Human Based Non-Animal Research Methods
Researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University, Harvard Medical School, and Children's Hospital Boston have created a device that mimics a living, breathing human lung on a microchip view video. The device, about the size of a rubber eraser, acts much like a lung in a human body and is made using human lung and blood vessel cells. The Wyss Institute team is also working to build other organ models, such as a gut-on-a-chip, as well as bone marrow and cancer models.
A microfluidic device using human colon cancer cells has been designed by researchers from the University of Massachusetts. It mimics the varying cellular conditions present in real tumours, which can limit the efficacy of many cancer therapies. The team believes that their device will be 'vital for understanding the behaviour of common cancer drugs in solid tumours and designing novel intratumourally targeted therapeutics'.
Work on computer cancer simulations has been ongoing for decades, and is highly useful for pharmaceutical and novel therapy development. A 2008 review by a researcher from the US National Cancer Institute stated: 'Applications and benefits of computer-aided drug discovery and development have been reviewed and demonstrated in a growing number of publications and supported by examples of drugs derived from the in silico approach'.
Computer models can also predict which patients may respond to chemotherapy. Researchers at the University of Virginia have developed a model that uses genetic analysis of tumours to predict which treatment will be of most benefit. In two large trials of drugs for breast cancer, the model predicted with 85 per cent accuracy which patients would respond to treatment. Yet another application is the prediction of cancer progression, and a resultant tailoring of therapy. A mathematical team from the University of California has demonstrated that cancer growth is not as erratic as previously thought, and depends in part on the tumour environment. Their model consistently reproduced cell invasion patterns observed in experiments and patient biopsy samples.
See how these human based methods are used in
Use of human-derived raw materials
Biopsy material from human cancers is immensely useful in the development of new therapies. German researchers have succeeded in culturing tissue from breast cancer patients for more than one year, during which the cells continued to proliferate. The team found that cancer drugs differed in their cell-killing effects between cultures from different patients. They say their work represents a reproducible screening platform to identify new biomarkers and to test new therapeutics in individual tumour samples'.
On a much grander scale, 'biobanks' can act as repositories for human bio-specimens. These can then be used by research groups to help understand the molecular mechanisms involved in cancer, and in development of targeted treatments for individuals. The Wales Cancer Bank aims to collect samples of tumour, normal tissue and blood from all patients in Wales who are undergoing an operation to remove tissue where cancer is a possible diagnosis. It hopes to have the consent of 5000 patients by the summer of 2011.
An excellent collection of expert-written, short papers on the use of human tissues can be found here:
Human tissue models
Scientists at the University of Nottingham are refining a 3D cell-culture model of tumour tissue to help develop anti-cancer drugs against solid epithelial malignancies. In the UK, 400,000 mice were used in 2008 for research in this area. The scientists say these xenograft models are inappropriate, with only a 30-40 per cent success rate (less than a tossing a coin) in predicting clinical efficacy in Phase II clinical trials. Their 'biomatrices' use human epithelial cells and material from human colon cancers that had spread to the liver.
Researchers funded by the Dr Hadwen Trust and the Lord Dowding Fund have developed unique 3D cultures of human breast cancer. These are being used to investigate a common pre-invasive stage of cancer that occurs in up to 40 per cent of patients. The development of a human-relevant model means that researchers can far more reliably investigate the earlier stages of the disease, as well as potential new breast cancer treatments. In 2010, the team further validated their model using sophisticated computer technology.
High resolution scanning
Today's sophisticated scans have an essential role in the diagnosis and treatment of the vast majority of malignancies. Earlier diagnosis has been shown to improve survival in many kinds of cancer.
A relatively new scanning technique, magnetic resonance spectroscopy (MRS), can demonstrate biochemical information about living tissues. This has applications in both research and treatment - improving the accuracy of lesion diagnosis, for example, or monitoring the response to new cancer therapiesin trials. MRS has been used most extensively in investigating breast, brain and prostate cancer.
Parts of this section have been adapted from the Safer Medicines Campaign - please see: http://www.safermedicines.org/ medicalresearch-news/index.shtml
Canadian scientists have developed microfluidics chips to enable fragile blood vessels to be studied easily and cheaply, without the highly skilled handling usually required to conduct such studies. The device will enable thousands of potential new drugs to be routinely screened for their effects on blood vessels, including, for example, changes in blood pressure.
Scientists initially used mouse arteries, though there seems no reason to suppose that they were essential. In fact, the lead engineer on the project said the next step is to make chips that could hold biopsy material from people. The chips could be used with vessels taken from individual patients to help doctors tailor blood pressure medication regimes.
Dr Peter Kohl, a researcher at Oxford University (where Professor Denis Noble pioneered the virtual heart) has developed computer models to improve heart surgery. His team aims to use heart scans combined with modelling to investigate surgical options for cardiac surgery, to ensure patients receive the best treatment. His research is part of an international drive to model the human body known as the Virtual Physiological Human initiative.
