HIGHLIGHT www.rsc.org/softmatter | Soft Matter Modular : engineering biological tissues from the bottom up

Jason W. Nicholab and Ali Khademhosseini*ab

DOI: 10.1039/b814285h

Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. To enhance the function of engineered tissues there is a need to generate structures that mimic the intricate architecture and complexity of native organs and tissues. With the desire to create more complex tissues with features such as developed and functional microvasculature, binding motifs and tissue specific morphology, tissue engineering techniques are beginning to focus on building modular microtissues with repeated functional units. The emerging field known as modular tissue engineering focuses on fabricating tissue building blocks with specific microarchitectural features and using these modular units to engineer biological tissues from the bottom up. In this review we will examine the promise and shortcomings of ‘‘bottom-up’’ approaches to creating engineered biological tissues. Specifically, we will survey the current techniques for controlling cell aggregation, proliferation and deposition, as well as approaches to generating shape-controlled tissue modules. We will then highlight techniques utilized to create macroscale engineered biological tissues from modular microscale units.

1. Introduction congenitally defected tissues. The field of Traditional tissue engineering strate- tissue engineering has emerged to fill the gies employ a ‘‘top-down’’ approach, in There is a substantial unmet demand for void where neither native physiology which cells are seeded on a biodegradable tissues to repair injured, degenerated or nor purely artificial implantable materials polymeric scaffold, such as poly(glycolic can sufficiently replace or repair these acid) (PGA)7–10 (Fig. 1). In top-down 1 aHarvard-MIT Division of Health Sciences and damaged tissues. While tissues such as approaches, the cells are expected to Technology, Massachusetts Institute of bone2 or skin3,4 can effectively repair populate the scaffold and create the Technology, CambridgeMA 02139 a small injury given sufficient time, many appropriate extracellular matrix (ECM) b Center for , tissues such as myocardium5 and carti- and microarchitecture often with the Department of Medicine, Brigham and Women’s 6 7 8 Hospital, , lage do not regenerate properly without aid of perfusion, growth factors and/or CambridgeMA 02139 intervention. mechanical stimulation.9,11 However,

Dr Jason W. Nichol is Dr Ali Khademhosseini is an a Research Fellow in Medicine Assistant Professor of Medicine at Harvard-MIT’s Division of and Health Sciences and Tech- Health Sciences and Tech- nology at Harvard-MIT’s Divi- nology. His research is in tissue sion of Health Sciences and engineering of cardiac and Technology and the Harvard vascular tissues, investigating Medical School. His research is the cell and tissue responses to based on developing micro- and mechanical stimuli. He is nanoscale technologies to currently developing biomate- control cellular behavior with rials with biomimetic mechanical particular emphasis on devel- properties combined with oping microscale microscale techniques to control for tissue engineering. He has Jason W: Nichol tissue microarchitecture. Select Ali Khademhosseini published 1 edited book, 65 peer- awards include the Whitaker Foundation Predoctoral Fellowship reviewed papers, 19 book chapters, 100 abstracts, and 14 patent (1999–2004) and the American Heart Association Postdoctoral applications. Select awards include Technology Review Magazine’s Fellowship (2006–2008), Finalist in the ASME Bioengineering ‘‘Top Young Innovator’’ (TR35) award (2007), the BMW Division student paper competition at the MS (2000), and PhD Scientific Award (2007), the ACS Victor K. LaMer Award (2008) (2002, 2003) level, as well as the BMES Poster Award in 2005. and the IEEE-EMBS Early Career Award (2008).

1312 | Soft Matter, 2009, 5, 1312–1319 This journal is ª The Royal Society of Chemistry 2009 2. Control of cell aggregation

Controlling cell aggregation is a key component of creating modular tissues with controlled microarchitecture. By restricting the geometry of cell aggrega- tion, engineers can create more controlled environments to study cell behaviors (Fig. 2, panels A–C).15,27–29 This control can be enacted in many ways, such as seeding cells in microwells30 or channels,31 micromolding cells in hydrogels16 or culturing cells in sheets.17,23

