3D bioprinted liver for predictive disease modelling and drug testing.

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“”Project Report”” — 2015/4/4 — 12:27 — page 1 — #1 ii

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Liver disease predictive modelling and drugdevelopment using 3D bio-printingBrijesh Chandrakar ∗, Diana Kshetrimayum ∗ , Eeshit Dhaval Vaishnav ∗ and Ajay Kumar ∗

∗Indian Institute of Technology Kanpur

Submitted to Prof. D. S. Katti for partial fulfilment of the course requirements for BSE 421A

Liver failure carries a high mortality rate and hepatic disease man-agement remains a major challenge. Currently, the only effectivetreatment option for patients with acute liver decompensation isliver transplant. There is a severe shortage of donor organs resultingin the death of one in seven patients before a suitable donor organcan be found. WHO estimates that over 650 million patients sufferfrom liver disease globally and with the alarming increase in obesityand an ageing population, liver disease is set to become an evengreater global health concern in the near future. The complexity ofbiological phenomena occurring in the liver microenvironment are notreflected in conventional 2D models of the liver system that are com-monly used. There are discrepancies between the in vitro predictionsand in vivo realities because when primary hepatocytes are isolatedand cultured in a 2D monolayer, they quickly lose some importantenzymatic properties which are crucial to predictive modelling. Thisloss of phenotypic features is a major deterrent to the developmentof new drug entities and the predictive modelling of liver biology ingeneral. Better in vitro models for studying the human liver biologywill have a great impact on hepatology and toxicology.1 Bio-printingis the process of generating spatially controlled cell patterns using 3Dprinting technologies. Generally, it involves a layer-by-layer approachto generate tissue-like 3D structures for use in the medical field oftissue engineering. Here, we propose a bio-printed 3D human liverconstruct consisting of parenchymal(human primary hepatocytes andhepatic cell-lines) and non-parenchymal cell populations(endothelialcells and hepatic stellates) for drug development and as a model forstudying liver biology. If a patient is already affected by a liver dis-ease or has genetic or metabolic disorders, the isolated autologouscell types may not produce the desired function in the 3D bio-printedconstruct. We then propose the use of reprogramming techniquesto generate induced pluripotent stem cells for use in 3D bio-printing.These cells, as shown in previous studies, would be proliferative, non-immunogenic (similar to autologous cells) and thus would be idealfor our proposed method of generating a model of liver disease anddrug development.2The attrition rate for drugs remains very high in-spite of efforts to improve the identification of toxicity of therapeuticcompounds.3. The objective is to demonstrate that the bioprinted3D liver tissues exhibit cellular composition closely mimicking humanliver tissue, a well organized and tissue-like architecture, sustainedmetabolic activities over time and a favourable response to someknown hepatotoxic agents (like diclofenac and APAP)4. We form as-sociations on scientific literature5 to show that the 3D model canallow biochemical interrogation of hepatotoxic agents and also thephysical examination of cells in tissue by histology, in order to ex-hibit a potential for liver disease modelling and drug discovery anddevelopment. A flexible 3D bio-printing method for rapid fabrica-tion of multi-cellular 3D liver constructs can thus enable both drugscreening and also athorough study of liver biology.

Liver Tissue Engineering | 3D Bioprinting

Introduction

T issues assume complex and well-organized 3-dimensionalarchitectures. One of the aims of tissue engineering is to

design an optimal analog of an in-vivo tissue. An importantaspect of tissue engineering approaches is a biocompatible net-work of synthetic or natural polymers called the scaffold thatmimics the extra-cellular matrix (ECM) of the cell. The scaf-fold, when seeded with cells, provides the appropriate biome-

chanical and biochemical conditions for proliferation and tis-sue formation. The biofabrication of a living structure withthe requisite functional and structural features requires a cre-ative and inter-disciplinary effort. There a lot of universitiesaround the world and even startups (like Organovo) that usenatural or artificial scaffolds, de-cellularized cadaveric extra-cellular matrices(ECMs) and now bioprinting. The recreationof native in vitro micro-environment is crucial for the successof biofabrication of living structures. The community is nowtrying to create scaffolds that are populated with stem cells ordifferentiated cells which is naturally increasing the complex-ity of the constructs. These scaffolds are susceptible to weak-nesses like eliciting immunogenic reactions, degradation, etc.The community is thus also focussing on self-assembly, self-organizing and regenerative capabilities of cells, tissues andorganisms. This is different from simply preparing a completetissue or a complete organ structure in-vitro and implantingit into the system.

In this project we discuss the fabrication of 3 dimensionalbiological structures using bioprinting. We will first attemptto give the reader a comprehensive understanding of the bio-printing process. We will then go on and talk about a fullybiological and print-based approach that uses bio-ink particlesand similar self-assembling multicellular units coupled withthe knowledge of morphogenesis and developmental biologyfor the fabrication of 3 dimensional biological structures6.

LiverIs Life Worth Living? It all depends on the liver! This wittyword play by 19th century American philosopher WilliamJames serves as a reminder that the health of the liver organis of utmost importance in living a healthy life. If you wantto live, you have to have a liver. Of the approximately 500functions of the liver, the most important include synthesisof amino acids and cholesterol; metabolism of carbohydrates,proteins and fats; and the production of bile which assists di-gestion in the small intestine. The liver plays several roles inthe regulation of the blood, breaks down insulin, breaks downtoxic substances and allows them to be excreted. In short, theliver supports almost every other organ in the body. The lo-cation of the liver is indicated in Figure 1. There is currentlyno way to compensate for the absence of liver function in thelong term, although liver dialysis techniques can be used inthe short term.

