Toward clinical translation of whole organ bioengineering

by Alexey Bersenev on April 16, 2016 · 0 comments

in tissue engineering

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The graph below illustrates the problem of donor organ shortage for transplantation in USA:

In the last two decades, scientists offered a few potential solutions to this problem: (1) whole organ bioengineering (WOBE) via decellularization – recellularization (decell-recell), (2) xenotransplantation and (3) generation of exogenic human organs in animal chimeras. Since introduction of CRISPR-based genome editing, interest to xenotransplantaion is getting hotter right now. Experiments with human-animal chimeras for generation of human organs got into ethical controversy and were depleted of governmental funding support. I think, as of today, the WOBE via recell-decell technology is the most realistic and quick way for moving into the clinic. In this post I’ll focus on some challenges in clinical translation of WOBE products for solid organs replacement. I gathered information mostly from talks and publications from Harald Ott, Doris Taylor, Angela Panoskaltsis-Mortari and Steven Badylak labs.

1. The best source for organs to decell
Whole donor organ is a source of extracellular matrix (ECM) for recell. Human deceased donors seem like the most appropriate source of organs for clinical use of WOBE products. However, there are three major problems with organs from cadavers: (1) availability, (2) quality/ suitability of ECM and (3) variability from product-to-product. Human organs will be used for WOBE product only if they are not suitable for conventional organ transplant and discarded. Due to scarcity of donors, organs as ECM source for WOBE will be rarely available. If you finally got an organ for decell procedure, it will be “low quality”. ECM from diseased/ old donors is not good and could negatively affect decell-recell procedure. Harald Ott wrote in his review:

… mechanical properties of ECM scaffolds derived from fibrotic lungs were different from healthy donor tissue, and elicited a profibrotic response in fibroblasts [6]. In discarded donor hearts, we found that atherosclerotic and structural heart disease lead to permanent alterations of the ECM scaffold, and therefore persist through the process of decellularization.

Potential solution: Organs from humanized pigs! You can set a manufacturing plant, where production of humanized pig organ ECMs will be standardized and scaled up. Progress in humanization  via gene-modification techniques (for example, described here), makes this alternative very attractive.

2. Generation of consistent product
Due to human donor-to-donor variability (different ECM quality), it is almost impossible to manufacture consistent WOBE product. This is a big obstacle in transition to clinical trials and commercialization. Product must be consistent!
Potential solutions:
(a) Automation of decell-recell process. Automation will be required in clinical WOBE product manufacturing. One of recently described automated devices allowed to cut lung decell process from 1 week to 1 day, making ECMs more consistent. Essentially device made of tubing sets, connecting perfusion buffers with organ vasculature through valves system. The software for this device is available as open code for academics. Automation is the only way to scale up the manufacturing and bring cost of the WOBE product down.
(b) Using clean pig facilities for the first step of processing (organ harvest and decell) will enable scaled manufacturing of high quality whole organ ECMs.

3. Availability of tools and clinical-grade bioreactors
You may see variety of bioreactors for organ bioengineering (see here, here and here) from research labs. All of them are custom made, sometimes in-house. As of now, tools manufacturers are not in hurry to make clinical-grade systems for donor organ perfusion and bioreactors for WOBE products culture. Perhaps, they don’t see market opportunity here yet and refrain to invest.
Potential solutions: Implement clinical systems for organ perfusion, such as TransMedics and OrganOx. Manufacturers of organ bioreactors for research should make clinical closed systems available. Perfusion solutions manufacturers have to make them clinical-grade.

4. Manufacturing release criteria of decell organ scaffold
In the future, perfusion-decell and recell steps of manufacturing could be done in different facilities. In case of centralized mass manufacturing of decell scaffolds, strict release criteria and certificate of analysis should be developed. The single most important specific criteria is efficiency of decell, measured by residual DNA/ cell content. An example of such criteria you can find in Badylak’s review:

Three relatively stringent criteria have been proposed to establish sufficient decellularization: specifically, the remaining ECM scaffold must have 1) less than 50 ng of dsDNA per mg of dry weight, 2) DNA fragments less than 200 bp in length, and 3) no visible nuclear material in histologic analysis with DAPI or H&E

If you’re using humanized pig organs, you have to test residual pig cells/ DNA, which can cause strong immune response. Unfortunately, there are not many antibodies and reagents available to ID pig cells. The next important test to include is assessment of integrity/ mechanical properties of scaffold (for example, collagen, elastin, GAG content – reviewed here). Of course, decell scaffold must be sterile. Pig-derived scaffold also must be tested for endogenous porcine viruses (for example, PERV detection test). Detection systems for porcine viruses must be commercially available and approved for biologic product release testing.