Meanwhile, in 2008, the US Food and Drug Administration entered into a partnership with Entelos, a company that specialises in modelling patients and even whole clinical trials. Their advanced computer simulations focus particularly on risks associated with the heart, to try to improve the safety of drugs released onto the market. It is believed that had the Entelos system been available at the time, the Vioxx painkiller tragedy, where tens of thousands of patients died of heart attacks and strokes, could have been avoided.
Use of human-derived raw materials
In 2008, Dutch researchers succeeded in growing large numbers of stem cells from adult human hearts into new heart muscle cells. It was previously necessary to use embryonic stem cells for this purpose. The stem cells were derived from material left over from open- heart operations. The cells grew into fully developed heart muscle cells that contracted rhythmically, responded to electrical activity, and reacted to adrenaline.
The principal investigator, Professor Pieter Doevendans, said: 'We're able to make heart muscle cells in unprecedented quantities, and on top of it they're all the same. This is good news in terms of treatment, as well as for scientific research and testing of potentially new drugs.' Doevendans planned to use the cultured heart muscle cells to study cardiac arrhythmias (abnormal heart rhythms). Stem cells from patients with genetic heart defects can also be grown into heart muscle cells in the lab, and be used to test new medicines. This could accelerate research into genetic heart conditions. In the future, new heart muscle cells could be used to repair heart tissue damaged during a heart attack.
Human clinical data and computer modelling
Norwegian researchers in 2009 studied the organisation of human ventricular fibrillation (VF), a heart rhythm disturbance that is lethal if not treated. They combined electrical recordings of the heart surface of patients undergoing cardiac surgery with simulations of VF in a detailed computer model of the human ventricles. They discovered that human VF is significantly different from that experimentally induced in dog and pig hearts, and concluded that 'the simpler spatial organisation of human VF has important implications for treatment and prevention of this dangerous arrhythmia'.
High resolution scanning
In 2009, the London Chest Hospital installed the first SOMATOM Definition Flash CT system in the UK, offering faster results and lower radiation doses than traditional models. The scanner has enabled experts at the Barts and The London Cardiovascular Biomedical Research Unit to undertake vital cardiac research. The system can perform a detailed scan of the entire heart in just 250 milliseconds - less than half a heartbeat. 'The Definition Flash will be an invaluable tool for helping in the research of new treatments for cardiovascular disease as it presents a much clearer picture of the workings of the heart', said Professor Anthony Mathur, head of advanced cardiac imaging at Barts and The London NHS Trust.
Microarrays are small membranes or slides containing samples of many genes. They have been extensively used to discover how heart diseases 'turn on' genes in cardiac cells. Such studies have thrown up interesting results. The biochemical profiles of failing heart tissue, for example, did not necessarily match a cause-based clinical classification. Researchers believe that this could have direct relevance to the type of drugs that might be successful in individual cases.
Someparts of this section have been abstracted from the briefing by the Dr Hadwen Trust entitled 'Developing human-related approaches to understand and cure Parkinson's Disease'. For more information, visit: www.drhadwentrust.org
In a recent study, researchers at Rutgers University have developed a computational model that shows how PD affects attentional performance during learning. The work demonstrated how the interaction of dopamine with the prefrontal cortex (a higher region of the brain) is key for attentional learning, whereas the interaction of dopamine with the basal ganglia (responsible for coordinated movement) is key for motor learning.
Systems biology uses the wealth of data already available to build an in silico approach to PD, combining mathematical modelling, systems analysis and associated measurement techniques. Computer models of the disease allow multiple in silico investigations of the origin and progression of PD, and provide a platform for researchers to design new experiments and trials.
Use of human-derived raw materials
Studies are conducted on human brain tissues from patients in order to better understand the disease. Such studies can only be done using human tissues, since the condition cannot be 'modelled' reliably in animals. Just one donated brain can be used in up to 50 different research studies. In parallel, researchers in Oxford have begun creating a bank of artificially grown brain cells from Parkinson's
patients. They are using a stem cell technique, developed by Japanese scientists three years ago, to turn a small piece of skin into a small piece of brain. Dr Michelle Hu of the John Radcliffe Hospital in Oxford, stated: 'We can look at what cellular processes are happening that make the cells die and learn why it is that the cells get sick. And we want to see if there are any treatments we can offer to reverse that process and help patients regain normal function'./www.bbc.co.uk/news/health-13810653
Human tissue models
Human cell culture experiments have suggested that distinct cell death mechanisms play a central role in the development of PD. These cells have been widely used for some years - one of the most popular lines was established in 1970 from a bone marrow biopsy of a four-year-old girl. It can generate fully mature nerve cells producing dopamine, a promising tool for the development of new drugs and cell based therapies.
