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Researchers Generate Decellularized Livers, Ready for New Cells and Transplantation

Decellularization is the most promising near term approach to generating patient-matched organs for transplantation. It is a fairly simple concept at root: researchers remove all of the cells from an organ, leaving the scaffold of the extracellular matrix with all of its intricate details and chemical cues. The challenge lies in building a reliable methodology that can be scaled up for widespread use. Much of the work on decellularization to date has focused on hearts and lungs, but in the paper noted here, researchers outline a method for reliably decellularizing whole livers.

Decellularization does of course require a donor organ as a starting point, unfortunately, but that can include a significant fraction of the potential donor organs that would normally be rejected by the medical community for one reason or another, as well as organs from other species, such as pigs. Given suitably genetically engineered pigs, a decellularized pig organ repopulated with human cells should contain no proteins that will provoke significantly harmful responses following transplantation. This and other options should roll out into availability in the years ahead, ahead of the range of more ambitious tissue engineering projects that aim to grow entire organs from a patient cell sample.

Decellularization is ahead of other methodologies for the creation for patient-matched organs because the research community has yet to produce a good method of generating the intricate networks of tiny blood vessels that are needed to support tissue much larger than a millimeter or two in depth - the distance that nutrients can perfuse in the absence of capillaries. Yet over the past few years many research groups have demonstrated the production of organoids, tiny sections of complex, functional organ tissue, for a variety of organs. Thus the actual production of organs from patient cells will be a going concern just as soon as the blood vessel question is figured out. Unfortunately, this has been the state of the field for years now, with many promising leads but no definitive end in sight. Meaningful progress in bringing decellularization to the medical community is to be welcomed in the meanwhile.

Decellularization of Whole Human Liver Grafts Using Controlled Perfusion for Transplantable Organ Bioscaffolds

The only therapy for liver cirrhosis is liver transplantation, but the shortage of organ donors imposes a severe limit to the number of patients who benefit from this therapy. With increasing shortage of donor organs and decrease of their quality, the development of novel procedures and alternatives for organ transplantation becomes essential. Thus, organ engineering, which involves the repopulation of acellular matrices, was explored with the use of polymeric scaffolds or three-dimensional (3D) printing of liver tissue to make scaffolds that can be seeded with hepatocytes or other cell types.

Although these are powerful tools worth exploring, it remains difficult to design and create artificial, yet functional liver tissue with functional vascular and biliary trees for clinical use. Alternatively, removal of cells from an existing organ, leaving a complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM), may provide a natural habitat for reseeding with an appropriate population of cells, and connected to the blood stream and biliary system.

Ideally, ECM is cell free, but remains the interlocking mesh of fibrous proteins (collagen, elastin, fibronectin, and laminin) and glycosaminoglycans (GAGs). Evidence from rodent models shows the feasibility of decellularization of whole liver organs that provides an excellent scaffold for reseeding liver (stem) cells for graft engineering. Also, porcine and sheep liver have been successfully decellularized to obtain ECM for transplantation. However, so far, there is very limited experience with decellularization of whole livers from humans.

Recently, researchers demonstrated efficient decellularization of a whole liver and partial livers to generate small cubes of human liver scaffold. Different decellularization methods have been described among which are physical force (freeze/thaw, sonication, and mechanical agitation), enzymatic agents (trypsin, endonucleases, and exonucleases), and/or chemical agents (ionic, nonionic, and zwitterionic detergents). Usually, combinations of these methods are used. In larger organs, such as human or porcine liver, perfusion through the intrinsic vascular beds is the favorable route to be able to reach all cells. So far, most experimental decellularization protocols include the use of sodium dodecyl sulfate (SDS) to generate full freedom of cells and translucency, but this also progressively destroys the ECM and hampers clinical translation.

In this study, we report successful decellularization of human livers to obtain transplantable whole organ scaffolds. We show proof of concept that these scaffolds can serve as feasible resources for future tissue-engineering purposes. Using a controlled perfusion system, a complete 3D acellular human liver scaffold was generated on a clinically relevant scale and free of allo-antigens. We present the feasibility of systematically upscaling the decellularization process to discarded human livers. Eleven human livers were efficiently decellularized by nonionic detergents by machine perfusion. A careful choice of the decellularization methodology is of great importance as methods described for decellularization may be well suitable for other organs than the liver, but may damage the composition of the matrix proteins.