2.1 Micromolds as templates to control cell aggregation

Adaptation of similar techniques was used to create modular tissues by seeding cells in microwells to form spheroids 15 30 Fig. 1 Bottom-up and top-down approaches to tissue engineering. In the bottom-up approach made by primary or stem cells and there are multiple methods for creating modular tissues, which are then assembled into engineered their own secreted extracellular matrix tissues with specific microarchitectural features. In the top-down approach, cells and (ECM). Researchers then created larger, scaffolds are combined and cultured until the cells fill the support structure to create an engineered more intricate modular structures such as tissue. linear channels31 to form more tissue-like structures. One major advantage of this despite advances in surface patterning12,13 for transplantation in diabetic rats.26 technique is the ability to create modular or use of more biomimetic scaffolding, Although these studies were not tissues of specific shape and geometry such as decellularized ECM templates,14 conceived as modular tissue engineering, where cells have created their own top-down approaches often have diffi- each cell-containing microcapsule microarchitecture through ECM remod- culty recreating the intricate microstruc- behaves as a tissue module performing eling and cell migration/aggregation. tural features of tissues. a particular function. Packing the However, one major limitation of this Modular tissue engineering aims to microcapsules created an artificial technique is that some cell types are address the challenge of recreating biological structure, however traditional unable to produce sufficient ECM, biomimetic structures by designing microencapsulation techniques did not migrate or form cell–cell junctions. structural features on the microscale to create tissue-like structures and lacked Researchers in the Morgan group build modular tissues that can be used as the microvasculature and architecture demonstrated that cell aggregation could building blocks to create larger tissues of native tissues. Current techniques be controlled to not only form spher- (Fig. 1). These modules can be created in for creating modular tissues draw from oids,32 but also individual and connected a number of ways, such as through research in microencapsulation and tori15 (Fig. 2C). The resulting connected self-assembled aggregation,15 micro- microfabrication as well as traditional cell tori yielded honeycomb structures, fabrication of cell-laden ,16 and tissue culture procedures. By creating demonstrating the potential for creating creation of cell sheets17 or direct printing modular tissues with more physiological larger modular tissues of defined geom- of tissues.18 Once created, these modules microarchitectural features, bottom-up etry using cells cultured in replica molds. can be assembled into larger tissues tissue engineering aims to provide more through a number of methods such as guidance on the cellular level to direct 2.2 Sheet-based tissue modules random packing,19–22 stacking of layers17,23 tissue morphogenesis. or directed assembly.24 There is a strong The following review will highlight the Another technique for creating modular biological basis for this bottom-up current techniques for creating modular tissues manipulates the envi- approach as many tissues are comprised of engineered tissues using bottom-up tissue ronment to create modular cell sheets. repeating functional units, such as the engineering principles. We will describe Through optimizing the conditions lobule in the liver.25 By mimicking approaches to engineering modular leading to , and ECM native microstructural functional units, tissues by classifying the techniques that production in cell culture, researchers bottom-up approaches aim to create more utilize only cells and cell-produced mate- have successfully created large cell sheet biomimetic engineered tissues. rials as well as approaches using cell/ modules. By stacking these cell/ECM The historical roots of the modular biomaterial combinations. Subsequently, sheet modules and allowing the modules approach trace back to microencapsula- we will highlight the approaches used to to remodel and interconnect over time, tion techniques, such as encapsulating direct the formation of larger engineered researchers have been able to create pancreatic islet cells in alginate tissues from microscale building blocks. tissues with robust mechanical properties

This journal is ª The Royal Society of Chemistry 2009 Soft Matter, 2009, 5, 1312–1319 | 1313 3. Techniques for assembling engineered tissue building blocks