1A novel in vitro three-dimensional bioprinted liver tissue system for drug development - Poster,Liver Presented at the American Society of Experimental Biology, Boston, MA, April 2013.2Liu, Hua, et al. ”In vivo liver regeneration potential of human induced pluripotent stem cells fromdiverse origins.” Science translational medicine 3.82 (2011): 82ra39-82ra39.3Bioprinted three dimensional human liver contructs provide a model for interrogating liver biology- Poster, Liver. Presented at the American Society of Biology, New Orleans, LA, December 2013.4Tissue engineering by self-assembly and bio-printing of living cells. Karoly Jakab, Cyrille Norotte,Francoise Marga, Keith Murphy, Gordana Vunjak-Novakovic and Gabor Forgacs, Biofabrication. 2,1-14 (2010).5Toward engineering functional organ modules by additive manufacturing. Francoise Marga, KarolyJakab, Chirag Khatiwala, Benjamin Shepherd, Scott Dorfman, Bradley Hubbard, Stephen Colbertand Gabor Forgacs, Biofabrication. 4 022001 (12pp) (2012).6Karoly Jakab et al 2010 Biofabrication 2 022001 doi:10.1088/1758-5082/2/2/022001

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Fig. 1: Liver

Self-AssemblyWe define self-assembly as the autonomous organisation ofcomponents starting from a given initial state to a terminalstructure or pattern in the absence of intervention externally.The processes of organogenesis and histogenesis are great ex-amples of self-assembly processes while a developing embryois a nice example of a living self-organising system. In organo-genesis, the cell-cell and cell-ECM interactions allow the de-veloping organs and their subunits acquire their final shape.Biofabrication approaches aimed at re-establishing the func-tion of damaged organs and tissues in the future will quitelikely be focussed on mobilising developmental morphogeneticprocesses coupled with adult biological requirements (whichis another way of saying that they will need to make use ofthe body’s regenerative ability). Interestingly, the liver is theonly internal organ that is capable of regeneration! The basicquestions of regeneration like why this is true or why a sala-mander can re-generate extensively while we cannot is yet tobe answered7

Here we describe a way of fabricating living structures of de-fined functionality and shape by implementing principles andprocesses of developmental biology and talk about a rapid pro-totyping technology based on bio-printing of self-assemblingmulticellular building blocks. We then go on to describe howwe can for now use our system of drug discovery and otherapplications8. The most advanced artificially manufacturedliver tissue is metabolically active for at most 42 days, so itsnot good enough for an implantation as of yet.

We tactfully avoid the major unsolved issues of biocompat-ibility and longevity that face any artificial organ or implantby focussing on using our device for use in drug discovery anddrug testing systems.

Materials and MethodsThis section focuses on the conceptualization, design, fab-

rication and assembly of the device and a description of thematerials used. Let us first start with an examination of 3Dbioprinting.

3D Bioprinting

3D printing was first described by Charles W. Hull in 1986.In his method, which he named ”stereolithography”, solid 3Dstructures were made by successively ”printing” thin layers ofa UV curable material, one on top of the other. In this pro-cess, a programmed movable beam of UV light shines on asurface/layer of a UV curable liquid and forms a solid crosssection at the surface of the liquid. The whole set-up is pro-grammed such that the solid surface is then moved away fromthe liquid surface by a thickness of one layer. When the nextcross-section is formed, it is adhered to the immediately pre-ceding layer and this process continues until the entire objectis formed9.This process was later applied for the formation of 3D scaf-folds that could be used for transplantation with or withoutseeded cells10. With the recent advances in 3D printing tech-nology, cell biology and materials science, 3D bioprinting asa form of tissue engineering was made possible. In 3D bio-printing, layer-by-layer precise positioning of biological ma-terials, biochemical and living cells, with spatial control ofthe placement of functional components, is used to fabricatetissue-like 3D structures, for use in the medical field of tissueengineering. There are several design approaches to 3D bio-printing which includes biomimicry, autonomous self-assemblyand mini-tissue building blocks, which are used singly or incombination11. The main challenges that the researchers facein fabricating 3D functional living human constructs are :

1. Adapting the technologies for the printing of sensitive, liv-ing biological materials

2. Reproducing the complex micro-architecture of extracellu-lar matrix (ECM) components and multiple cell types insufficient resolution to replicate biological functions.

Design approaches for 3D bioprinting

There are three general approaches to 3D bioprinting, all ofwhich are discussed briefly below :

1. Biomimicry,2. Autonomous self-assembly3. Mini-tissue building blocks

Biomimicry. : Biomimicry is an approach to innovation thatseeks sustainable solutions to human challenges by emulat-ing nature’s time-tested patterns and strategies ( BiomimicryInstitute). The application of this biologically inspired engi-neering to 3D bioprinting involves the manufacture of identicalreproductions of the cellular and extracellular components of atissue or organ12, which can be achieved by replicating specificfunctional components of biological tissues on the micro-scaleresolution.Thus for this approach to succeed, understanding the microen-vironment - the specific arrangement of functional and sup-porting cell type, the composition of the ECM, the biologicalforces in the microenvironment, the gradients of soluble and

7Whitesides GM and Grzybowski B, 2002, Self-assembly at all scales Science 295 2418-218Whitesides GM and Boncheva M, 2002, Beyondmolecules: self-assembly of mesoscopic and macro-scopic components Proc. Natl Acad. Sci. USA 99 4769-749Hull, C.W. Apparatus for production of three-dimensional objects by stereolithography. US4575330 A (Google Patents, 1986)10Nakamura, M., Iwanaga, S., Henmi, C., Arai, K. and Nishiyama, Y. Biomatrices and biomaterialsfor future developments of bioprinting and biofabrication. Biofabrication 2, 014110 (2010).11Kamei, M. et al. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442,453?456 (2006).12Guillemot, F. et al. High-throughput laser printing of cells and biomaterials for tissue engineering.Acta Biomater. 6, 2494?2500 (2010).

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insoluble factors, is crucial. Researches in the field of engi-neering, imaging, biomaterials, biophysics and medicine canhelp with this regard of understanding the microenvironment.

