A HUGE collaborative project led by Los Alamos
National Laboratory (LANL) and Vanderbilt University is bringing
together human surrogate organ constructs married with highly sensitive
ion-mobility mass spectrometry in a bid to revolutionize the way that
compounds are screened.
Scientists from several different institutions are working together in the hope of developing a benchtop human, called Homo minitus, where miniaturized heart, lung, liver and kidney constructs are brought together and interconnected in a project called ATHENA (Advanced Tissue-engineered Human Ectypal Network Analyzer).
It is hoped that eventually Homo minitus will be able to mimic the way that multiple human organs respond to novel drugs and chemicals, providing far more information than an animal model can.
The current system for drug development first involves studies on cells in tissue culture followed by tests in non-human animals. By law, no drugs are allowed to enter human trials without first being tested in animals for possible adverse effects.
If approved, the novel compound may then proceed into phase I clinical trials on humans, but astonishingly around 40 per cent of trials fail in this stage, costing billions each year. This is because what happens in an animal does not necessarily mirror what may happen in a human due to physiological differences, therefore unexpected toxic effects may appear.
Although synthetic livers are currently being tested in the hope of reducing the need for animal testing, this is the first project aimed at connecting numerous different organ constructs in order to give a much more comprehensive picture on how a compound interacts with the body and produces side-effects. Senior scientist Rashi Iyer from LANL said “By developing this ‘Homo minitus’ we are stepping beyond the need for animal or Petri dish testing: There are huge benefits in developing drug and toxicity analysis systems that can mimic the response of actual humans.”
The scientists are not aiming at developing exact organ replicas; instead they have been miniaturizing them in such a way that will retain their functional capacity and key features necessary to behave in a manner similar to that of actual human organs. If successful, the organs will ultimately be hooked up via a blood surrogate in a way that imitates actual bodily connections.
It is hoped that this system could also be used in the field of toxicology, since a huge percentage of the tens of thousands of chemicals used in commerce are untested, and even those that have been tested have not been extensively investigated for long-term chronic effects.
Researchers from Vanderbilt University led by Professor John Mc Lean combined this miniature organ system with an ion-mobility mass spectrometer, enabling the detection and identification of the thousands of molecules that living cells produce, allowing them to monitor fluctuations in what is both consumed and produced in response to compounds being tested.
The first results to be reported from this system were in a presentation by Professor John Wikswo, which described the surrogate liver developed by a team of scientists also at Vanderbilt University. A small perfusion device was created, only a few inches in size, that was capable of keeping human liver cells alive for extended periods. They tested the effects of different dosages of the well known liver toxin acetaminophen. They found that the liver cells responded in the same way as a normal liver by first forming metabolites, then tryptophan levels began to increase as the cells became compromised. “After that we saw decreased production of bile acid, a clear indication that something was going very wrong with the liver, as expected when exposed to seriously high doses of acetaminophen,” said Wikswo.
The next stages of the project will hopefully involve hooking up the heart developed in Harvard to the liver, followed by the lung developed at LANL and the kidney from the University of California San Francisco and Vanderbilt University.
In recent years, a cadre of scientists and clinicians around the world has begun to develop more relevant and advanced laboratory tests for drug efficacy and toxicity: small bioreactors that can form human organ structures and are equipped with sensors to monitor organ health.
The ATHENA project combines the skills and insights of some of the top researchers in this pioneering field of research. The liver construct is being developed by Katrin Zeilinger, head of the Bioreactor Group and her colleagues at the Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charite UniversiUitsmedizin, Berlin. Kevin Kit Parker, Tarr Family Professor of Bioengineering and Applied Physics at Harvard University, is leading the heart effort. Shuvo Roy, director of the Biomedical Microdevices Laboratory at the University of California, San Francisco (UCSF), and Associate Professor of Medicine William Fissell of Vanderbilt are developing the kidney construct. In addition to leading the project, Iyer is directing work on the lung organ at LANL. Wikswo and his VIIBRE group are building the hardware platform and a heart test system.
Andrzej Przekwas, chief technology officer and senior vice president for research of CFD Research Corporation (CFDRC), a technology company in Huntsville, AL, and the LANL and Vanderbilt groups are creating a blood surrogate to sustain the four devices. CFDRC is also building a mathematical model of ATHENA to guide system design and data analysis.
One of the key questions for human organ construct developers is scale: What size should they make their artificial organs? Different groups have selected a variety of scales ranging from microhuman (one-millionth of size of human organs) to millihuman (one-thousandth the size).
“Scale is extremely important,” said lyer. If the scale is too small, she pointed out, then it is difficult to recapture the physiology because you need a quorum of cells before they act as an organ and it is difficult to get enough effluent to analyze. If the scale is too large, the costs of fabrication and human cell acquisition make the devices prohibitively expensive.
The ATHENA team at Charite started with a patient-support liver bioreactor with the volume of a human liver and scaled it down to a four-layer, three-dimensional device with a volume of only one-tenth of a milliliter. Zeilinger noted that “the cell mass of the final design was optimized based on metabolic performance and enzyme release and cell structures now resemble native human liver tissue.”
Charite’s original organ perfusion system cost $80,000 and was the size of a small refrigerator. Using simple microfluidics, the VIIBRE team created a 5x4x3.5-inch perfusion device that costs about $2,000 to make, Wikswo reported. They have validated its basic characteristics and demonstrated that it can keep human liver cells healthy for an extended period of time- the goal is a month.
Scaling is also important to determine the relative sizes and function of each organ represented on the platform. So if one were to have a liver that represented one thousandth of a human with a lung that represented one millionth of a human, the outcome would be very skewed. It’s just like having the heart of a 10-pound infant pumping to a liver of a 300-pound adult- it’s a no-go.
“We have picked a scale that is between microhuman and millihuman- one-tenth of the millihuman,” Iyer said. “I think the success that we are having with our liver device means that we have hit the sweet spot.”
In addition to successfully shrinking the organ platform, researchers in the Vanderbilt lab of John McLean, Stevenson Associate Professor of Chemistry, have introduced another important innovation by connecting the organ platform to a powerful, highly specialized instrument called an ion mobility-mass spectrometer, which can simultaneously detect and identify minute quantities of thousands to tens of thousands of different biological molecules simultaneously.
Other human organ construct/organ-on-chip research projects have reported tracking the variations in concentrations of a few well-known chemical compounds that are expected to change, but this is the first to successfully monitor the fluctuations of the thousands of different molecules that living cells produce and consume.
The researchers have used this capability to monitor the liver cells’ response to different dosages of a well-known liver toxin, the drug acetaminophen.
“We could actually see what the acetaminophen is doing to the liver cells,” said Wikswo. “In the beginning we saw an increase in the drug and its metabolites. Then, over the next 24 hours, we recorded a steady increase in tryptophan as acetaminophen began to interfere with normal liver metabolism. After that we saw decreased production of bile acid, a clear indication that something was going very wrong with the liver, as expected when exposed to seriously high doses of acetaminophen, and a decreased ability to detoxify penicillin.”
According to Iyer, this rich level of detail confirms that the ATHENA organ platform coupled with mass spectrometry technology can provide a more sensitive and effective method for screening both new drugs and toxic agents than is available today.
The team plans on hooking up their liver device to the Harvard heart this winter. They expect to add the lung construct being developed at Los Alamos next year and the UCSF/Vanderbilt kidney the year after.
The research is funded by Defense Threat Reduction Agency agreement # CBMXCEL-XLl-2-0001.