High resolution scanning
Non-invasive methods such as magnetoencephalography (MEG) have been useful in studying neurological disorders such as PD. The Dr Hadwen Trust and the Lord Dowding Fund for Humane Research are funders of the MEG system located at the recently launched Aston Brain Centre, a Centre of Excellence. Aston University has a 40-year track record of leadership in clinical neurophysiology. MEG is an extremely powerful tool for improving understanding of drug/brain interactions, with a particular relevance for PD.
The Californian researcher who co-authored the first published paper on DNA microarrays recently commenced a project to discover biomarkers (chemical indicators of disease) for PD. The study involved the collection and analysis of samples from well- characterised Parkinson's patients using microarray technology. Experiments already underway had enabled rapid and efficient sample preparation of specimens from Parkinson's Disease patients, an important step in the discovery of molecular markers for the disease. In a ''wo-pronged approach', a whole human genome microarray was used to study 25,509 genes. A second microarray, comprising monoclonal antibodies against human plasma proteins, was used to identify low abundance biomarkers in the blood
of Parkinson's patients. Used together, the two microarrays can identify mRNA and protein molecules, and how they are differentially expressed in Parkinson's versus normal subjects.
Epidemiological studies can provide essential information about the causes of Parkinson's Disease, predisposing risk factors, protective factors and preclinical characteristics. Information gleaned can then be investigated in the laboratory or in clinical trials. Epidemiological investigations are also critical for public health planning.
Genetic risk factors can be uncovered as well as environmental influences. In 2011, Californian researchers published the results of the largest single PD genome-wide association study to date. They studied more than 3,400 cases and 29,000 controls. Two novel genetic associations were described, and 20 previously described associations were validated.
Volunteer studies are extremely useful to investigate subtle differences between patients. A collaborative project at Oxford Parkinson's Disease Centre is studying DNA from patients, in combination with MRI brain imaging, to identify potential biomarkers for the earlier detection of PD. This might permit the protection of healthy nerve cells and delay the onset of the disease.
Researchers from the University of California, Irvine have grown human neural stem cells on a microfluidic platform. These cells (obtained from peripheral tissues with informed consent) turned into specialised nerve cells called astrocytes, and remained healthy throughout the entire culture period. The device could be used for a wide range of basic and applied studies into diseases such as Alzheimer's and Parkinson's.
Bioengineers from the University of California, San Diego, have developed detailed computer models of brain metabolism, which could explain why some types of neurons die sooner in Alzheimer's patients. The group used their model to 'knock out' a gene known to be damaged in Alzheimer's Disease and found that the consequences agreed with clinical data. The model could be useful for drug development and the prediction of side effects.
Researchers from the Massachusetts Institute of Technology have developed a computer-based approach to identifying protein structures found in Alzheimer's. They generated all likely structures of the protein tau, including a mutant form associated with an increased Alzheimer's risk. One chemical structure was more common in the mutant form and represented
a possible target for new drugs. Several more tau mutants could be analysed in the same way.
Use of human-derived raw materials
The most useful human materials for Alzheimer's research are donated brains, both of patients with memory impairment and normal controls. A network of brain banks exists to supply tissue to the neuroscience community. Brains for Dementia Research (funded jointly by the Alzheimer's Society and Alzheimer's Research UK) helps to collect brains from individuals that have been assessed regularly during life. Studies can then correlate cellular and chemical changes with pre-mortem clinical symptoms. Large scale genetic studies can be performed, and tests that would give greater accuracy in diagnosis can be investigated.
As Dr John Xuereb (director of the Cambridge Brain Bank from 1991-2002) acknowledges: 'Alzheimer's, Parkinson's and other neurodegenerative diseases occur in humans and it is in human tissue that we will find the answers to these diseases.'
Human tissue models
Scientists have recently created human nerve cells, including a variety that is lost early in the development
of Alzheimer's, from human embryonic stem cells. A further breakthrough came with the generation of fully-functioning nerve cells from the skin of a 30 year old woman. Cells such as these could be helpful in screening chemicals to identify those that might be helpful in Alzheimer's. A 2011 paper by one of the stem cell researchers commented: ‘Mechanistic studies of AD generally rely on autopsy samples, which are limited in supply and contain the disease aftermath, or on animal models, which do not fully recapitulate AD pathogenesis. Consequently, it has been very difficult to elucidate the initiating events of AD. Furthermore, recent clinical trials for AD have been largely disappointing. A proper understanding of the initiating events of AD and the existence of live disease models that accurately recapitulate the pathogenesis would lead to a much better informed therapeutic development effort.'
High resolution scanning
Magnetic resonance imaging (MRI) is a widely used research and diagnostic tool in neurological disorders. Functional MRI (fMRI) is a specialised scan that can measure changes in blood flow related to neural activity. It can be used to investigate alterations in brain function related to the earliest symptoms of Alzheimer's Disease, possibly before development of significant irreversible structural damage.
