Tissue-Engineered Lungs for in Vivo Implantation
Thomas H. Petersen, Elizabeth A. Calle, Liping Zhao, Eun Jung Lee, Liqiong Gui, MichaSam B. Raredon, Kseniya Gavrilov, Tai Yi, Zhen W. Zhuang, Christopher Breuer, Erica Herzog, Laura E. Niklason1,
Introduction:
Human lungs usually do not repair beyond the cellular level, and currently the only method of replacing damaged lung tissue is through lung tissue transplantation, an expensive procedure that achieves less than 20% survival rate after ten years. Techniques to decellularize organs and use them as an acellular matrix scaffold to generate lung tissue have recently been tried with promising results. However, regenerating viable lung tissue is a tall order as the tissue should: contain lung specific cells, have branching structure of airways, separate blood and air, and allow for respiration. In an attempt to construct functional lung tissue using a rat model, lung tissue was decellularized and the resulting scaffold was repopulated with neonatal lung epithelial cells. The populated lung scaffold was cultured in a bioreactor simulating fetal lung environment. Lastly, the lung was tested for functionality.
Materials and Methods:
Preparation of a decellularized lung scaffold
Lung tissue from adult Fischer 344 rats was harvested and treated with detergent solution for roughly 2-3 hours. The resulting lung matrices, as verified by micro-CT scans indicate an intact microcellular matrix. The protocol was effective in preserving the micro structural properties of the native lung, yet removal of any antigenic components that may interfere with culturing of tissues.
Properties of the lung bioreactor
A bioreactor was used to culture the cells on the acellular scaffold. Cells were injected into the vasculature and airway compartments, to which they adhered. Subsequent tests indicated healthy proliferation and low incidences of apoptosis. Culture medium was fed though the pulmonary artery at physiological pressures and a negative pressure breathing loop with air was implemented to simulate respiration. The breathing loop was essential for ensuring cells did not block the airways and cleared the airways of any obstructions. In addition, exposure to air increased the numbers of epithelial cells, which was one of the essential criteria for a functional lung tissue graft. Compliance tests yielded high similarities between native tissue and engineered tissue, and an immunohistochemical staining indicated the seeded endothelial cells were extensively distributed with tight junctions between them. It was noted that extended culture periods resulted in a cellular distribution more similar to that of native lung tissues. In order to check if such scaffolding techniques are also compatible with human tissue, human lung tissue underwent a similar protocol of decellularization to obtain a matrix that was repopulated with lung cells. The cells adhered to the vasculature suggesting that such techniques may be compatible with human lung tissue, but an extensive study was not performed.
Implantation of engineered lungs into rats
The cultured engineered lungs were implanted and replaced the left lungs of four animals. Within minutes blood flow was established in the engineered lung vasculature. X-rays showed that inflation in the engineered lung was less than that of the right native lung. Additionally minimal bleeding was observed.
Results:
Data collected primarily consisted of testing of the engineered lung properties before testing for ability for gas exchange. Compliance testing for the elasticity of the engineered lung yielded similar stress-strain values between the native lung and the engineered lung. However, it was noted that the engineered lung was noticeably less elastic than the native lung; however, the difference was considered minor and did not alter the performance of the engineered lung.
Implantation of the engineered lung – Blood gas analysis of the blood taken from the engineered lung indicated that oxygen and carbon dioxide gas exchange did occur. Partial pressure of oxygen increased from 27 +/- 7mmHg in the pulmonary artery to 283 +/- 48 mmHg in the left pulmonary vein indicating hemoglobin oxygen saturation. However, this partial pressure is quite different from the native lung’s (634 +/- 69 mmHg. Additionally, levels of carbon dioxide exchange fell from 41 +/- 13mmHg in the pulmonary artery to 11 +/- 5 mmHg in pulmonary vein indicating efficient carbon dioxide removal.
Discussion
Overall, performance of the engineered lung was quite promising, although the tissue could not perfectly emulate that of native tissue. The engineered tissue overall was not as pliable and did leak some blood. However, performance was sufficient for the oxygenation of blood. Despite this, the success of the engineered tissue demonstrates a strong feasibility in the use of micro architecture of native lung tissue for regeneration of functional lung tissue. Still, many issues must be overcome for long term engineered function to persist. Ideally, the air-blood barrier must be improved to prevent further bleeding and production of certain proteins and surfactant needs to be increased. Lastly, other technologies for obtaining compatible epithelium such as lung stem cells is still required to make this a feasible clinical procedure.
Critique
The focus of this paper was to test the efficacy of the use of their engineered lung tissue as a functional tissue transplant. Considering that such functional lung tissue needs to emulate so many properties of native lung tissue in order to even function, and then for these researchers to be able to create such a working model is quite impressive. Based on the performance of the tissue, the researchers laid out clear goals to tackle in the future for improving the efficiency of the tissue. Aside from the mentioned technical improvements for tissue function, potential future projects include culturing of human lung tissues and improving incubation techniques.
