Perspective: The Promise of Multi-Cellular Engineered Living Systems
Perspective: The Promise of Multi-Cellular Engineered Living Systems
Perspective: The Promise of Multi-Cellular Engineered Living Systems
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Brain organoid-on-a-chip: A next-generation human brain avatar for recapitulating human brain physiology
and pathology
Biomicrofluidics (November 2022)
I. INTRODUCTION
Over the past several decades, bioengineers, biophysicists, and biologists have made steady
progress toward the creation of “multi-cellular engineered living systems” (M-CELS). These
systems are composed of living cells and tissues organized in a way that produces novel func-
tionalities by design. For present purposes, we consider the subset of M-CELS composed of
mammalian cells and used primarily for biomedical applications and exclude, for example,
other potentially important applications such as those in plant systems, energy harvesting, or
the microbiome. Defined in this way, it includes organ-on-chip or tissue chip systems being
developed for drug screening or disease models1,2 with the potential to expedite drug discovery
and provide important new insights into fundamental disease processes. It also encompasses
implantable “hyper-organs,” ones that, for example, sense a biological signal and synthesize
and secrete a biologic product in response. Also included are biological actuators or bio-robots
that have applications in various fields. These M-CELS might be assembled in vitro from
a)
Author to whom correspondence should be addressed: [email protected]
TABLE I. Disciplines needed for progress in M-CELS and brief description of their respective contributions.
While steady progress in each of these disciplines fuels our ability to create M-CELS, several
major recent advances are particularly enabling and noteworthy. First, the ability to reprogram
adult, committed cells to generate induced pluripotent stem (iPS) cells5 has freed us from reliance
on embryonic stem cells to form new organs or other systems. We can now differentiate induced
human pluripotent stem cells (iPSCs) into a variety of cell types and begin to construct complex
systems with a consistent genotype. Second, recent success in the growth of organ-like structures—
organoids—from embryonic stem (ES) or iPS cells has shown that it is possible to co-differentiate
cells into multiple cell types that begin to show the form, and in some cases, the function, of a real
organ.6 Third, with the help from several new government programs worldwide, these technologies
have been brought to bear on the growing field of tissue- or organ-on-a-chip development.
As we develop the enabling technologies for M-CELS, at least two approaches for fabricat-
ing M-CELS can be envisioned (Fig. 1), here viewed in the context of a simple muscle-actuated
sphincter or pump that requires a vessel lumen for flow, muscle to contract or collapse the vessel
locally, and neural control, possibly by the use of optogenetically modified cells and activation
by light. Both approaches begin with a concept and detailed mapping of the various cell types
needed to produce the end-product. In one approach, differentiated pluripotent or primary cells
are seeded or plated in a specified spatial pattern in two or three-dimensional constructs to pro-
duce the elements of the system. These may include, for example, an endothelial monolayer, a
pancreatic islet, a muscle strip, or a cluster of neurons. These elements are then arranged within
a device or substrate in such a way that they interact with each other in a desired manner. We
liken this approach to current “top-down” engineering design and assembly of complex systems
FIG. 1. (a) Pathways to building a M-CELS. (b) Key steps to achieve the two distinct pathways. The wavy line in (a) shows
that integration across the two pathways could also be possible.
040901-4 Kamm et al. APL Bioeng. 2, 040901 (2018)
from simpler components, all according to a master plan for the complete, assembled M-CELS.
In contrast, one could fabricate a M-CELS beginning with a disordered collection of pluripotent
cells that are subjected to a variety of guidance cues—chemical, mechanical, electrical, and
genetic—either globally or locally, which induce the cells causing them to co-differentiate into
multiple cell types and self-organize into a new multi-cellular system. We define this to be an
“emergent engineering” approach. Figure 1 shows these two pathways and the potential for inter-
actions or cross-over between them.
At this early stage in the development of M-CELS technologies, we recognize the need to
look ahead and consider the eventual scientific, commercial, and ethical impacts of our
approaches and actions. The purpose of this paper is to provide a review of recent accomplish-
ments, assess current state-of-the-art in M-CELS, and point to future needs and challenges. We
also seek to foster a wider conversation of these issues, initiated in a Workshop on Engineered
Living Systems, held in Chicago, IL, in August 2016 and continued in August 2018.
either during or after initial assembly, representing an important design challenge for a syn-
thetic morphology.
A key issue for the future of M-CELS and regenerative medicine is to find the appropriate
paradigm with which to enable direct modification of the shape for engineering and biomedical
applications. Currently, the assembly of correctly shaped and patterned tissues is limited by our
ability to manage their construction at a micro-level (e.g., cell type, cell position, and scaffold
used)—a major barrier to progress in the repair of complex organs such as hands and eyes.