Scientists at the Maudsley Hospital have been first in the UK to use automated MRI software to detect early signs of Alzheimer's Disease. The package automatically compares a scan against 1200 others showing varying stages of the condition, and delivers an 85 per cent accurate diagnosis.
The Neuroimaging Research Group at Aston University, Birmingham, is exploring alternatives to animal research in the study of brain, behaviour, and drug properties. They use magnetoencephalography (MEG) imaging, which yields direct neurological measurements in human subjects. It gives 1000 times more precise time-related measurements than a fMRI scan.
Genome-wide association studies, in which genetic data from large populations is analysed and compared, can offer new insight into the underlying causes of Alzheimer's disease. Recently, in the largest study of its kind, researchers from a consortium of 44 US universities and research institutions identified four new genes linked to the condition. 'This is a major advance in the field thanks to many scientists across the country working together over several years, 'said Dr. David Bennett, director of the Rush Alzheimer's Disease Center. 'These findings add key information needed to understand the causes of Alzheimer's Disease and should help in discovering approaches to its treatment and prevention.'
There are many well-established human based non-animal research techniques that already play an important role in developing therapeutic interventions.
The results-based analysis we present demonstrates that animal research does not complement good science. The options for studying human disease in humans are growing all the time, and are supported by solid science and, increasingly, commercial and government funding.
Human-derived raw materials
Human-derived raw materials can be obtained and used in a range of ways. From donated human cadavers down to human DNA, all levels of tissue sample can be gainfully employed. Intact slices of human tissue, ethically obtained from patients who undergo operations or biopsies, can be maintained in the laboratory so that they retain their function. Tumour biopsies, for example, can be used to see whether a drug has bound to its intended molecular target. Comparing healthy and diseased donated organs can provide important information on disease processes. Stem cells of human origin also have enormous utility.
Human tissues or organ systems
Human tissues or organ systems can also be recreated in laboratories. A Cardiff University team led by cell biologist
Dr Kelly Bérubé has grown human lung cells in the laboratory to form three-dimensional tissue-like structures. These can be used to test substances for the potential to cause damage if inhaled.
Human lymph nodeshave been created in the laboratory, and can be employed to test vaccines and biologically-based drugs, like the TGN1412 monoclonal antibody, which - having been passed as safe on the basis of tests on monkeys - went on to cause catastrophic injuries to human trial subjects.
Human systems, from individual organs to the whole body, can be simulated using highly sophisticated computer programs. These are created using data obtained from people. Computer simulations have been developed, for example, to predict the behaviour of a drug in the digestive system. These simulations are likely to predict such effects in humans more accurately than animal models, and in a much more efficient way.
Microdosing involves giving a tiny amount of a substance - less than one hundredth of the quantity expected to have a noticeable effect - to a volunteer or patient. This dose is sometimes labelled with a safe amount of a radioactive chemical. Body fluids are then analysed to see how the body has responded, or PET imaging is employed to ascertain how the substance behaves in specific organs. This technique has already been used successfully to test drugs for cardiovascular disease, pain, Alzheimer's disease and gastrointestinal disorders.
Cell components, including DNA, RNA and protein molecules, can be arranged in a microarray, which is, typically, a tiny chip or slide made from silicon or glass, or it can be a membrane. The signals produced by microarrays are read by scanners and the data generated are analysed by computer. The technology can be used for drug development, both to identify potential drug targets and to test for efficacy and toxicity. Thousands of genes can be monitored simultaneously.
There is a wide range of scanning technologies that can reveal processes in living humans. The images produced are now truly remarkable and are especially useful in neurodegenerative conditions like Parkinson’s and Alzheimer's.
Microfluidic devices contain human tissue samples in tiny chambers linked by microchannels. Fluids and chemicals flow in a natural way between different compartments, simulating conditions in the human body. As with microarrays, microfluidics can produce large amouts of information very quickly. The technology can help scientists to understand how cancers spread, for example. Microfluidics can investigate human tissues and organ systems, with the creation of 'bioreactor' designed to supply nutrients and remove waste products. One team of researchers has developed a system in which huma liver, brain cortex and bone marrow are interconnected through a circulatory system mimicking blood flow. These models can be used to predict the effects of substances as they move between these organs.
Epidemiology involves the study of significant numbers of people over a period of years, comparing their lifestyles, genes, medical interventions, enviroments, social status, etc. It remains a powerful tool with huge potential, and has already produced enormously valuable findings, including the link between smoking and lung cancer.
Clinical data and observation are grealy under-used, inluding the information gathered from mimally or non-invasive procedures (such as blood or urine sampling). Data from this type of benign intervention, undertaken in consenting patients already unergoing procedures, could be collated much more efficiently than is currently the case.