Developing a patterning substrate that generates gut organoids from human pluripotent stem cells

In 2015, Katsunori Tsuchiya was startled to see stem cells differentiating into several types of gut cells and becoming intestinal organoids within just 90 days on a patterning substratum developed by his research and development team at Dai Nippon Printing Co., Ltd. (DNP).

The miniature organoids made by Tsuchiya's team are three-dimensional cell cultures that incorporate key features of the human intestines and measure up to one centimeter long. They have absorption and secretion functions and even exhibit Peristaltic motion. *

Development of the organoids has been carried out by the National Center for Child Health and Development (NCCHD) in collaboration with Tohoku University and DNP, and the research results were announced in the Journal of Clinical Investigation, a U.S. medical journal, in January 2017. It was the first time ever that a three-dimensional organoid with intestinal functions had been produced in vitro from human pluripotent stem cells, both embryonic stem (ES) and induced pluripotent stem (iPS) cells. Behind this world-class achievement are Tsuchiya and other DNP researchers who contributed to the research and development through the provision of DNP's technologies to make thin, multilayered films, which are based on its printing know-how to coat several functional materials thinly and evenly.

* Peristaltic motion is a movement seen in an animal's digestive organs and accompanies muscle contraction waves like the locomotion of earthworms and other worms.

Pluripotent stem cells are master cells. They have the capacity to differentiate into all kinds of cells in the human body. In recent years, extensive research has been conducted in regenerative medicine using human pluripotent stem cells, which can make, in vitro, cells useful for studying new treatments or new drugs. Researchers have recently been attempting to make an organoid also in vitro.

But there are some major hurdles to clear in making organoids like intestines, which have one of the most complex structures and functions among human organs. Intestinal tracts have digestive, absorption and immunity functions derived from cells ­­– intricately functioning in concert. The intestinal tract also causes peristaltic motion. It had been considered difficult, if not impossible, to make a functional intestinal organoid in vitro.

The process of generating intestinal organoids on cell-culture substrate for patterning

DNP's technology for thinly coating multilayered materials helped researchers overcome these hurdles. The technology harnessed DNP's expertise in making printing plates, coating and patterning over many years as a printing giant in Japan.

The substratum is made as follows: On a glass or plastic surface, a special material is coated with a thickness of several nanometers, on which several nanometers of a polymer that does not allow the adhesion of cells is coated. This is subsequently exposed to ultraviolet light to make patterns of two areas – one for cell adhesion and the other for cell rejection. Cells adhere to the areas where the top polymer layer is removed by UV light, while avoiding the surface coated with the polymer layer, allowing cells to accumulate in patterns.

The 54th day of stem cells on a patterning substratum

The NCCHD and DNP aim to commercialize production of the intestinal organoids, which can be stored in vitro for an extended period when given nutrients, as a revolutionary bio-tool for testing new drugs. The NCCHD and DNP aim to perfect the technology to stably make intestinal organoids by the end of 2021, after which DNP plans to develop technology to mass-produce intestinal organoids for pharmaceutical and clinical testing companies by the end of 2022.

The mechanism for generating intestinal organoids remains shrouded in mystery. DNP plans to conduct further research with the NCCHD, one of the few institutions researching stem cells in Japan, to unlock this mystery.

Cell-culture substratum that was made based on DNP's technology to coat multiple, thin layers of materials

DNP is focusing on life science businesses development, optimizing its printing technologies to mass-produce products whose thickness measures micrometers or even nanometers. The genesis of DNP's life science businesses dates back to 1985, when the company started producing urine examination kits. The technology to coat enzymes just like inks was then applied to products to test for saliva as well as to pregnancy test kits. DNP also commercialized packaging materials for pharmaceutical products and medical equipment.

DNP started research and development in the medical sphere in earnest in the early 2000s. In 2004, DNP developed a technology to make capillaries measuring 10 micrometers in diameter on a pattering substrate by transferring patterned cells cultivated on a substrate to a different film or other materials in what is an application of its photomask (plates for making semiconductor products) technology, in collaboration with Tokyo Medical and Dental University.

In 2006, DNP set up its life science laboratory at the Institute of Advanced BioMedical Engineering and Science at Tokyo Women's Medical University and started actively conducting joint research on regenerative medicine. In 2008, DNP developed a technology to produce a cell-culture substrate, in which a patient's cells are cultured, for transplantation. By further advancing the technologies it developed with the university, Japan's first cell-culture substrate for patterning, which provided the basis for optimizing the substrate for generating intestinal organoids.

These outstanding achievements prompted the NCCHD to approach DNP regarding a tie-up. DNP technologies until then had been involved in culturing blood vessel, heart and bone cells taken from the human body by developing substrates in which the adhesion of cells was a main feature. The NCCHD thought DNP's substrates would be capable of controlling cell differentiations in unprecedented ways by culturing and patterning pluripotent cells. They started joint studies in 2011.

The Regenerative Medicine Promotion Act enforced in 2014 has given a tailwind to DNP and other private firms pushing ahead with research in life science areas.

DNP is aiming to stably supply intestinal organoids to pharmaceutical companies developing new drugs to treat congenital intestinal diseases and chronic intestinal inflammations, such as ulcerative colitis and Crohn's disease, whose causes are unknown.

Using organoids would allow pharmaceutical companies to slash the cost of developing drugs. During the screening of candidate chemical compounds, they can gather chemical reaction data from the organoids, which share properties with real human organs. It also will eliminate the use of animal experiments to test a drug's efficacy and safety before clinical trials.

Developing a new drug requires a huge amount of time and money. According to a survey on drug development published in Research Paper Series No. 59 by the Office of Pharmaceutical Industry Research in July 2013, researching and developing a single drug costs an average of ¥50 billion. But only one out of every 5.6 new drug development projects that advanced to human clinical testing after clearing pre-clinical trials with animals was approved later by the Ministry of Health, Labor and Welfare for its efficacy and safety. In short, drugmakers see many development projects fail despite a massive investment.

DNP has been conducting experiments mostly on iPS cells to develop know-how for stable mass-production at DNP Cell Processing Facility (CPF) in Kashiwa City, Chiba Prefecture, since 2017. Germs are so strictly controlled that researchers must change clothes twice before entering the CPF. A maximum of two people at a time are allowed to enter the room.

Tsuchiya is general manager of the Third Department, Research and Development Division for Converting Technology. The division is under DNP's Research & Development Center.

Originally a chemistry expert, Tsuchiya was transferred to his current position in 2010 because of his experience in dealing with a U.S. bio-venture firm during his 4 1/2 year stint in New York in the late 2000s.

“At first, I didn't even know what researchers were talking about,” Tsuchiya said. “But gradually I became fascinated with cells because each behaves differently. They are very sensitive and difficult to control, which really appealed to me.”

Tsuchiya also is a key member of the Forum for Innovative Regenerative Medicine, an association dedicated to promoting regenerative medicine. This role involves visiting other companies and government ministries and agencies for talks. “Through these activities, I would like to elevate DNP's presence in the life science business field,” Tsuchiya said.

In addition to developing technology to stably generate organoids, Tsuchiya has another mission for his team members, who mostly have bioscience backgrounds, including biology and pharmaceutical sciences. “We have to further incorporate DNP's printing technologies in our life science technologies to ensure DNP makes original products,” Tsuchiya said. “It is essential that each researcher understands DNP's printing technologies and thinks about how best to infuse DNP technologies into the life science business.”

April 16, 2018 by DNP Features Editorial Department