One of the major challenges of bottom-up tissue engineering is to assemble modular tissues with specific microarchitectures into macroscale biomimetic engineered tissues. The challenge is to retain the microarchitecture and cellular behavior of modular tissues, while creating engineered tissues with robust mechanical properties to withstand clinical implan- tation and interact appropriately with the native tissues. Some of the specific issues facing the field are: creating materials with biomimetic mechanical properties, integration of modular units to form robust integrated tissues, creation of functional microvasculature, improving manufacturing techniques to scale up production to tissue-level dimensions and successfully testing these tissues in the appropriate in vivo environment. Here we highlight five examples of promising techniques for creating and assembling modular tissues into engineered biological tissues, examining the advantages and disadvantages of each Fig. 2 Control of cell aggregation. Patterned stamps are used to create confined regions for cells to technique. These techniques highlight the aggregate and interact (A, B, C), scale: 25 mm, reproduced with permission from Journal of Cell most current research in creating modular Science.29 Use of larger geometric patterns such as connected tori (D) create modular tissues of tissues with improved higher order specific cell and ECM architecture, scale ¼ 400 mm.15 Copyright 2007 by Fedn of Am Societies for structures and assembly, functional Experimental Bio (FASEB). Reproduced with permission of Fedn of Am Societies for Experimental Bio (FASEB) in the format Journal via Copyright Clearance Center. vasculature, and the ability to reliably manufacture and evaluate these tissues in vivo. and cell–cell contacts. Using cell sheets with precursors, inserted into as the building blocks to create engineered molds and polymerized either by using 3.1 Additive photocrosslinking of tissues layer-by-layer, researchers have UV light16 or by incubation.50 Alterna- cell-laden hydrogels successfully created functional tively cell-laden hydrogel modular tissues vessels,17,23 as well as in vivo patches to can be created directly by passing UV Additive photocrosslinking uses 2D repair ocular,33 bladder34 and other light through a photomask, initiating the arrays of cells and ECM/ as the defects.35 crosslinking process within the building blocks to create tissues layer- only within the areas where the UV light by-layer.54,55 Using additive photo- penetrates through the mask.51 This polymerization, dual layer modular 2.3. Cell-laden hydrogels technique bypasses the need for creating tissues were created to fabricate living Since many cell types cannot produce a mold and is also amenable with high tissues of cellular hydrogels for hepatic sufficient ECM to create robust micro- throughput microfluidic techniques.24 tissue engineering. were tissues, researchers began mixing cells One example of this technique created isolated from adult female Lewis rats then with natural and artificial hydrogels to modular rings of primary neonatal rat mixed with liquid PEG functionalized form modular tissues of specific geome- cardiac cells in collagen or matrigel. These with RGD motifs. This mixture was tries and mechanical properties.36–42 The rings were then further cultured in contact inserted into a sealed chamber and most common techniques embed cells with multiple other engineered cardiac polymerized by UV exposure through within photopolymerizable polymers bands to form a composite graft, which a photomask in the shape of an array of such as poly(ethylene glycol)43,44 and could be conditioned to further improve 3 pointed stars (Fig. 4A1). The spacer other materials,45–47 sensitive cell alignment and contractile function height was then doubled and a 2nd layer hydrogels48 or self assembling peptide gels (Fig. 3). The resultant cardiac grafts was created on top of the first extending which can mimic ECM structure.38–42,49 significantly improved cardiac function in the full spacer height (Fig. 4A2). Finally To create microtissues, cells are mixed rat myocardial infarct models.5,52,53 the spacer was increased again, and the

1314 | Soft Matter, 2009, 5, 1312–1319 This journal is ª The Royal Society of Chemistry 2009 architecture combined with the RGD motif. While this technique successfully creates modular tissues with geometries similar to the native environ- ment, one potential drawback of this technique is the inability to restrict the thickness of subsequent layers. As the cell–hydrogel mixture must fill the entire volume of the chamber, and it is not possible to restrict the depth of UV polymerization, each newly created layer must be the full height of the chamber, potentially limiting the number of layers achievable. However, if the goal is to create layered structures of the same geometrical pattern, this restriction would be of minimal impact.

3.2 Random packing of encapsulated modules

A major limitation in many engineered tissues is the lack of functional micro- Fig. 3 Modular cardiac tissue engineering. Native cardiomyocytes are mixed with ECM (collagen vasculature to ensure proper blood or Matrigel) in ring shaped molds. These rings are then assembled together on a cyclic stretching perfusion and connection with device to condition the rings and allow them to integrate with the other rings creating an aligned surrounding tissues. Despite progress in tissue for implantation on the cardiac surface, scale: 10 mm.52 Adapted by permission from Mac- using microfabrication techniques to millan Publishers Ltd: Nature Medicine, copyright 2006. improve the vascularization of engineered tissues56,57 this limitation is still present in mask was changed to create a honeycomb crotissue. This technique successfully many engineered tissues. enclosure around each individual 2-layer increased hepatocyte adhesion and urea In an alternative approach to creating unit to create an engineered hepatic mi- production with the created micro- capillary networks, a random packing system was developed to create tortuous channels of perfusable modular tissues. Cylindrical, perfused tissues were created by assembling cell-laden collagen units together within perfused tubing.19–22 First, modular constructs were created by mixing collagen with or without HepG2 cells and allowing the mixture to reach gelation to create cylindrical tissue modules. The gels were then collected in media and seeded with HUVEC cells until a confluent layer formed around the modules. The HUVEC-HepG2 modules were then collected into tubing with a porous plug then perfusion was initiated to allow the cell-laden modules to compact, remodel and form a porous, perfusable tissue. Tissue constructs were perfused with whole blood to demonstrate the ability to withstand clotting, while maintaining high viability Fig. 4 Hepatic tissue engineering using additive photopolymerization. Multiple layers are depos- of the HUVEC and encapsulated HepG2 ited sequentially (red, then green, then blue, A) to create multilayer hepatic tissues (B), scale: 1 mm, cells (Fig. 5). (C), scale: 500 mm, (D).25 Copyright 2007 by Fedn of Am Societies for Experimental Bio (FASEB). This simple solution to creating Reproduced with permission of Fedn of Am Societies for Experimental Bio (FASEB) in the format a perfusable tissue would be very effective Journal via Copyright Clearance Center. in tissues whose primary function is