Repopulation of a complex organ such as the liver poses numerous challenges. Using the extracellular matrix of the native liver obviously helps to create the most optimal niche for cells to repopulate, but the types of cells to be infused to create fully functional liver tissue remains to be elucidated. In addition to the liver-specific matrix proteins, the still present vascular and biliary system may also provide entry routes for the different cell types needed. Obviously, efficient recellularization is a complex process in which hepatocytes or other parenchymal cells need to pass the remnant basement membrane of the decellularized blood vessels or bile ducts to enter the parenchyma after vascular or biliary administration, respectively. In addition, cell numbers that are required for efficient recellularization are highly dependent on cell type and volume of the scaffold.

Reendothelialization is a pivotal step to prevent thrombosis as a result of the massive collagen contact surface that blood will encounter upon reperfusion, and which cannot be prevented by coating with heparin. We demonstrated, like others did in animal models, that matrix sections can be reseeded with endothelial cells and these cells end up at the location of the decellularized blood vessels and pave the basal membrane. In our studies, HUVEC were used as a source of endothelial cells, as in most studies in rodents and pigs, but other sources such as endothelial progenitor cells are also used and show similar results. The next hurdle to be taken toward clinical application is to choose a cell source for liver parenchyma repopulation. An adult liver contains ∼150-350 billion cells of which the largest part (70%-85%) is made up by hepatocytes. However, adult primary hepatocytes of high quality are scarce and therefore limit tissue-engineering applications. Ideally, autologous cells, isolated from the patients themselves, are used as these cells will have a low risk to trigger an immune response. Alternatively, (autologous) pluripotent stem cells that self-renew and are able to differentiate into all cell types needed could be seeded.

In summary, human cadaveric livers can be successfully decellularized using machine perfusion and nonionic detergents, and can be repopulated with endothelial cells. The next steps toward clinical application involve finding a cell source or combinations of cell types to reseed the matrix, including the vascular and biliary system, to gain functional liver tissue.

Comments

As a layperson I can't see why they don't use CRISPR-cas9 to germline engineer a gene for half a prodrug that causes cell apotosis on administration of a small molecule drug, then use that to decellularize a pig liver. There is probably more to it than that though.

Posted by: Jim at October 13th, 2017 11:00 PM

The Biotink team who won the 2016 iGem (international genetically engineered machine) contest have an interesting approach to bioprinting - genetically engineering the cells to be printed with molecules that cause the cells to stick together.

http://2016.igem.org/Team:LMU-TUM_Munich

"We are living in an aging society that is facing a decreasing supply of donor organs for medical transplantation. To confront this pressing issue, we developed a game-changing approach to bioprint tissues for biomedical applications. Our interdisciplinary work aims to create a unique ink, named bio(t)INK, to revolutionize bioprinting. The printing process uses a hijacked 3D printer and two components of biotINK to induce an instantaneous polymerization reaction, creating three-dimensional multi-cellular structures in a user-definable manner. The principle of this two-component glue relies on the rapid and specific interaction of biotin and its tetrameric binding protein avidin. To make use of this high biotin-avidin affinity for cell-cell cross-linking, we engineered cells presenting biotin moieties or biotin-binding proteins on their surfaces as well as recombinant matrix proteins, which co-polymerize upon printing. Furthermore, we explored genetic circuits which allow us to functionalize the bio-synthetic tissue and install biosafety mechanisms. Altogether, we are confident that our system provides the necessary means to advance the SynBio community to the next level - the tissue level."

https://www.youtube.com/watch?v=MscvWuAuAK0

Posted by: Jim at October 13th, 2017 11:13 PM

@Reason,

I have a question, Reason. When your readers write in and have suggestions to help solve problems, have you or Steve Hill (and others) been able to use these suggestion to further advance the field in which the reader commented on? Has there ever been at least one idea borne by a reader that was of a significance to a researcher?