Autonomous self-assembly. : This approach is a ’scaffold-free’version and it relies on understanding the embryonic organdevelopment as a guide. In autonomous self-assembly, the cellis used as the primary driver of histogenesis, directing thecomposition, localisation, functional and structural propertiesof the tissues. Cellular spheroids undergo fusion and cellularorganisation to mimic developing tissues and hence, an inti-mate knowledge of the embryo tissue genesis and organogene-sis, their developmental mechanism and along with it how tomanipulate the environment to drive embryonic mechanismsin bioprinted tissues is crucial.

Mini tissues. : Mini tissues are the smallest, functional build-ing blocks of a tissue, such as the kidney nephron. If thesemini-tissues can be fabricated, they can be assembled into thefunctional macro tissue by rational design or self-assembly orcombination of both the strategies. One example of such ap-proach is the self-assembly of vascular building blocks to formbranched vascular networks13.To print a complex 3D biological tissue with multiple func-tional, structural, mechanical properties, combination of theabove strategies are used. The main steps in bioprinting pro-cess is imaging and design, choice of materials and cells andprinting of the tissue construct, which can then be trans-planted after a period of in vitro maturation or can be re-served for in vitro analysis. 11 gives a brief picture of thevarious approaches.

Imaging

To provide information on 3D structure and function at thecellular, tissue, organ and organism levels, imaging is done bytissue engineers with the help of medical imaging technologies,most important of which are computed tomography (CT) andmagnetic resonance imaging (MRI), computer aided manufac-turing tools (CAD-CAM) and mathematical modelling. Thedata collected are processed using tomographic reconstructionto produce 2D cross-sectional images and further analysis iscarried out to produce the 3D anatomical representation. Thisprocess has been described as the transformation of analyticalanatomy into synthetic anatomy5. Each of the methods hastheir own set of advantages and disadvantages and hence, theyare chosen depending on the tissue to be reconstructed. Theinformation contained in the 2D horizontal slices provides thebioprinting device with layer-by-layer deposition instructions.Variations in the technologies also affect tissue and organ de-sign. Instead of trying to create an organ or tissue model fromthe ground up, researchers and engineers can use a CT scanor MRI to create a 3D model to print. For example, the Uni-versity of Louisville, when creating a 3D-printed model of ayoung boy’s heart so doctors could use it for his surgery, theresearchers used the CT scan from his doctor to make the 3Ddesign model. Websites like Instructables even have tutorialsto describe how to turn a CT scan into a 3D printable modelto print14.

Tissue Bioprinting strategies

The most common technologies used for deposition and pat-terning of biological materials are inkjets(as shown in Figure12) 15, microextrusion16 and laser-assisted printing17.

Factors such as surface resolution, cell viability and the bi-ological materials used for printing are important and should

be considered for different features of these technologies, asgiven in the Figure 13.

Organovo. made the first commercially used bioprinter, calledNovoGen MMX, which is the world’s first production 3D bio-printer (Figure 2). The printer has two robotic print heads.One places human cells and the other places a hydrogel, scaf-fold, or other type of support.

Fig. 2: Using Organovo’s NovoGen MMX bioprinter, cells arelayered between water-based layers until the tissue is built.

Scaffolds and Materials

One of the main challenges in the 3D bioprinting field hasbeen to find materials that are not only compatible with bi-ological materials and the printing process but can also pro-vide the desired mechanical and functional properties for tis-sue constructs. Materials currently used are predominantlybased on either naturally derived polymers (including alginate,gelatin, collagen, chitosan, fibrin and hyaluronic acid, often

13Kamei, M. et al. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442,453?456 (2006).14Lyndsey Gilpin. 3D ’bioprinting’: 10 things you should know about how it works. TechRepublic,US (April 23, 2014).15Klebe, R.J. Cytoscribing: a method for micropositioning cells and the construction of two- andthree-dimensional synthetic tissues. Exp. Cell Res. 179, 362-373 (1988).16Cohen, D.L., Malone, E., Lipson, H. and Bonassar, L.J. Direct freeform fabrication of seededhydrogels in arbitrary geometries. Tissue Eng. 12, 1325?1335 (2006).17Guillemot, F. et al. High-throughput laser printing of cells and biomaterials for tissue engineering.Acta Biomater. 6, 2494-2500 (2010).

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isolated from animal or human tissues) or synthetic molecules(polyethylene glycol; PEG). The advantages of natural poly-mers for 3D bioprinting and other tissue-engineering applica-tions is their similarity to human ECM, and their inherentbioactivity and as for synthetic polymers, they can be tai-lored with specific physical properties to suit particular ap-plications. Synthetic hydrogels, which are both hydrophilicand absorbent, are attractive for 3D bioprinting regenerative-medicine applications and for our project, we are going to usesynthetic hydrogel. Materials must have suitable crosslinkingmechanisms to facilitate bioprinter deposition, must be bio-compatible for transplantation over the long-term, and musthave suitable swelling characteristics and short-term stability.Short-term stability is required to maintain initial mechani-cal properties, ensuring that tissue structures such as pores,channels and networks do not collapse. As bioprinted tissuesdevelop in vivo, they should be amenable to remodeling, fa-cilitating the formation of structures driven by cellular andphysiological requirements. Most importantly, materials mustsupport cellular attachment, proliferation and function. Cur-rent options for printing cells involve either the deposition ofmultiple primary cell types into patterns that faithfully repre-sent the native tissue or printing stem cells that can proliferateand differentiate into required cell types. The cell type chosenshould be able to expand into sufficient numbers for print-ing. Precise control of cell proliferation in vitro and in vivo isimportant for bioprinting. Too little proliferation may resultin the loss of viability of the transplanted construct, whereastoo much proliferation may result in hyperplasia or apoptosis.In addition, the timing of cellular proliferation is important.Better understanding of the nature and composition of en-dogenous stem cells will be beneficial in engineering tissuesthat can maintain their long-term function after transplanta-tion18.

