Hepatotoxicity is one of the primary reasons why drugs are recalled drugs from the market, a result principally due to shortcomings in preclinical hepatotoxicity testing.
Current ADME/Tox testing protocols use human hepatocytes, which survive for only a few days in vitro, or rat hepatocytes, which may not respond to drugs in the same way that human hepatocytes do.
Scientists at the Massachusetts Institute of Technology hope their recent invention may sidestep these issues. The researchers used a microtechnology-based process and elastomeric stencils to culture living human liver cells in a standard multiwell format that maintains function for up to six weeks.
The polydimethylsiloxane, or PDMS, stencils comprise 300-µm-thick membranes with through-holes in the bottom of each well in a 24-well mold. The mold was sealed against a polystyrene plate, collagen-I was adsorbed to exposed polystyrene, the stencil was removed, and a 24-well PDMS “blank” was applied.
The selective adhesion of hepatocytes to collagenous domains yielded “micropatterned” clusters, which the researchers surrounded with supportive mouse 3T3-J2 fibroblasts. The micropatterned configuration comprised 500-µm colonies with 1,200-µm center-to-center spacing.
Sangeeta Bhatia, an associate professor in the Harvard-MIT division of health sciences and technology and MIT’s department of electrical engineering and computer science, and Salman Khetani, an HST postdoctoral associate, published their work in the Nov. 18 online issue of Nature Biotechnology.
Bhatia and Khetani formed a startup company called Hepregen that is currently in the process of obtaining an exclusive license for this technology, which is called micropattern co-culture.
Khetani spoke with CBA News this week about the technology and his plans for Hepregen.
Did you use human primary hepatocytes in your work?
Yes. The main cell type we used is the primary hepatocyte and the supporting cell type is a fibroblast cell line. These were primary hepatocytes harvested from those who had passed away, and … their livers were not suitable for transplantation.
Could you do this work with stem cells?
Yes. The basic idea would be that instead of primary cells, you would use stem cells that had first been differentiated into liver cells and then use the liver cells on our platform.
You can do this one of two ways: You can have stem cells that have first been differentiated into liver cells by some other method, and then once they are differentiated into hepatocytes, you use them with our platform. Or, you can use our method initially with a supportive cell type that induces both differentiation of the stem cell into the hepatocyte and supports the hepatocytes survival and functionality.
However, that is an area where we would need to do more research and iron out the details.
Could you give me a little background on your work?
Our primary motivation is the recognition by those in the field that primary hepatocytes are sort of the gold standard when it comes to ADME/Tox screening in drug discovery and development.
Liver toxicity is a major problem for drugs being withdrawn after they have hit the marketplace. Animal [models aimed at identifying liver toxicity in preclinical trials] are not sufficient. You need human responses when screening drugs [but] we do not want the marketplace to be the human screening area.
The idea is using liver models in a drug-discovery program where the … toxic candidate compounds would be screened out early on in the process. The gold standard for that kind of screening work is the freshly isolated hepatocyte.
Stem cells may in the future become a source of these cells, but the field, in general, is not there yet. However, the problem that has plagued the field for over two decades now is that primary hepatocytes are wonderful cells in the body, but as soon as they are outside the body, they lose their function and quickly die off.
People have tried many different modifications, including culturing them on a collagen gel or a Matrigel. Out of those modifications, you may gain a few extra days survival … compared to 24 hours. But their function will be constantly declining.
This innovation is primarily geared toward creating a multiwell system in which … the hepatocytes maintain their functions [in each well] close to their natural physiologic state for not just days, but for four to six weeks.
How does this technology work? What do you do once you get the liver cells?
Primary hepatocytes that have been isolated from the human donor are arranged in a micro pattern. They are arranged in colonies of 0.50 mm or 500 µm in size. They are very regularly spaced — these 500 µm colonies are spaced exactly 1,200 µm apart, center-to-center. Surrounding these colonies are the supportive fibroblasts.