These constructs are difficult to assemble directly but are routinely regenerated in vivo by some
model species. Alternatively, the self-assembly of organoids leverages endogenous patterning
cascades but comes at the cost of losing control over the final shape and pattern of the M-
CELS. Ultimately, a solution lies between these extremes, as a kind of guided self-assembly
that provides judicious tweaks to an otherwise self-directed endogenous cascade of morphogen-
esis,27,28 as further discussed in Sec. III. Advances in the field will depend on finding the mini-
mal amount of input required for a system to progress towards a desired complex outcome.
Promising approaches include activating master regulators of developmental programs (e.g.,
“build an eye here”) and learning to modulate biochemical and mechanical signals that trigger
pattern formation above the cell level (e.g., organ size and spatial relationship between organs).
Ever finer-scale reductionist analyses of the mechanisms of signaling at the cellular and sub-
cellular levels, which are progressing at a very rapid pace, are complemented by theoretical and
experimental approaches that quantitatively investigate the top-down, computational aspects of
pattern regulation employed in tissue shape generation and maintenance.29–31
It is essential to understand and exploit the inherent processes that direct patterning of com-
plex structures.32 Cells and tissues need to make many decisions about what to build, where,
FIG. 2. Methods for controlling emergence in M-CELS. Following the design specifications (left), a collection of proce-
dures can be applied in a spatiotemporal manner to induce differentiation and organization of the cells (middle) to produce
the desired form and function (right).
Given the degree of plasticity revealed by bioelectric controls of growth and form, advances in
this field will be an invaluable aid in the top-down programming of the shape in bioengineering
applications.
Genetic engineering of cells allows us “from the inside” to modify and extend the biologi-
cal programs that underlie cellular behavior, through the design and implementation of synthetic
gene regulatory networks.52 With the advent of the Cas9/CRISPR system,53 it is now possible
to edit the genome and dynamically regulate specific genes. Synthetic biology has evolved from
demonstrating a simple gene circuit in prokaryotes54,55 to large multi-input circuits in prokar-
yotes56 and eukaryotes.57 These circuits usually comprise genetically encoded sensors that
detect levels of intracellular and extracellular biomarkers, a computational core that processes
sensory information and makes decisions about which specific actions to take, and actuators
that affect the cell state and the surrounding environment. Initial emphasis in synthetic biology
focused on the creation of gene circuits that operate orthogonally to the cell, attempting to min-
imize bidirectional interactions and dependencies between the circuit and the host. In contrast,
genetic circuits fashioned for M-CELS must at their core be conceptually and practically
embedded within the host and tissue, responding to dynamically changing cellular and extracel-
lular conditions and controlling the cellular milieu towards desired phenotypes.
Unlike many traditional approaches in engineering, M-CELS must develop the capability
to cope with or exploit the heterogeneity of cell types, states, environmental conditions, and
fluctuations in gene expression (or “noise”). Engineering noise of M-CELS will require address-
ing noise at multiple-scales throughout development. Predictive modeling and advanced tools to
modulate the noise of genetic and regulatory circuits within the cell will be needed.58–61
Addressing these issues within an ensemble includes accounting for the coupling of fluctuations
IV. ORGANOIDS
Several recent scientific advances have helped seed the nascent field of M-CELS, but none
more so than those in organoid culture systems. Organoid, in this context, refers to an aggregate
of multiple organ-specific cell types structured similarly to the in vivo organ and sharing its key
functions. Research in organoids, built on work that started in the early 1900s,66 is now
040901-8 Kamm et al. APL Bioeng. 2, 040901 (2018)
emergence, but more computational methods need to be developed in order to identify and pre-
dict desired features of organoid development, especially if large industrial-scale manufacturing
of these systems is needed for the next generation of drug development.
V. ORGAN-ON-CHIP MODELS
Building on the success of microfluidic technologies and iPS and stem cell engineering and
driven by the widely recognized need for transformative changes in the process of drug discov-
ery and development, a variety of new assays have been advanced which enable certain aspects
of the organ function to be replicated with in vitro models. While the “organ-on-chip” (OoC)
technologies are still at a relatively early stage in development, nascent versions of cardiac mus-
cle,88,89 liver,1,90 brain,91–93 lung,94 skin,95 placenta,96 and various other tissues have been
reported (Table II). Similar systems have been created for the purpose of modeling and gaining
new insights into fundamental disease processes such as cancer97 and Alzheimer’s disease.98
Given the increasing availability and reduced cost of generating iPS cells, the prospect of devel-
oping patient specific assays to screen for optimal personalized therapies is on the horizon.
