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Procedia CIRP 5 ( 2013 ) 276 – 281

The First CIRP Conference on Biomanufacturing Scalable Biofabrication of Tissue Spheroids for Organ Printing

R.A. Rezendea,*, F.D.A.S. Pereiraa, V. Kasyanovb, D.T. Kemmoku, I. Maia, J.V.L. da Silvaa, V. Mironova

a Center for Information Technology Renato Archer (CTI), Division of 3D Technologies (DT3D), Rodovia D. Pedro I (SP - 65) Km 143,6, Amarais, Campinas, SP, 13.069-901, Brazil b Riga Stradins University, Riga, Latvia, European Union. * Corresponding author. Tel.: +55-19-3746-6203; fax: +55-19-3746-6204.E-mail address: [email protected].

Abstract

Organ printing technology or robotic additive biofabrication of 3D functional tissue and organ constructs is based on using tissue spheroids as building blocks. In order to bioprint human organs it is necessary to develop technology for scalable production of millions tissue spheroids. Ideally, these tissue spheroids must have standard size and shape suitable for bioprinting process. The scalable biofabrication of large volume of standard size tissue spheroids could be achieved only by maximal employment of robotics and automation technologies. The three main competing groups of emerging tissue spheroid biofabrication technologies include: i) modified handing drop method, 2) molded non-adhesive hydrogel technology and iii) digital microfluidic technologies. The comparative analysis of emerging scalable tissue spheroid biofabrication technologies has been performed. Our data indicates that all these technologies have potential for robotization and automation. The molded non-adhesive hydrogel technologies provide best outcome for standardization of tissue spheroid size. The microfluidics technology has strong advantage in accelerating of tissue spheroids biofabrication (theoretically, up to 10 000 droplets per second). New emerging approaches for biofabrication tissue spheroid using nanopatterned biomimetic surface and technologies for tissue spheroid encapsulation and functionalization will be also presented. The tissue spheroid biofabrication technologies are still evolving and represent hot area in biofabrication research. Moreover, these technologies are already subject of ongoing commercialization. Thus, it is safe to predict that scalable robotic tissue spheroid biofabricators must be integrated parts of organ biofabrication line.

© 20132012 The The Authors. Authors. Published Published by Elsevierby Elsevier B.V. B.V. Selection and/or peer-review under responsibility of Professor Mamoru Mitsuishi andSelection Professor and/or Paulo peer review-review Bartolo under responsibilty of Professor Mamoru Mitsuishi and Professor Paulo Bartolo

Keywords: Tissue Spheroids; Bioprinting; Scalable Robotic Biofabrication; Tissue Engineering.

self-assembling tissue spheroids at large scale is an 1. Introduction important technological imperative.

Organ printing is a computer-aided robotic layer by layer additive biofabrication using living tissue 2. Why tissue spheroids? spheroids as building blocks. [1-4] The fundamental biophysical principle of organ printing technology is 2.1. Tissue spheroids as building blocks intrinsic capacity of tissue spheroid for tissue fusion driven by surface tension forces. Tissue fusion is an Tissue spheroids have the small size and ideal ubiquitous process during the embryonic development. geometric form for bioprinting. Tissue spheroids are Implementation of organ printing is not possible without formed by cell aggregation and have the maximal development of technology for large scale production of possible initial cell density. They have an intrinsic living tissue spheroids. Thus, robotic biofabrication of capacity for tissue fusion which make them an ideal

2212-8271© 2013 The Authors. Published by Elsevier B.V. Selection and/or peer review-review under responsibilty of Professor Mamoru Mitsuishi and Professor Paulo Bartolo doi: 10.1016/j.procir.2013.01.054 R.A. Rezende et al. / Procedia CIRP 5 ( 2013 ) 276 – 281 277 building material or some sort of living building blocks. know what are the material properties of building They can have complex composition and be pre- material. vascularized. Most importantly, as it will shown here To measure quantitatively and precisely the material tissue spheroids could be produced automatically using properties of tissue spheroid is not a trivial thing. robots in large amounts and of uniform size and shape However, there are already several approaches for study [4]. material properties of tissue spheroids which have been developed and successfully implemented (Figure 1). 2.2. How many tissue spheroids are necessary in order to print human organ?