5. Thrombogenicity of decellularized ECM
The biggest short-term problem after WOBE product implantation is thrombosis. Decade of research have demonstrated that denuded vasculature of organ scaffold is highly highly thromobogenic.
Potential solution: No-brainer here – if you want your engineered organ work properly in vivo, you must re-endothelialize it! Anti-coagulants are useless in this situation. Endothelium seeding must be stable and cover whole vascular system.

6. The best strategy to recell
Recellularization itself is bringing a number of challenges. What is the best cell source for recell? What is the best strategy to do it? How to ensure cell retention? How many cells is enough? There are no easy universal solutions here, but there is a lot of research studies, specific to each organ.
(a) Cell types. Due to low proliferative and differentiation capacity, primary cells is not good source for recell. Progenitor and stem cells, including iPS are currently studied as source for recell. However, the use of autologous iPS-based recell, is not as easy as many people claim.
(b) Strategy. Re-endothelialization could be the most clinically important part of recell. Some researchers recommend re-endothelialization as the first step. The second step is to make WOBE product stroma. Mesenchymal stromal cells is the most widely used source for stroma re-creation in decell scaffold. Finally, the last step is using progenitor/ stem cells for expansion and differentiation to highly specialized organ-specific cell types. For example, auto- iPS cell-derived endoderm progenitors for the lung. Ideally, expansion and differentiation of stem/ progenitor cells is completed in situ.
(c) Cell retention. Good cell retention within WOBE product is a big problem. An example from Ott’s review:

Vascular perfusion as a different seeding approach was used when decellularized mice hearts were repopulated with human iPSC-derived cardiac progenitor cells…
This utilized the ability of progenitor cells to spontaneously migrate from the vascular bed to the ECM. After terminal cell differentiation in situ, cardiomyocytes were found in the ventricular wall and ventricular septum. However, 85–90% of cells were washed out from the vascular conduit within the first 7 days.

In his recent study, only about 5% of recellularized lung was covered by iPS cell-derived endothelium after few days in culture.
(d) Cell number. One may think that in recell the more cells the better. Yes, solid organs made of many many billions of cells. For example, human heart will require ~4 billions cardiomyocytes for complete recell (as of today, max # of recell cardiomyocytes = 0.5 billions for human heart was achieved by Ott’s group). However, after full re-endothelialization, significant part of organ maturation and remodeling could be completed in vivo.  Few researchers now bet on the concept of recell organ as “inductive template”, which will be remodeled by endogenous cells and fully matured in vivo.
(e) Seeding. There are many cell seeding techniques described, but seem like combination of vascular perfusion of cell suspension with direct intra-parenchymal injections will work the best.

7. Immunogenicity
The host immune response to decellularized and recell ECM remains understudied. Implantation of commercially available decell ECM causes profound immune response with shift from pro-inflammatory M1- to tissue remodeling M2 macrophages with degree of decell. It is important to recognize the difference between host immune rejection response and “tissue remodeling” response:

With removal of cells, native ECM scaffolds are depleted from major histocompatibility complex class I and II antigens and therefore would not elicit cell-mediated graft rejection. However, retained cell debris can result in a host-induced inflammatory cascade.
… an M2 macrophage-mediated immune response toward acellular matrix may have positive effects on in-vivo ECM remodeling.

In the recent study, Ott’s group compared macrophage infiltration of human cadaveric heart tissue, human decell heart ECM and porcine decell heart ECM in rat host. Overall, infiltration of CD163+ macrophage was profound without significant difference between 3 types of scaffolds. The humoral immune response, which could be disastrous for implanted WOBE product, will totally depend on efficiency of decell (quality of ECM) and nature of recell components (allo- cells, culture in xeno+ conditions). Immunnosupression may be required, analogously to organ transplant.

8. Regulation
Decell ECMs are currently approved on a market as devices and used for regeneration of soft tissues. However, there is no regulatory framework for WOBE product. Theoretically, it could be regulated as combination product by FDA CDRH (device) and CBER (cells/ tissues) centers. Seem like the most practical approach is to split manufacturing and regulation for 2 parts: manufacturer #1 makes decell organ ECM (device) and sells it to manufacturer #2, who does recell part (tissue/ cell drug) and send it to transplant center.

Overall, still a lot of work should be done before the first clinical trials of WOBE products. My best estimation will be 2-5 years from now. Obviously, decell-recell tissue patches, sheets, hollow organs and parts of solid organs (liver or lung lobe) will be ahead of whole solid organs, but we will get there!

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