In the case of our product, a bioprinter is used to createliver tissue, which is one of the original experiments in bio-printing by the company. Using a NovoGen MMX bioprinter,the cells are layered between water-based layers until the tissueis built. That hydrogel in between layers is sometimes usedto fill spaces in the tissue or as supports to the 3D printedtissue. Collagen is another material used to fuse the cells to-gether. Spheroids of parenchymal (or fundamental) liver cellsare loaded into a syringe. In another syringe, nonparenchymalliver cells and the hydrogel, which fuses together to create abio-ink, is loaded. The bio-ink makes a mold in the cell dish,and the liver cells fill up the rest of the dish. When the cellsare put in an incubator, they fuse together even more to formthe full liver tissue (Figure 3)

Fig. 3: Cells are fused together with hydrogel, creating tissue.

Cell sourceTypically, an organ or even a tissue consist of multiple celltypes that are specific in their biological functions. To havean accurate 3D print, these cell types must be re-constructedwith those functions. Not only that, some cell types providesupportive, structural and mechanical support and functions.Obvious choice for cell types would be to use multiple pri-mary cell types into patterns that mimics the native tissueor printing stem cells that can proliferate and differentiateinto required cell types. The important thing is, cells cho-sen should mimic the physiological state of cells in vivo andshould maintain their in vivo functions under given conditions.Apart from that, cells should be proliferative. This prolifera-tion should be optimum. One of the major advantage of 3Dbioprinted liver model is it can be individualised i.e. it canbe exact replica of a particular patient. So, for example if onewants to study the effect of drug on a particular patients liver,one can generate 3D printed liver of the same patient by us-ing autologous cell source. One more advantage of autologoussource is the possible future application of bioprinted liver intransplantation. Transplantation of other bioprinted organshave already been achieved, (http://rt.com/news/202175-3d-bioprinted-organ-transplant/). So, to avoid possible rejectionby immune system autologous cell sources are desirable. Foran ill patient, it may not be possible to undergo surgical pro-cedure to procure healthy primary cell types. And also, itsdifficult to isolate hepatic cells for large scale applications.Hence, stem cells are promising cell type for bioprinting ap-plication. Liu et al., 2011 have shown that, ’human inducedpluripotent stem cells (iPSCs)-derived hepatic cells at variousdifferentiation stages can engraft the liver in mouse transplan-tation model.’ Human embryonic stem cells (hESCs) are de-rived from the inner cell mass of fresh or frozen embryos atthe blastocyst stage of development.

As with any transplanted tissues or organs, rejection of bio-printed constructs by the host immune system is a potentialproblem that can be overcome by using an autologous sourceof cells or by using tolerance-induction strategies. Advancesin cell culture techniques as well as in reprogramming and di-rected differentiation methods will be important for providinghighly proliferative, functional, non-immunogenic and robustcell populations that are suitable for bioprinting applications.So, we decided to work around all of that for our project. Wedecided to not implant all of this into patient bodies rightaway, and instead use our system for drug development.

As much as hESC are desirable properties of the cell source,its derivation remains ethically controversial and challenginglogistically due to limited supply of donor human embryos.Hence, we propose the use of hiPSCs derived from ’reprogram-ming’ of somatic cells to a pluripotent state through overex-pression of a key of transcription factors.

Based on the findings of Liu et al, we suggest the use ofhiPSCs as cell source for 3D bioprinting of liver. Proper char-acterization of bioprinted liver will be performed to test theefficiency and functionality of the bioprinted liver from iPSCscell source. Now we follow this up with some experimentaldetails for the sake of completeness. These results have beenbacked by literature cited with this paper.

18Sean V Murphy and Anthony Atala.3D bioprinting of tissues and organs. Nature32, 773-785(2014)

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Fig. 4: Schematic of human embryonic stem cell (hESC) andhuman induced pluripotent stem cell (hiPSC) derivation pro-tocols. (a) ESCs are derived from the inner cell mass (ICM)of the blastocyst, whereas (b) iPSCs can be derived from avariety of somatic cell types using a variety of reprogrammingtechniques. Commonly used assays to determine the equiva-lence of hESCs and hiPSCs at the molecular and functionallevels are listed in the table on the right(Narsinh et al., 2011)

Protocol for generating the cell source.

• Human iPSC lines derived from human hepatocytes (en-doderm), bone marrow mesenchymal stem cells (meso-derm), and liver fibroblasts (mesoderm) were generated us-ing retroviruses expressing genes encoding the transcriptionfactors Oct4, Sox2, Klf4, and c-Myc 19.

• Hepatic differentiation of human iPSCs (standard protocolsfollowed in previous work of Liu et al) iPSCs derived fromstep 1 is cultured in standard ESC maintained media.

• Human ESC lines WA09 (H9) and WA01 (H1) (WiCell)are cultured on irradiated mouse embryo fibroblast (MEF)feeder layers in the ESC medium.(This study can be donein accordance with Johns Hopkins Institutional Stem CellResearch Oversight regulations and following a protocol ap-proved by the Johns Hopkins Institutional Review Board.)

• Confluent cultures (50 to 60%) are placed in RPMI medium[supplemented with GlutaMAX and 0.5% defined fetalbovine serum (FBS) and activin A (100 ng/ml) (RnD Sys-tems)] for 5 to 6 days to induce DE stage cells.

• DE cells are passaged with 0.05% trypsin? 0.53 mMEDTA and plated on collagen I?coated dishes in minimalMDBK-MM medium (Sigma) supplemented with Gluta-MAX and bovine serum albumin (BSA) (0.5 mg/ml), fi-broblast growth factor 4 (FGF4) (10 ng/ml), and hepato-cyte growth factor (HGF) (10 ng/ml). Day 10 HP cells areto be used for experiments.

• HP cells are switched to complete hepatocyte culturemedium containing 5% defined FBS, FGF4 (10 ng/ml),HGF (10 ng/ml), oncostatin M (10 ng/ml) (PeproTech),and 10-7 M dexamethasone (Sigma). Differentiation is tobe continued for another 10 days to generate MH cells.

These cells can be used as a source for the further processsteps for liver bioprinting.

Now that we have established some background for Bioprint-ing, let us talk about the type of bioprinter that our proposeddevice shall use.