If we assume that the approximate size of average adult human kidney is 100 mm x 50 mm x 50 mm then the estimated volume of such human organ will be 250 000 mm3. If we assume that average diameter of tissue spheroid is 0,2 mm or 200 micrometers the in order to bioprinter human organ of kidney size we must biofabricate 6.250.000 (6,25 mln) of tissue spheroids. It Fig. 1. Methods of measurement of material properties of tissue have been reported recently that it is possible to spheroids. a) tensiometry; b) aspiration; c) spreading; d) microfluidics robotically biofabricate using non-adhesive molds based squeezing. placed in standard 96 multiwells up to 5000 tissue spheroids per multiwall. Thus, we will need 1.250 Historically the compression of living tissue spheroid multiwells with 5000 tissue spheroid in each in order to using parallel plate or tensiometer was one of the first bioprint one human organ. It is also conceivable that one method of quantitative estimation of their material middle size dispensing robot can effectively handle properties [6]. However, it is subject of some criticism approximately up to 50 multiwells. If it will be that case now because technology of measurement is based on then in order to robotically biofabricate one human some unconfirmed assumptions [7]. More recently organ we will need just 5 dispensing robots which can aspiration assay is becoming increasingly popular for produce 250 multiwells. This simple calculation study tissue spheroid [8]. However, measurement by demonstrates that even at recent level of technology aspiration assay reflects more local material properties development the transition from low scale laboratory rather than desirable global material properties. Another method of biofabrication to large scale industrial method is based on tissue spheroids spreading [9]. biofabrication of tissue spheroids in desirable volume is Finally, recently simple and elegant microfluidics technologically feasible. We must not forget that method for study tissue spheroid material properties commercially available robotic fluid dispensor and have been developed and experimentally tested [10]. multiwall handling robots are also of the different scale. Microfluidics method as well as indirect method based Robotic fluid dispensor will be able to produce enough on non-invasive but carefully calibrated impedance tissue spheroid for bioprinting human organ like kidney. measurements could be employed for high throughput screening of tissue maturation factors and also for 2.3. Material properties of tissue spheroids. quality control of tissue spheroid at industrial scale.

Why it is important to know material properties of 3. Method of scalable robotic biofabrication of tissue tissue spheroids? The material properties of the tissue spheroids spheroids are essential to know because of several reasons. Firstly, capacity of tissue spheroids to undergo 3.1. Modified hanging drop methods. tissue fusion directly depends on their material properties. The more rigid tissue spheroid fuses more Handing drop method is a classic biological method slowly. The level of maturation of tissue spheroids such which was used by Prof. Aleksander Maximow as some as chondrospheres and osteospheres is manifested in sort of minibioreactor for cell culturing in the beginning their material properties and could be used as one of of XX century. According our personal experience the objective criteria of their functional maturation. main limitation of hanging drop method was low Materiall propertis of tissue spheroid could be also used reproducibility due to satellite formation and laborious for their standardization. Finally, if tissue spheroid is a handling. However, two companies InSphero and 3D building block [4] or even is a specific living form of Biomatrix recently modified this method by making biomaterial [5] and we want to build complex hanging drop droppable and thus allow applying robotic construction like human organ, it is not a bad idea to automated dispensing (Figure 2) [11,12]. 278 R.A. Rezende et al. / Procedia CIRP 5 ( 2013 ) 276 – 281

3.4. Tissue spheroids biofabricating commpanies

One of the best objective indicators of the maturation of any technology is initiation of technology transfer or commercialization of technology. Both modified hanging drop method and non-adhesive hydrogel molding method of tissue spheroid biofabrication have been commercialized. It is safe to predict that microfluidics will be next. There are some tissue spheroids such as chondrospheres and cardiospheres which are already in clinical trial (Table 1).

Fig. 2. Hanging drop method of scalable biofabrication of tissue spheroids. a) scheme of the method ; b) robotic fluid dispenser.

