. These are generically called somatic stem cells. It has also become possible to produce stem cells capable of differentiating into all types of cells, by manipulating cells taken from an embryo during the development from fertilized egg to an individual. These are called embryonic stem cells (ES cells), as opposed to somatic stem cells.”
“With these trends in mind,” says Ito, “I began an extensive study of engineering-based techniques for culturing stem cells of limited availability, and building a bridge between the proliferated cells and regenerative medicine.”
Ito had been involved in developing cell culture techniques. Usually, cell culture is performed using a culture broth supplemented with proteins that promote cell growth, known as cytokines. However, Ito’s technique comprises culturing cells on a layer of cytokines immobilized on the bottom of a Petri dish. “Cytokines simply added to the culture broth are incorporated by the cells and quickly consumed. In contrast, immobilized cytokines escape from cellular incorporation, so that their growth-promoting effects persist for a long time. When I discovered this fact, I said to myself—I did it! However, it took a long time for my achievement to be appreciated. Now, five or six years after my discovery, other researchers are using this culture method.”
This method, however, posed a problem. Because there are a wide variety of cytokines, many different methods must be used to immobilize them onto Petri dishes. The all-round immobilization method was developed through trial-and-error attempts. “All-round immobilization is actually a by-product of my research into cell culture,” says Ito. “Although cytokine immobilization has been proven to enable efficient cell culture, it will take much time to find practical applications in regenerative medicine.” Ito says that he began developing protein chips with the belief that the all-round immobilization method would find immediate application for diagnostic purposes. “This is in line with my own philosophy of creating something that is practically useful.”
Success with stem cell culture using human cells
Now returning to the story of stem-cell culture. Somatic stem cells and ES cells can be infinitely propagated while retaining their potential to differentiate into all types of cells if cultured under appropriate conditions. For human stem cells, however, this approach does not apply when they are cultured alone. Hence, it is common practice to culture human stem cells in the presence of mouse cells. Ito points out, “As long as non-human cells are used, the resulting stem cells cannot be used for human medicine, partly because of the risks of infection by unknown pathogens. It is essential to establish a method to culture human stem cells.”
Hence, Ito attempted to culture simian ES cells on a layer of human amniotic epithelial cells immobilized on the bottom of a Petri dish (front cover of this issue). “In Japan, the use of human ES cells for research purposes is subject to legal regulations, so we use simian ES cells, which can be taken as equivalent to their human counterparts in terms of research value because both species belong to the same order of primates. We have succeeded in propagating ES cells in the undifferentiated state for more than six months, even using human amniotic epithelial cells.” This represents a major step toward regenerative medicine. Ito’s laboratory has also succeeded in culturing human hematopoietic stem cells from umbilical blood.
However, Ito is looking at a goal beyond this. “I believe that an unknown protein contained in amniotic epithelial cells could make it possible to culture the stem cells. I have started identifying it.” He says that in the future, he plans to immobilize just these proteins on the bottom of a Petri dish without the use of human cells, and culture stem cells on them.
In addition to the protein chips and stem cell cultures based on the all-round immobilization method described above, Ito has implemented a broad range of technical innovations. Described below is one such achievement that represents a particularly interesting approach.
A number of polymers are biodegradable but have different times to degradation in the body. “Assume that drugs are placed in multiple holes made in a chip about one centimeter square,” Ito begins. He continues to explain that the holes are covered with various polymer films that have different degradation rates. Thus, the release of the drugs can be programmed as the films dissolve in sequence over time, and the different drugs can be made to act in sequence, by a one-time ingestion of the chip formulation. “The action pattern can be set freely, for example, at 12-hour intervals over three days or at one-week intervals over one month,” says Ito. It is very useful to have this kind of formulation because it eliminates any risk of forgetting to take the drug.
“Manufacturing based on a biotechnological approach—I want to create useful articles by combining biomaterials and artificial materials,” Ito concludes. And with this goal in mind, Ito’s laboratory will continue to develop a series of new technologies.