This journal is ª The Royal Society of Chemistry 2009 Soft Matter, 2009, 5, 1312–1319 | 1315 Fig. 5 Assembly of modular tissues in perfusable engineered tissues. HepG2 cells are encapsulated in collagen, cast into modular units then coated with a confluent layer of HUVEC cells and packed inside a perfused tube (A). The resultant tissue consists of interconnected cell modules (B–E) which are perfusable and non-coagulative due to the endothelial (HUVEC) cells.20

filtration, such as the liver or kidney, tant cell aggregate is cultured to allow the While this technique could be effective especially when envisioned as an external cells and ECM to integrate into tissue in creating modular 2D tissues, one device or a self contained implantable structures. This technique can be used to disadvantage of this technique is the device. However, as a technique for create individual patterned cell groups, or impact on cell viability in the time elapsed creating microvasculature there are more intricate 2D arrays of cells between creation of subsequent tissue potential shortcomings such as the lack of (Fig. 6).18,42 In addition, it is possible to layers. Also, as in sheet based tissue mechanical integrity in the absence of an print subsequent layers once the printed engineering, layer to layer binding and enclosure. Also, as the secondary cell layers have sufficient culture time, integrity could be difficult to achieve. types must persist and remain effective creating 3D arrays of cells and tissues. Additionally, not all cell types will when fully encapsulated by endothelial cells this could potentially limit the secondary cell types for this application.

3.3 Tissue printing

Similar in concept to additive photo- polymerization, printing creates 2-D arrays of cells and ECM/polymer42 which can be built layer by layer into tissues.18,58–60 While photopolymerization creates layers all at once, deposits cells in small groups using similar technology to traditional printing systems. The advantage of this technique is the high potential level of control in cell and ECM placement and alignment to create engineered tissues with a wide array of properties and geometries. Fig. 6 Organ printing. The desired pattern is input into the computer attached to the cell printing Replacing ink with cells suspended in device (A, D, E), individual cells and ECM are deposited (B) in larger geometrical patterns (C, F, liquid ECM or self assembled ECM G).18 Reprinted from TRENDS in , 21/4, Mironov, V., T. Boland, T. Trusk, G. mimics, a specific pattern is designed and Forgacs and R. R. Markwald, Organ printing: computer-aided jet-based 3D tissue engineering, printed onto a substrate, then the resul- pages 157-61, copyright (2003), with permission from Elsevier.

1316 | Soft Matter, 2009, 5, 1312–1319 This journal is ª The Royal Society of Chemistry 2009 respond positively to the conditions different aspect ratios through photo- limits to the size of structures achievable, potentially required for this technique. polymerization with a photomask. These and as seen in Fig. 7, the potential for microtissue modules were then collected random or uncontrolled structures still in hydrophobic mineral oil causing the exists. The development of controlled 3.4 Directed assembly of tissue hydrophilic hydrogels to aggregate , as well as optimal module sizes modules together to form tissues of varying and shapes, are promising approaches to Recent research from our group has dimensions, which could then be solidified solve some of these challenges. demonstrated the ability to use directed through a secondary UV polymerization assembly techniques to create higher step. Varying the aspect ratio of the 3.5 Cell sheet technologies order tissue structures using UV cross- modules demonstrated the ability to linked hydrogel modular microtissues.24 control the ultimate size and shape of the One technique that has successfully ad- This technique addresses the challenges of resulting aggregate tissues as the number dressed the insufficient mechanical prop- creating higher order structures when of hydrogels per tissue increased propor- erties and cell alignment in engineered the tissue modules used are fragile or tionally with the module aspect ratio. modular tissues is cell sheet engineering. otherwise difficult to manipulate, while Future applications of more intricate By creating sheets of cells under condi- also suggesting potential methods for structures were demonstrated with lock tions that encourage ECM production, microfluidic or automated techniques for and key shapes, indicating the potential engineered modular tissues made using creating engineered tissues from modular versatility of this technique. these techniques have shown mechanical microtissues. Some potential advantages of this properties on the order of native tissues.17 By harnessing the surface tension system are the ability to reproducibly One of the pioneering works in cell characteristics of hydrophilic hydrogels direct the structure of tissues based on the sheet tissue engineering created blood we assembled modular tissues into higher module geometry without the need for vessels over a decade ago.17 In this work, order structures (Fig. 7). First, cell-laden complicated assembly and manipulation smooth muscle cells and fibroblasts were rectangular hydrogels were created in procedures. However, there are practical cultured separately under overconfluent

Fig. 7 Directed assembly of modular tissues. Rectangular cell/hydrogel modules were created directly by photopolymerization using UV through a photomask, then allowed to aggregate and self-assemble in a hydrophobic media (mineral oil), scale: 200 mm. After assembly a 2nd UV polymerization solidified the structures, which varied in size and organization based on the module geometry.24

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