3.2. Non-adhesive molded hydrogel. Fig. 3. Scalable robotic biofabrication of tissue spheroids using non- Non-adhesive hydrogel molding has been adhesive hydrogel molding technology. a) mold ; b) scheme of the commercialized by Brown University start-up company method; c) robotic fluid dispenser (EpMotion-5075, Eppendorf, [13]. It is also gravity based approach Germany). and it is based placing cell suspension into microrecession in non-adhesive agarose hydrogel. We 3.5. Novel emerging methods of tissue spheroidds recently modified this method by using more dense mold biofabrication and robotization approach [14] (Figure 3). The improvement of tissue spheroid biofabrication is 3.3. Digital microfluidics. ongoing process. Recently, several interesting approaches for biofabrication of tissue spheroids based Digital microfluidics based method of scalable on creative employment of novel physical and chemical biofabrication of tissue spheroids theoretically can principles have been developed which include using produce up to 10 000 spheroids per second. Coaxial thermo-reversible hydrogel [16], cell surface droplet generator has been developed and tested [15]. modification [17], dielectrophoresis [18], magnetic thus, digital microfluidics is one of most perspective levitation [19], nanopatterned surface like pillared sheet method of scalable biofabrication of tissue spheroids. based on biomimetic principle of so- However, the issue of low cell density in hydrogel [20]. It clearly reflects the ongoing progress in the droplets generated by microfluidics droplet generating development of novel more sophisticated method of devices must be addressed and technology must be tissue spheroid biofabrication. However, from the optimized (Figure 4). position of criteria of scalability not all of these sophisticated methods have been proven potentially useful or suitable for clinical translation and not all have strong potential for scaling up. For example, because of R.A. Rezende et al. / Procedia CIRP 5 ( 2013 ) 276 – 281 279 single cell level precision and elegant simplicity of 4. Post-fabrication processing: functionalization and dielectropheresis principle deserves special attention. cryopreservation of tissue spheroids The emerging nanotechnology such as nanopatterned pillared sheet and patterned nanosurface from thermo- 4.1. Cryopreservation of tissue spheroids sensitive hydrogel could be also scalable because surface area coated with attachment permissive and thermo- This issue is still under discussion and there is no reversible hydrogel can control number of attached cells. clear consensus between researchers - do we need to From another side, using sophisticated method of cell develop cryopreservation technology for long term surface engineering and cell modification with DNA storage and transportation of biofabricated tissue doubly could be clinically relevant method as well as spheroids or they must be fabricated only locally (ideally method based on intensive using nanoparticles or other inside hospital or even operation room) and used for potentially toxic nanomaterials for cell levitation and organ printing only freshly fabricated. It is a complex scalable production of clinically relevant tissue biological, technological and also economic question spheroids. and heavily based on using only autologous or also allogeneic cells, using integrated or separated robotic systems, and selected centralized or decentralized (local) business model. What is important from biological and biotechnological point of view that the efficient cryopreservation technologies with clinically relevant and sufficient level of viability of tissue spheroids after thawing have been already developed.

4.2. Harvesting and translocation of tissue spheroids

Tissue spheroids as so- bioprinter, which automatically implies that from technological point of view the bioprinter must have either cartridge loaded with bioink like desktop inkjet printers or it must have a direct connections with multiwells or digital microfluidics systems which biofabricate tissue spheroids. In first case scenario we must developed cartridge and probably deal with cryopreserved tissue spheroids. In second case scenario we must develop effective methods of translocation of freshly biofabricated tissue spheroids directly into bioprinter dispensing head or nozzle. More traditional pressurized or gravity based approaches could be and must be initially explored on feasibility. However, possible technological platforms exploring acoustic, electric and magnetic levitation of tissue spheroids as the Fig. 4. Scalable biofabrication of tissue spheroids using digital way of their rapid translocation and dispensing in robotic microfluidics. a) digital microfluidics based on droplet generator; b) bioprinter also deserves special attention and systematic scheme of the digital microfluidics based droplet generator developed exploration on feasibility of such approaches. at CTI; c) digital microfluidics based coaxial droplet generator Only one original paper was specially devoted to issue (10). of harvesting and it have been reported the method of tissue spheroids harvesting based on squeezing hydrogel Table 1. Some commercial tissue spheroids biofabricating companies. where microrecession for biofabrication of tissue spheroid have been made. Another advantage of this Company Name Country Method method that is potentially suitable for highly desirable 3D BioMatrix USA Hanging Drop automation and robotization [21]. Microtissue USA Mold In any case, tissue spheroids translocation from zone InSphero Switzerland Hanging Drop of their biofabrication toward bioprinter is not a trivial CyFuse Japan Hanging Drop biological and engineering task and still basically either Co.don Germany Hanging Drop ignored or just overlooked. 280 R.A. Rezende et al. / Procedia CIRP 5 ( 2013 ) 276 – 281