However, there are several minor points in the paper that could be improved. Based on the protocol, a total of four engineered lungs were implanted into four rats. Because the researchers were simply testing the efficacy of the transplant, this number is adequate, although small. Future, improved engineered lungs could be tested with more rat models. Testing with human lung tissue was briefly mentioned but whether the cells could actually be cultured was not pursued. Given that this was not the purpose of the experiment, more tests need to be done before assessing whether human lung tissue could truly be created via similar techniques. Additionally, the researchers mentioned that the engineered lungs were functional for short periods of times, up to two hours, but little additional information was given on why this time frame was chosen. Whether the rats died or were simply euthanized after a period of time or that the tissues quickly lost efficiency is unknown. Lastly, much more qualitative data may be provided in terms of lung tissue properties. A small plot for the stress-strain characteristics of the tissues is provided albeit lacking details. However, the mention of reduced surfactant production and protein expression such as aquaporin-5 could be paired with relevant data.
12 comments:
While the author's results do seem to be a good step forward for tissue-engineered lungs, I agree that a much more thorough evaluation needs to be done before considering this in humans. The authors need to examine a longer transplantation in mice to ensure the safety and consistency of the lung. Also, authors should address the fact that engineered lung was less elastic and contained much less pressure than a native lung. Overall, I found the paper to be interesting, especially the design of the bioreactor which they used to culture the lung.
I also agree that a longer transplant time would be more convincing that this is a viable option for lung replacement therapy! The fact that they were only in vivo for 2 hours sounds awfully suspicious to me!!
They should have explained in the discussion why the engineered lungs were not as elastic as the native lungs. Could it be the cause of less oxygenation? Similar to emphysema. Were the mice treated with the immunosuppressant after the transplantation?
Also, as Hayley and jkao asked, why after 2 hours?
I think its suggested that the lungs failed, which is why the implantation time was so short. There was a part in the closing discussion that implies this. Either way they really should have documented a longer time span for implantation--it'd be nice to know for sure what exactly happened. Given the lungs reduced elasticity and it's small size, I wouldnt be surprised if the lung collapsed shortly after implantation.
I agree that the transplants should have been evaluated for a longer period of time. I was also wondering about the economic feasibility of the tissue-engineered lung. If this were put into clinical practice, would there have to be a separate bioreactor for each patient? The process seems very costly.
As with most studies, further evaluation of lung function in the long-term would yield more convincing results. The stark differences between native lung partial pressure compared to the engineered lung pp's are worrisome. Do the large differences indicate the lung's inability to function after days? weeks? Also, it would be cool and informative if the researchers were able to measure ventilation rates and oxygen deliverance capacities of the subject mice by running them on a treadmill!
I am particularly interested in this research because of the potential impact it may have on lung cancer patients. Lung related diseases, as you mentioned, often have low survival rates because of the specificity of the requirements of the lung tissue substitution. Because the research was performed on small scale (with rats) only, I am curious whether they expect any drastic differences with sizing up to human scale. For instance, are there dimensions that increase exponentially such as surface area to volume ratios that may be important? Overall I am impressed by the research and hope it makes a big positive impact for lung cancer patients.
Though I am excited by the promise of tissue engineered lungs, it seems very suspicious. I would think that if there was bleeding into the implanted lung, that there was some kind of failure. The lung is such a complex and porous organ, and though it is impressive that the authors' got the lung replacement to work in vivo for 2 hours, the complexity of the lung and the sheer size of the human lung seems extremely hard to replicate.
While this implant that they proposed is quite interesting, I do think that their results have not shown enough evidence to really say that their device is very feasible. In addition to what others have said about increasing the in vivo transplantation time, I think they should run other tests to check how well integrated are the implants within the body, are there any signs of inflammation, vascularization, etc.? It's not very convincing when they only showed the compliance test and blood gas analysis.
This is an interesting article! However, as you people have mentioned above, there are some shortcomings but I think that can be improved through future research. Firstly, the researchers need to show that the implementation can work in long term because immune response will be a big issue. Once they overcome this issue, they can move on to improve other functions of natural lungs, such as self-cleaning, cough, etc. Then they need to think about how to enlarge the engineered lung to desired scale (mostly human, but that can be improved to other mammals too)
I would like to see some more info on the biocompatibility of the engineered lung, as many of you have said. This would again require longer in vivo time scales. It would also be interesting if the authors gave some sort of cost analysis, since it seems to be quite expensive at this stage. Would they expect the cost to decrease dramatically in the next few years? Decade?
It is an interesting paper and I like the idea to culture the cells using bioreactor. However according to the paper the mechanical property of the engineered lung, such as elasticity and pressure is still much lower than human lungs. Besides, the fact that there was bleeding occured indicated the endothelial layer was not well established in the engineered lung. Immune response is another issue that this design must encounted. Maybe using other scaffold, instead of decellulerized lung scaffold can be used to avoid such effects.
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