Inkjet. In summary, in inkjet technology, either individual cellsor smaller clusters are printed. This is a rapid, cheap and ver-satile method but it is not without its disadvantages. We doneed to ensure high cell density for the fabrication of solid or-gan structures, which is not easy using this method. Also, thethe speed of cell deposition is very high and thus there is a pos-sibility of causing considerable damage to the cells (althougha lot of new developments in this field have led to the survivalof cells for a longer time). Achieving an appropriate struc-tural organization and achieving functionality still remains achallenge.

Micro-Extrusion.Mechanical Extruders basically place ’bio-ink’ particles and multicellular aggregates of defined compo-sition onto a supporting environment called a ’bio-paper’, ac-cording to templates generated by a computer that are con-sistent with the topology of the desired biological structure.The post-printing fusion and the sorting of cells within bio-ink particles results in the formation of the bio-ink particlesresults in the formation of organoids. This technology has theadvantage that the bio-ink particles themselves represent 3Dtissue fragments. Thus, cells in them are in a more physiolog-ically relevant arrangement, with adhesive contacts with theirneighbors, which assures the transmission of vital molecularsignals. The method employs early developmental mechanismsas stated earlier in this paper, such as tissue fusion and cellsorting. The major disadvantage of the method is the rela-tively high cost of the printers.

Now we attempt to look at each of the steps involved indetail. These procedures have been adapted from existing lit-erature to suit or purpose for the project.

Bioprinting-Manufacturing of our liver tissueFirst have a look at Figure 5 and orient yourself to the sys-tem. This figure gives an overview of all the basic componentsof a print-based tissue-engineering process20. Bio-ink blocksare normally spherical or cylindrical in shape and are pre-pared from cell-suspensions. The extrusion-based bioprintingthat we implement describes an automated deposition methodthat allows the building of 3D custom-shaped tissue and organmodules without the use of any scaffold. We generate a fullybiological construct this way that is functionally and struc-turally very close to the native tissue. The bio-ink units aredelivered according to a computer-generated template alongwith hydrogel (which we like to call as the ’biopaper’) thatserves as a support material. Look closely at figure 6, this iswhat was initially used to establish proof of concept. A lot ofresearch21 went into developing the rapidity, reproducibilityand scalability of the technique.

19Liu, Hua, et al. ’In vivo liver regeneration potential of human induced pluripotent stem cellsfrom diverse origins.’ Science translational medicine 3.82 (2011): 82ra39-82ra39.20Jakab et al. Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication 2(2010) 022001 (14pp) doi:10.1088/1758-5082/2/2/02200121Jakab K etal. 2008. Tissue engineering by self-assembly of cells printed into topologically definedstructures. Tissue Eng.

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Fig. 5: Components of the print-based tissue-engineeringtechnology. (a) The bio-ink-filled micropipette printer car-tridge filled with multicellular building blocks that can bespheroidal (left) or cylindrical (right) depending on themethod of preparation. (b) The bio-printer. Three-dimensional printing is achieved by displacement of the three-axis positioning system (stage in y and printing heads alongx and z (top: Neatco, Carlisle, Canada; bottom: Organovo-Invetech, San Diego)). (c) Spheroids are delivered one byone into the hydrogel bio-paper (itself printed) according to acomputer script. (d) Layer-by-layer deposition of cylindricalunits of bio-paper (shown in blue) and multicellular cylindricalbuilding blocks. The outcome of printing (spheroids in panel(c), multicellular cylinders in panel (d)) is a set of discreteunits, which postprinting fuse to form a continuous structure.

Fig. 6: Layer-by-layer deposition. (a) A sheet of biocompati-ble hydrogel is printed, and the building blocks are embeddedinto the hydrogel. (b) and (c) The alternate deposition of lay-ers of hydrogel and building blocks is pursued according to thepredefined blueprint of the desired 3D structure (here, a tubu-lar construct). (d) Fusion of the building blocks and removalof the hydrogel result in a hollow tube after a few days.

Limitation. : For the benefit of the reader, we have includedsome limitations of this system. The success of printing de-pends strongly on the control of the gelation state of thecollagen-hydrogel layers and uneven gelation compromisesthe spatial accuracy. Another limitation arose during print-ing constructs of larger size and more complex pattern (i.e.branching tubes). The preparation of spheroids in large quan-tities (>1000 for branched tubular structures) became exces-sively time consuming. Finally, the manual filling of the mi-cropipette printer cartridge with the cellular spheroids (oneby one) represented a serious challenge.

To overcome the above limitations, we replaced the colla-gen sheets with agarose rods (figure 6) and we used cylindricalmulticellular building blocks instead of spheroids. The agaroserods are formed in situ and deposited by the bioprinter auto-matically, rapidly and accurately. Agarose is an inert and bio-compatible hydrogel that cells neither invade nor rearrange.The agarose rods kept their integrity during post-printing fu-

sion, and were easily removed to free the fused multicellularconstruct.

Fig. 7: Horizontal layer-by-layer deposition of the build-ing blocks. (a)-(e) A possible deposition scheme for a tubu-lar structure built of agarose rods and spherical multicellularbuilding blocks. (f ) The same tubular configuration printedwith cylindrical multicellular building blocks.

The construct that results from the printing process andthe post-printing fusion of the bio-ink particles is placed inan incubator where it achieves its final 3D structure and ma-tures to develop the appropriate biomechanical properties. Ithas been established by scientific literature22 that no biolog-ical functionality was lost in the above described bioprintingprocess.

In conclusion, this novel print-based tissue-engineering tech-nology has many distinguishing features and a huge potentialfor the generation of tissue and organ structures:

1. It represents an approach for producing fully biological(scaffold-free) small diameter vessels;

2. It utilizes natural shape-forming (i.e. morphogenetic) pro-cesses, that are present during normal development;

3. It can provide organoids of complex topology (i.e. branch-ing tubes)

4. It is scalable and compatible with methods of rapid proto-typing

Having looked at the biofabrication, let us now turn our at-tention to drug-delivery techniques. We first include a descrip-tion of different techniques before talking about drug testingtechniques.