4.3. Functionalization of tissue spheroids. hydrogel for 3D printing or rapid prototyping of acellular synthetic scaffolds is not an organ printing. 3D Functionalization of tissue spheroids is directly printing of cells suspended in hydrogel with low cell related to their storage in cartridge and movement or density is also not an organ printing. Organ printing is translocation. In order to prevent preliminary and layer by layer deposition of tissue spheroid which can undesirable tissue spheroid fusion in cartridge during fused and which have maximal possible initial cell bioprinting process they must be encapsulated in highly density. Now let consider issue of feasibility. Could non-adhesive and lubrificated hydrogel such as situation when there are already at least five commercial hyaluronan which is usually used for coating companies in different countries around the world endovascular device for interventional cardiology. There producing tissue spheroids and there are already five are several commercial companies producing companies producing robotic bioprinters be defined such encapsulators. The commercial encapsulators have been term as science fiction doma initially designed for immunoisolation of pancreatic In really we are already islands but they could be also relatively easy adapted for facing dramatic translation from research to encapsulation of not natural but biofabricated tissue commercialization. Finally, is it possible to find spheroids in desirable non-adhesive and lubricating compromise or common ground between traditional hydrogel. solid scaffold based tissue engineering approach and The second aspect of tissue spheroid relatively new bottom-up modular microtissue based functionalization is providing them with additional tissue engineering or it is completely incompatible properties suitable for their translocation. For example, research direction. Our concept of tissue spheroid tissue spheroid could be either encapsulated into encaged in interlockable solid microscaffold or hydrogel containing magnetic nanoparticles or it could be encage in microscaffold also loaded with magnetic question. The lockyball concept allows combining all nanoparticles. In both case functionalization of tissue biomimetic advantages of tissue spheroids approach with spheroid enables their magnetic levitation. advantage of solid-scaffold based approach. However, it We recently developed a design concept of encaging is important to underline that basic fundamental tissue spheroids into interlockable microscaffold or principle of bottom up approach is not compromised. Tissue spheroid encaged in solid interlockable functionality - they enable rapid bottom up bioassembly microscaffold and encapsulated in think layer of of cartilage or bone tissue from tissue spheroids. lubricating hydrogel could be used for bioprinting. Lockyballs enable tissue spheroid retention and maintain It is also very important to indicate that tissue shape of 3D construct and tissue spheroid will later fuse spheroid could have complex internal structure and and form tissue. composition and be pre-vascularized and prebuild in Finally, functionalization of tissue spheroid could be with high level of histological authenticity [22]. Finally, directly related with tissue assembly process during tissue spheroid could be printed together with solid or bioprinting. For example, tissue spheroids encapsulated hydrogel scaffold and thus be incorporated into 3D solid in hydrogel loaded with thrombin will be very easy and scaffold of bioprinted cell-free hydrogel woodpile [23]. practically instantly be sintered by spraying fibrinogen solution and formation of fibrin hydrogel which will 6. Conclusion work as glue. Thus, presented data strongly indicate that scalable 5. Some misconceptions about tissue spheroids based robotic biofabrication of tissue spheroids are not only organ printing technology. technologically feasible but are already a subject of ongoing commercialization. It is a direct manifestation There are three main misconceptions about tissue and reflection of general trend in biomedical spheroids in context of their using as building blocks in robotizations and automation as the way to improve organ printing technology. First misconception is about productivity and control the cost in biomedical industry. revolutional novelty of solid scaffold free approach in From another side, there are specific requirements for tissue engineering, second misconception is about emerging bioprinting technology such as standardization practical feasibility of organ printing technology and of tissue spheroids size and shape, processibility, storage associated still persistent believe that organ printing is and large volumes which must be carefully addressed either still in science fiction domain or in infancy state. during development of new and optimization the Finally he is misconception about principal existing scalable robotic biofabrication technology. incompatibility between solid scaffold and solid scaffold free approach in tissue engineering. Using polymer and R.A. Rezende et al. / Procedia CIRP 5 ( 2013 ) 276 – 281 281

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