Just curious as I have been reading your blog for well over a decade and I really appreciate you advancing this cause and your readers for putting in their thoughts as well.

Robert

Posted by: Robert at October 14th, 2017 1:10 PM

@Robert: I think it more a matter that connections are made, and the blog may or may not be a direct vehicle in that process. Usually not, as there are more efficient channels of communication.

You might look at https://www.fightaging.org/archives/2016/01/the-present-state-of-progress-towards-clearing-glucosepane-cross-links-a-contributing-cause-of-degenerative-aging/ to see an example of a previously unknown researcher coming out of the woodwork and making a connection to our community.

There have been a number of other cases over the years where researchers or angel investors made contact through other channels and then went on to become more involved, but that is rarely manifested on the site itself.

Posted by: Reason at October 14th, 2017 1:15 PM

I have noticed in my lifetime of 2 males whose skin looked 25 at around 50 years of age. These 2 males had normal rural agricultural backgrounds/lifestyles, but must have had a genetic reason why they were so resistant to skin aging. My mother told me of another 80 year old male whose skin looked about 50. Why don't geneticists look into these rare cases to try to identify why their skin does not age normally. Geneticists look into Centenarians to see what genetic variants allows them to live so long, but of course few if any of them have markedly reduced skin aging, so one must set up a research model to study only those who show little or no skin aging. As far as I know, those rare cases who show little skin aging die of other causes at normal death ages

Posted by: Biotechy at October 15th, 2017 7:36 AM

@Biotechy

Hi Biotechy ! Just a 2 cent,

I think it's a myriad of things. But, one, if not the biggest, is oxidative stress. Aging is characterized by oxidative stress increase. And oxidative stress is at the heart of everything that means 'intrinsic aging'. People who age fast (HGPS/Werner progeria syndromes, diabetes, cancer, atherosclerosis, alzheimer's, CVDs, hypertension, hepatitis, and more), often display oxidative stress (this is manifested as loss of Redox and loss of 5-methylcytosine (irreversible oxicatiion of important DNA residues and amined acids. Irreversible because it really is; it is permanent, this in turn changes the 'biological' signature/phenotype of the person - they are effectively, and permanently, 'older' biologically (you see that in DNA methylome/epigenetic signature/epigenetic clock can cannot 'rebound' anymore (the epigenome is like an elastic, it can 'rebound' so long as you don'T stretch to break point (irreversible chemical reactions and transcription loss/epigenetic drifting. As such, officially 'older' (in bioclock)))). Down the line, this translates as 'accelerated' replicative senescence and shorter time to 'DDR spontaenous or oncogenic' senescences. They don't ever reach Hayflick limit. SuperCentenarians die because Hayflick has been reached/most cells are in the low 2-3Kb region telomeres (strong signal for replcative senescence) and they die of other complications (like transthyretin). I say they could probably last a 20 years extra; and 150 it is pretty much the total barrier (Hayflick full on).

People that like 25 years old in the skin and are 50 have kept a high redox and very low oxidative stress. And most centenarians, if you look at pictures of them younger, were 'baby face' looking (meaning they had high collagen levels/plump skin like a young person; which means little oxidative stress. If you have a strong 'young' ECM with no AGEs in it - no matter what you chronological age is, it helps a lot for the secreted enzymes help tp fight off oxidative stress and means you are keeping a homeostatic skin redox). One study used human skin fibroblast and demonstrated taht when it was exposed to UVs and others ROS oxidative stress inducers; the fibroblast senesced much quicker and did not even reach their Hayflick limit. These fibroblasts showed elevated mitochondrial ROS emissions - unscavenged (by redox), a faster accrual of lipofuscin (aging pigment manifestation in lysosome and extra-cellularly),
accelerated epigenetic DNA methyl loss, accelerated 8-oxodG, y-H2AX insults/oxidative lesions, accelerated telomere loss (up to 10 times faster loss of bp/per pd; just like in Progeria HGPS) , chromosome loosening/dyfunction, sluggish division/cell replication, and many more 'hallmarks'. So basically what this means, is like these people who are 'Ageless' ageless baby skin looking; they keep healthy and keep their antioxidative stress powers that be in check; that is genetics of course (such as stronger activating NRF2/ARE/EpRE in these people and FOXO/DAF genes related to mTOR/IGF).