Drug Delivery TechniquesThe three types of cells that can be targeted for drug deliveryin the liver are : The Hepatocytes, The Kupffer cells and TheHepatic Stellate cells.23

Hepatocytes

Targeting the asialoglycoprotein receptor on hepatocytes.The asialoglycoprotein receptor is specifically and abundantlypresent on hepatocytes and this receptor has been used todeliver all kinds of therapeutic compounds ranging from ther-apeutic proteins, antiviral agents to anticancer drugs into hep-atocytes. Although this has not led to any clinical applicationyet.24

Gene delivery to hepatocytes using viral systems.Aden-oviruses are a natural ligand for the coxsackie- and adenoviralreceptor, and integrin receptors present on hepatocyte. Ade-noviruses accumulate therefore massively in hepatocytes. Al-though gene delivery to hepatocytes has already been widely

22Jakab K etal. 2008. Tissue engineering by self-assembly of cells printed into topologically definedstructures. Tissue Eng.23Drug targeting to the diseased liver. Klaas et al.24X.-Q. Zhang, X.-L. Wang, P.-C. Zhang, Z.-L. Liu, R.-X. Zhuo, H.-Q. Mao, K.W. Leong. Galacto-sylated ternary DNA/polyphosphoramidate nanoparticles mediate high gene transfection efficiencyin hepatocytes. J. Control. Release, 102 (2005), pp. 749?763

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explored in clinical trials, many problems, such as immuno-genicity of viral vectors, transduction efficiency and off-targeteffects still need to be solved.

Kupffer and sinusoidal endothelial cells

By Phagocytosis. Kupffer cells represent the largest phagocy-totic activity within the body, phagocytose non-cellular par-ticles. For this reason many drug delivery systems like lipo-somes, micelles and viral particles end-up in these cells vianon-specific uptake mechanisms. In particular when the par-ticle size is larger than 100 nm, uptake by the RES is mostprominent.

Use of negatively charged drug delivery vehicles. Uptake inKupffer- and endothelial cells can also be mediated by spe-cific receptors. These cells bind negatively charged moleculesvia scavenger receptors that are abundantly expressed on theirmembrane. A high negative surface charge may cause rapiduptake of liposomes, polymers, oxidized LDL and proteins viathese receptors. Chemical modification of a neutral plasmaprotein or lipid may also yield a substrate for this receptor.

Mannose/fucose-modified proteins for Kupffer cell targeting.The cells of the RE system are also equipped with sugar re-ceptors that are important for recognition of foreign particles,like bacteria and yeast. The mannose receptor directly bindsto prokaryotic cells that have high mannan content in the cellwall.

Hepatic stellate cells (HSC)

Modified albumin-based carriers for HSC targeting.Cell-specificity for activated HSC in fibrotic livers was obtainedby using albumin-based carriers that bind to receptors whichare highly upregulated on activated HSC: the mannose 6-phosphate/insulin-like growth factor II receptor, the colla-gen type VI receptor, and the platelet derived growth fac-tor B (PDGF-B) receptor. To reach these receptors, albuminmolecules were substituted with mannose 6-phosphate (M6P)or with peptides that recognized either the PDGF-receptor orthe collagen type VI receptor. For all these carriers, exten-sive hepatic accumulation was found in experimental modelsof fibrosis in animals (approx. 70% of the dose accumulated infibrotic livers) and the main target cells responsible for this ac-cumulation were identified as activated HSC, although someuptake in other liver cells was also found depending on thecarrier used. This was confirmed in human tissue.

Specific delivery of therapeutic cytokines to HSC. The target-ing to HSC via the M6P homing device was also used to delivera cytokine to HSC. Interleukin-10, a cytokine endowed withanti-inflammatory and anti-fibrotic activities was successfullydelivered to HSC by coupling M6P-residues to it.

Gene delivery to HSC. Not only drugs but also genes are tar-geted to HSC. In most of the studies on gene delivery to HSC,adenoviral vectors are used. However, adenoviruses predomi-nantly transduce hepatocytes due to their CAR expression

Delivery systems to hepatocellular carcinoma cells. In well-differentiated forms of HCC, hepatocytes express the asialo-glycoprotein (ASPG) receptor and many drug delivery systemshave already been developed to deliver drugs to this receptorusing lactosaminated or galactosamine substituted drug carri-ers. In particular polymers have been applied for the purpose

of drug delivery to HCC but also modified albumins have beenexplored. The ASGP-receptor has also been employed as atarget receptor for the delivery of genes with anti-neoplasticeffects to the hepatocytes by making complexes of plasmidsand polymers coupled to ASGP-receptor binding ligands. Alsoother drug delivery systems such as cationic liposomes, vi-rosomes and adenoviral vectors have been exploited for thedelivery of anticancer drugs and genes in HCC. However, themajor flaw of this approach is lack of specificity for the cancer-ous hepatocytes versus the normal hepatocytes because bothcell types express ASGP-receptors.

Drug Testing TechniquesAfter the drug has been administered to our liver, we wouldlike to observe the effects it has on it. For that, a few tech-niques that could be used are25 :

1. ELISA : The enzyme-linked immunosorbent assay(ELISA) is a test that uses antibodies and color change toidentify a substance. This is a powerful technique that canhelp us analyse the metabolites of our administered drugand pretty much any protein whose presence(location andconcentration) we would like to test in our liver pre/postdrug administration.

2. Histological analysis : Histology is the study of the mi-croscopic anatomy of cells and tissues of plants and animals.It allows us to directly perceive through vision, the changesthat take place inside the organ upon drug administration.

3. Cell viability : The toxicity of our drug can be estimatedby determining the cell viability or the live cell count. Thiscould be done using techniques like the various colorimet-ric assays e.g. WST assay. For this test, cells must beextracted from the concerned portions of the organ for anal-ysis.

