A perfusion bioreactor to maintain bone cell viability ex vivo
Date
2016
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
The existing model for drug development involves pre-clinical studies that can take years to complete and cost millions of dollars for a single compound. Adding to the drawbacks of this model, are the numbers of animal lives lost in the process. Animal models often fail to predict human responses to a drug because they do not precisely replicate human anatomy and physiology. Therefore, there is a requirement for an alternative to test drugs in a laboratory setup. The human skeletal system may hold the key to this dilemma. Bones are not only a great source of stem cells and bone cells, but also contain bone marrow that is extremely sensitive to compound toxicity. Bone is rigid organ and doesn’t require any external support to prevent it from collapsing, as is the case with other organs. Finally, testing drugs and observing its effect directly on a human organ ex vivo will provide better answers to questions regarding the efficacy and outcome of future clinical trials. Taking the advantages of using bone as an organ to test drugs, a perfusion bioreactor was designed that would maintain bone cell viability ex vivo. The bioreactor design was based on the concept of a perfusion bioreactor that directly flows media through the vasculature of the bone thereby providing it oxygen, nutrients and ideal conditions required for keeping the bone alive. A bioreactor is an engineered system that maintains a biological environment to keep a specimen alive. Factors that can be controlled using bioreactors are fluid shear stress, loading, and flow of media, sterility, flow of gases into the system, nutrients, pH and temperature. Using the model, the bioreactor system was designed to maintain optimal perfusion pressure, maintain viability of cells in the bone and perfuse the femoral head continuously with media. The bioreactor was subsequently tested for its efficacy. The perfusion pressure was maintained at the physiological pressure of blood in the femoral head, which ranges from 0.1 to 1.1psi. This pressure range was achieved by using mannitol. Mannitol is a sugar alcohol used to reduce intracranial pressure. Testing different concentrations of Mannitol showed that using Mannitol at a 10% (w/v) concentration in DMEM would allow the system to maintain physiological pressure. Perfusion was proved first in bovine femoral heads and subsequently in human femoral heads. The successful perfusion of femoral head was proved in both models for a time period of 12 hours. In testing the human femoral head, we also demonstrated the presence of live cells 12 hours after perfusion of the femoral head with DMEM containing Mannitol. Cell viability was tested by adding the dye: Calcein Red-Orange AM. In the human femoral perfusion model, the effect of perfusion versus diffusion was compared. Results showed that perfused bone had more stain than a diffused sample, thereby demonstrating media was perfused through the entire bone. Testing perfusion also verified the viability of cells in the human femoral head after a 12-hour bioreactor run. Parameters such a pressure, pH and glucose were tested at intervals to ensure there was no contamination and the system worked seamlessly. Pressure consistently remained between 0.1 and 1.1 psi over the 12 hour run of the bioreactor and pH remained at 7.45, indicative of physiological pH of blood. Glucose tests were inconclusive as no significant change in glucose levels was observed. In establishing this model further, this model could be used to test drugs, reduce the longevity of drug discovery by observing drug effects on human organs prior to clinical testing, as well as in observing progression of bone related diseases and its pathology.