The point is if currently you look yougner than your age, it is a good sign (it is not an assurance) that you may possibly have a chance of reaching centenarian age; for skin collagen levels (measure in mm thickness and also can measured by subsurface scattering or gradient calcul from a photo of their skin) describe 'overall' what is going in most organs. It is a good surrogate of 'overall' oxidative stress (but studies that you use skin, must use the parts that are not 'exposed' (such as arm pit or below groin skin) because this skin is not exposed to daily UVs and tells a different 'misleading' story than other skin parts such face (crows feet/wrinkles/sagging/collagen loss/AGE crosslinks), spots, where there there was obvious acceleration of aging by being exposed to daily sun UVs radiation; but it does not represent the overal aging in the body; not like 'unexposed/protected skin patch' does - where it is much better predictor of the actual epigenetic age of a human since it is a correct representation of the body's biological age (not a 'baked/UV maillard filled' tanned-skin patch). I think geneticists have to concentrate on longer than supercentenarians, these humans are going to be pitted against longer-lived animals; it is a necessite to understand why other animals live 5 times longer than a Supercentenarian. With SENS and/or stem-cell replacement/nice stem cell replacement I hope we can twarth the replicative senescence problem; but I don'T think it is very feasible - only with a ruse/circumventing the replicative senescence. I.E. keeping the telomeres long, keeping the redox, keeping that biologically 20-year old body you had in 'repeat' fashion; it is going to be hard because aging continues (Even in the oldest people, death is coming since they hav accumulated irreversible DNA residue insults, the Hayflick is soon and their lysosomes are filled with rising lipofuscin).

Just a 2 cent.

Posted by: CANanonymity at October 15th, 2017 9:39 PM

PS: one more thing is being born with an extra 1kb chunk of telomeric DNA. Certain children are born with longer telomeres - than others (thus are 'biologically 'younger' right from the start thus have an 'extra' years for later on), this is due of inherited genetics and 'in-family' longevity (a family history of Centenarians); oftenly they have children late and this late effect is translated as offspring that live long too (because they obtain a higher telomere size from birth, thus have 'more to work with' later on down the line since their telomeresize is higher thus less replicative senescence (more telomeric DNA size when they are old decades later)). This can been seen in late-father conception, elderly fathers give longer telomeres to their children despite the DNA defects in their sperm (sperm telomere size rises with age by testicular telomerase); this extra telomere length in spermatozoid is transfered to child when it enters ovule egg; whom thus benefits from a longer lifespan and can become a centenarian too. So, yes again, with the inherited genetics being a large part of why certain people are gifted with these longevities (and haelthy 'young looking' skin). The fact that elderly fathers give this and that sperm telomeres rises with age, is a testament that the survival of the father - living to an old age is what matters. I.e., the fact the father lives long is a 'survival benefit' for evolution/specie - thus benefits the child too (it is also the same wit the 'grand-mother' theory where grand-mothers helped to increase 'parenting/grand-parenting' and increased longevity of humans by transfering longevity genes to their children/little children down the line (since they Did Living Long, they reached old age as elderly Grand Mothers; thus Survived/Compensation (evolution compensates you for living long when you reproduce very little as humans do (unlike mice where it is short-life/hyper-reproduction to counter short-life high mortality for specie survival/continuity).

Posted by: CANanonymity at October 15th, 2017 10:48 PM

Thank you Just a 2 cent & CANanonymity! You gerontologists have Encyclopedic knowledge of Aging Science! It will take me a while to digest all of the above information.

Posted by: Biotechy at October 16th, 2017 9:03 AM

Thanks Reason for your perception on how your blog connects people. This blog gives me hope that we can eradicate aging within a few decades.

I have always dreaded getting old and now that I am over 50, it is hitting close to home. Given that my mom died a month ago does not help either.

As I live in the bay area, I would love to visit SENS in Mountain View and meet some of the people who work there.

Posted by: Robert at October 16th, 2017 12:45 PM

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