4. Biopsy : Finally, a biopsy could be performed on the organfor a detailed analysis of the disease.

Results and ApplicationsLet’s talk about the applications of our proposed 3D bio-

printed liver models.

3D bioprinting and its applications in in Vitro physi-ological models

Normally, cells are slowly and gradually made to grow on adegradable scaffold designed specifically for that particular tis-sue. This is achieved by a programmed design (explained in3D bioprinting section). The scaffold is a temporary syntheticextracellular matrix that is provided with chemical, biologi-cal, mechanical cues to guide cell’s differentiation and assem-bly into a 3 dimensional tissue. One of the most importantapplication of in vitro bio-printed liver is in the field of phar-maceutical drug and toxicology screening for drug discoveryand development. Currently, liver cell-based drug screeningmostly rely on several rounds of animal testing. Studyingdrug action involves 3 phases:

1. Cell-based assays2. Tissue based assays3. Animal and human testing.

25P.C.N. Rensen, L.A.J.M. Sliedregt, M. Ferns, E. Kieviet, S.M.W. van Rossenberg, S.H. vanLeeuwen, T.J.C. van Berkel, E.A.L. Biessen. Determination of the upper size limit for uptake andprocessing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo. J. Biol.Chem., 276 (2001), pp. 37577?37584

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The 3D bioprinted liver will not only bridge the gap be-tween cell based assays and animal testing but can also reducethe dependence on animal models. Also, the animal stud-ies are limited due to variations between animal and humanliver specific function. Currently, mostly 2D culture of tissuesare mostly used for in vitro drug testing. But the biosensorswhich are used to detect the cell responses are limited by the2 dimensional nature of the culture. Also it undermines thehigher order 3 dimensional processes in the tissue. A better 3dimensional tissue model may be used to quickly detect biolog-ical and chemical responses when studying tissue metabolismand drug response. These responses will be more accurateand more rapidly generated26 27 28. Another application ofbioprinted liver is in vitro disease pathogenesis. For exam-ple, one can re-create a cancerous liver tissue and metastasisconditions, which will be highly helpful in understanding andtargeting drug action.

Applications in biopharmaceutical industry and drugdevelopment

Due to various complexities (lack of volunteers, regulations,complexity of disease), the cost of drug development processhas been ever increasing as can be seen from Figure 8

Fig. 8: Cells are fused together with hydrogel, creating tissue.

On average (from data in 2005), companies spend an esti-mated 1.3 billion dollars on RnD over a 10-15 year span foreach approved drug29 30. Approximately half of this invest-ment takes place during the preclinical testing phases, whilethe balance is invested during clinical studies. Liver is the or-gan mainly responsible for drug metabolism and detoxificationin human body. Therefore, pre-clinical stages of drug testingtypically involves in vitro liver toxicology tests followed byextensive tests on animal models to predict the effect of thedrug. Currently, toxicity of a substance are tested on live an-imal livers over long durations. However, 50 percent of thedrugs that don’t render liver damage in animal tests are in-fact found to cause liver injury during clinical trials31. Thereason being differences in complexity of human and animalmodels. Also, there are individual variations due to genetics,pathophysiology and environmental factors. Clearly, an accu-rate in vitro modality to predict acute and chronic toxicity tovarious drug doses in liver will be a lot of help. Previous 2dimensional monolayer culture models32 are limited as they

can be examined for over a few days as these models don?tprevail liver functions for longer durations but rather undergocell de-differentiation. These in vitro models are highly inac-curate and unreliable as 90 percent of the candidates passedby these in vitro tests fail to pass the clinical trials33. Thus,there is a need for a 3 dimensional liver model that can sustainliver specific functions for a longer duration. Over proposal isto develop such a 3D bioprinted liver model which can sustainliver functions for a longer period.

Drug Delivery and Testing on 3-D Liver TissueDespite efforts to improve the ability to identify the toxicityof therapeutic compounds, the attrition rate for both exper-imental and approved drugs remains very high. Cardio- andhepatotoxicity remain primary reasons for late stage failuresand post-market withdrawals. Therefore more robust human,in vitro models of these organ systems are needed. We have de-veloped a bioprinted three-dimensional(3D) liver system thatcaptures several key features of in vivo tissue, in a multi-well format suitable for drug screening. Using our bioprint-ing technology we have fabricated 3D liver constructs contain-ing architecturally- and physiologically-relevant features fortwo hepatic cell lines and primary hepatocytes, within stan-dard multi-well culture plates. Bioprinted, 3D hepatic neotis-sues were further enhanced in complexity with the additionof endothelial and hepatic stellate cells. Biochemical studiesdemonstrate that several critical liver functions are presentincluding cytochrome P450 activity. Tight junction proteinexpression was observed throughout the 3D tissue. Analysisof cell death and proliferation following in vitro maturation re-vealed the constructs were viable. These results demonstrate aflexible bioprinting method to rapidly fabricate multi-cellular3D liver constructs in a multi-well format enabling both drugscreening and interrogation of liver biology.

Fig. 9: Schematic of multi-compartment micro-organ devicefor drug metabolism studies

26Bratten, C.D.T., Cobbold, P.H., and Cooper, J.M. 1998. Single-cell measurements of purinerelease using a micromachined electroanalytical sensor. Anal Chem 70:1164-1170.27Chen, A.A., Tsang, V.L., Albrecht, D.R., and Bhatia, S.N. 2006. 3-D Fabrication Technologyfor Tissue Engineering In: Springer US, BioMEMS and Biomedical Nanotechnology: 23-28.28Meyvantsson, I. and Beebe, D.J. 2008. Cell culture models in microfluidic systems. Ann RevAnal Chem 1: 423-449.29DiMasi, J.A., Hansen, R.W., and Grabowski, H.G. 2003. The price of innovation: new estimatesof drug development costs. J Health Econ 22: 151?185.30DiMasi, J.A. and Grabowski, H.G. 2007. The cost of biopharmaceutical RnD: is biotech different?Managerial and Decision Economics 28(4): 469?479.31Kaplowitz, N. 2005. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov 4: 489?499.32Baudoin, R., Corlu, A., Griscom, L., Legallais, C., and Leclerc, E. 2007. Trends in the develop-ment of microfluidic cell biochips for in vitro hepatotoxicity. Toxicol In Vitro 21(4): 535-544.33Sivaraman, A., Leach, J.K., Townsend, S., Iida, T., Hogan, B.J., Stolz, D.B., Fry, R., Samson,L.D., Tannenbaum, S.R., and Griffith, L.G. 2005. A microscale in vitro physiological model of theliver: predictive screens for drug metabolism and enzyme induction. Curr Drug Met 6: 569-591.