So this biological model has two components to it: One is that we organize the architecture of the cell by arranging the cells in a specific architecture using tools that we borrowed from the semiconductor industry.
Form follows function. We found that by creating single-cell colonies spaced 1,200 µm apart, we could change the function of the cells.
As the colony size grows, cell-cell interactions increase. These cell-cell interactions lead to better function. But that architectural modification is not enough. For the cells to survive for several weeks, you need a supportive cell type that provides some nutrients, some signals which we have not yet elucidated. That is ongoing research in our lab.
You need to put these supportive cells around the colonies. The combination of these architectural modifications and these supportive cells leads to this robust biological model, which we call micropatterned co-culture.
To create the micropattern, we use a technique similar to that used by children to draw their letters using a stencil. It’s a rubber stencil, and basically each well is a thin membrane with holes in it. The dimensions of these holes are optimized to provide the best function.
Each hole is 500 µm and spaced 1,200 µm apart. You basically attach the stencil to a 24-well plate. You put the extracellular matrix protein through the holes and then peel the stencil off. You put in its place a placeholder, just to maintain the multiwell structure of the plate.
When you seed these hepatocytes they will go to the ECM spots. Then you can surround the colonies with supportive fibroblasts. This is a very rapid way of creating a system of micropattern co-cultures.
The goal of this technology is to take this technique from the hands of the academic and bring it out into the marketplace.
Hepregen is in the process of obtaining an exclusive license for this technology. Dr. Bhatia and myself are the academic co-founders of Hepregen. It’s a virtual company at this point. We are currently looking for seed funding.
You are in the process of obtaining seed funding for your company. In what form?
We are keeping our options open. We are doing two things: looking for seed funding and we are applying for a bunch of SBIR grants through the NIH and the NSF. Those grants are actually due within the next week to 10 days.
Everything in the paper is really a proof of concept. We have shown that the cells can survive in culture and we have demonstrated that they maintain functionality, but as far as their applicability to drug discovery and development, we have shown proof of concept.
Now we need industrial-level data, industrial-scale data. The SBIR grants and seed funding are to set up shop, first of all, then to actually use industrial level parameters to show the power of the system in collaboration with the pharmaceutical industry. So it would have to go into beta testing, which is what we hope to do within the next year.
So, in terms of a timeline, by when are you hoping to secure funding?
Our grants are due by Dec. 5 and it takes about six months to get the money. That money will mostly be used for R&D. In terms of seed funding to set up shop, we are actually presenting to investors this month and next month and then again in January. We are hoping that by the spring of next year, we will have an operational company.
And then all next year, we will be doing beta testing of this platform. We are in negotiations with pharma partners. I cannot discuss this further except to say that they are major pharma companies.
Can you discuss how much money you are seeking?
It will be a balance between how much partnership money we get and what SBIRs we get. In terms of seed funding, it’s probably on the order of one or two million dollars. As I said, that number can be as low as half a million, if we got a lot of money from pharma partners, or it can be as high as several million, because we really need to establish a fairly large infrastructure. That is something that we are actively figuring out right now.
In terms of our target market, the screening market, the ADME/Tox market, is approaching close to $500 million to $750 million per year in the US. I got that estimate from a Frost and Sullivan report.
In addition, right now we have a model of a healthy liver. But what if we could create a model of a diseased liver? What if we could infect it with hepatitis C or anything else that affects the liver? Once you create a model of a diseased liver, then you could do drug discovery on it. And that’s a very big market — billions of dollars.
The market numbers vary depending on which segment you are focusing on. Right now, since we do not have a model of a diseased liver, we are focusing on the drug screening, ADME/Tox market.
What would be the next step of this project? Obtaining seed funding?
The first thing is to get seed funding to set up shop. The second thing is to get this platform into the hands of customers — biotech and pharma companies for beta testing and ADME/Tox. We are simultaneously approaching investors and pharma companies so that by the spring of 2008, we will have collaborative agreements with the pharma companies and seed funding so that we can begin the beta-testing phase.