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A Useful Schematic

We believe that this top level view will make the basic appli-cation of our proposed 3D bioprinted liver model extremelyclear. The multi-chamber device shown in the Figure 9 hereis simply a flow-diagram schematic showing how a pro-drug(which is just a biologically inactive compound which can bemetabolized in the body to produce a drug.) is fed into themicrofluidic circuit towards the first liver chamber where thedrug is metabolized and hence either activated or deactivatedto a metabolite that can subsequently exert a downstream ef-fect on a target epithelial tissue that can be quantified. Thisschematic just gives the uninitiated a top-level idea of one waya drug metabolism study may be carried out.

Characterizing the drug effect : Fluid Dynamics andMass Transfer in Liver Model

Now once, we have developed a 3D bioprinted liver which mim-ics in vivo liver capillary architecture, we can study the effectof drug flow through those channels.Drug media can affect thetissue function by either enhancing the mass transfer (e.g. ofgases, nutrients and drugs) or by direct physical stimulationof cells or combination of both. Since, we want to test theeffect of dosage of drug media on bioprinted liver, we needto have accurate determination of the media flow parameters(drug concentration, shear stress, mass transfer rate) whichwill flow through the bioprinted liver. These measurementswould help to design and manipulate the media flow to mimicin vivo parameters faced by the liver. Following figure showsschematic of the drug media flow through bioprinted liver cap-illary.

Fig. 10: Characterising drug effects : Fluid Dynamics andMass Transfer

Now to model fluid dynamics and mass transfer one can soft-wares such as COSMOL Multiphysics software. The softwarerequires few parameters as inputs like: drug concentration atthe inlet, kinetics of drug reaction, diffusion coefficient of drugacross cross-linked cell. And it can model and give drug con-centration at the outlet. COSMOL can also model the shearstress experienced by cellular matrix due to the drug fluidflow. Knowledge of which is important to mimic the in vivoconditions (stress experienced by in vivo liver).

In summary, our human tissue model is multi-cellular, func-tional and dynamic models for pre-clinical testing and drugdiscovery research.

Discussion

Three-dimensional (3D) printing is a 20-year-old technologythat is now gaining currency. Interest in the equipment rosesharply last year; in 2012, the market for 3D products reached777 million dollars and predictions suggest that this couldreach 8.4 billion dollars by 2025 as medical uses for these print-ers are being developed. In printing liver tissues, a 24-wellplate is printed in about 45 minutes and is usable for testingin just two days. The above image depicts the evolution of op-eration theatre from early 20th century (top left) to modernday operation theatre (middle) to to possible operation the-atre after few decades. In near future, we will be able to printliver according to the requirement and need of the patient andwill hopefully be able to have successful transplants which willovercome the limitation of donor shortage. Research indicatesthat the cost of drugs that fail is estimated at about 40 per-cent of all drug spending. So, even if the drug spending ismore than 50 billion dollars per year, there is an opportunityto save more than 20 billion dollars every year. So, even ifusing 3D bioprinting for drug testing only causes a modestimprovement, this is still a huge potential benefit.

ConclusionWe have seen how bioprinting enabled highly reproducible fab-rication of structurally and compositionally defined 3D tissuesinto standard tissue culture formats. Bioprinted 3D liver tis-sues exhibited several key features that remained stable overtime:

1. Cellular composition closely mimicking human liver tissue2. Development of well organized, tissue-like architecture

>250 micro-m in thickness - Sustained metabolic activitiesover time

3. Demonstrated response to known hepatotoxic agents (di-clofenac and APAP) - Reproducible, multi-well format

4. Tissue-like cellular density, with high viability and devel-opment of well-organized microarchitecture (microvascula-ture, tight junctions) indicative of substantial intercellularcommunication.

5. Cell type-specific compartmentalization, with establish-ment and retention of user-defined spatial localization ofparenchymal and non-parenchymal components.

The tissue provides the ability to measure analytical protein,gene expression, histological and toxicological endpoints withfar greater relevance than traditional cell and tissue modelsover an extended in-vitro lifespan of over 40days. At the veryleast, this is an advancement in pre-clinical toxicology testingscience.

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Fig. 11: Bioprinting Process in Brief : Imaging of the damaged tissue and its environment can be used to guide the designof bioprinted tissues. Biomimicry, tissue self-assembly and mini-tissue building blocks are design approaches used singly andin combination. The choice of materials and cell source is essential and specific to the tissue form and function. Commonmaterials include synthetic or natural polymers and decellularized ECM. Cell sources may be allogeneic or autologous. Thesecomponents have to integrate with bioprinting systems such as inkjet, microextrusion or laser-assisted printers. Some tissuesmay require a period of maturation in a bioreactor before transplantation. Alternatively the 3D tissue may be used for in vitroapplications.

Fig. 12: Bioprinters : a) Thermal inkjet printers electrically heat the printhead to produce droplets in the nozzle and acousticprinters uses pulse formed by piezoelectric or ultrasound pressure (b) Microexrusion uses pneumatic or mechanical dispensingsystem (c) lasers focused on an absorbing substrate is used to generate pressure in laser-assisted bioprinters.

Fig. 13: Comparison of Bioprinter Types

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3D bioprinted liver for predictive disease modelling and drug testing.

Engineering

Transcript of 3D bioprinted liver for predictive disease modelling and drug testing.