Practical challenges include phenotypic instability, low throughput associated with system
complexity, material-drug incompatibilities of commonly used device materials such as PDMS,
and biomaterial inconsistencies and limitations. A fundamental question for OoC technology is,
will we be able to create microscale constructs that adequately recapitulate the macroscopic
organs? There is a danger, even if we construct OoC using human cells with sufficiently mature
phenotypes, that the resulting system will have the physiology more reminiscent of a small rodent
rather than the humans from whom the cells were originally derived. Two major scaling issues
TABLE II. Example organ-on-chip designs of different physiological functional units. A wide range of organ mimics have
been reported which recapitulate the basic functionality of that organ.
and even faithfully constructed minimal functional tissue units would metabolize at mouse-like
rates rather than human-like rates. While there are potential solutions such as limiting oxygen
availability,105 effective implementation in OoC systems is still a challenge.
Even if one were to achieve human-like physiological parameters for each organ module,
another challenge arises when connecting multiple OoCs together with a common medium that
might be circulated by our muscle-actuated pump example. If we shrink all linear dimensions
proportionally, the multiple OoC system will not reflect human proportions in terms of function.
For example, if a lung is isometrically miniaturized by a factor of 100, the surface area avail-
able for gas exchange falls nearly 10 000-fold. The same change in linear dimension for muscle
would reduce oxygen consumption rates by a factor of 106. Some potential solutions include
“functional scaling” of organs where miniaturization factors would be assigned to different
organs based on whether the organ function depends more on its surface area or its vol-
ume.104,106 While these and other potential solutions have been proposed,107–109 effective
implementation and validation in OoC systems remain a significant challenge.
While the long-term goal may be the construction of an OoC that reproduces all aspects of
human physiology, the near-term opportunity may be in fit-for-purpose OoCs that test for specific
aspects of efficacy or toxicity. Such efforts will require close collaboration with target end-users
to develop OoCs with “just enough complexity” to provide a valuable physiologic readout while
also allowing for a robust and high throughput assay. The systems will also have to be extensively
validated against existing gold-standard animal models using a large panel of reference chemicals.
A helpful resource with various examples of validating non-animal technologies is The European
Union Reference Laboratory for Alternative to Animal Testing (eurl-ecvam.jrc.ec.europa.eu).
Finally, the continuing difficulties in predicting adverse immune responses and the emergence of
small scale113 and phenotypic assessment of neuromuscular disease.114 A variety of muscle sys-
tems have been explored to achieve multi-dimensional and complex motion and deformation
from muscle contractions on 2D and 3D substrates.115 These engineered systems serve as a test
bed for studying muscle force, their temporal dynamics, longevity, and overall performance in
contrast to their in vivo counterparts, with the ultimate goal of providing muscle repair, analysis
of muscle disease, and evaluation of drug efficacy. More interestingly, it is now possible to use
human iPSCs to form patient specific self-organized muscle strips to develop disease models
for prognosis, to interrogate disease mechanisms, and to study drug effects116 for individualized
medicine. Recognizing that muscles are often activated by neurons for both voluntary and
involuntary motions, recent work with M-CELS also involves muscles innervated by neurons,
forming functional neuromuscular junctions.102
The force and deformation attributes of engineered muscle systems have recently been
employed to generate locomotion of small structures. They include swimming in fluids mimick-
ing flagella dynamics at small scale (low Reynolds number),117 coordinated flapping dynamics
at larger scale (high Reynolds number),118–120 and walking using leg-like structures.115,121–124
These small robots move autonomously with cardiomyocytes as actuators, or they are stimu-
lated to move by light or an electric field when optogenetic or regular skeletal muscle cells are
used to actuate them. Through these elementary systems, we are learning to imitate different
capabilities of natural organisms, including locomotion and active transport, which can lead to
new applications.125 These include future in vivo applications in drug delivery and micro-
surgery. However, major challenges need to be overcome, including the issues with biocompati-
bility, imaging and control of trajectories, viability, and their removal after the desired function.
In the near term, they serve as test beds for studying emergent properties of biohybrid robots
organismal) and the need to integrate different control modalities. In biology, as in other areas,
the system function is tightly interwoven with the structure. The structure/function relationship
manifests itself at different scales of living systems, and it is also impacted by the numerous
ways in which cellular systems exert control over themselves and their surroundings. In the
efforts to engineer living systems, we have at our disposal a large set of proven methods for
manipulating biological behavior. It can be modulated “from the outside” via micro and macro-
scale methods described in Sec. III.
To fully realize the potential of M-CELS, these control methods can serve as composable
functions. These functions should have inputs and outputs and perform some transformation on
the living systems. The functions operate in space and time and at defined scales. While they
always perform operations physically, their impact can be both physical and regulatory/informa-
tion-processing. Functions have specific energy usage, operate at different modalities, and may
require coordination among multiple cells and time scales. While the concept of a function is
relatively straightforward, implementing a design framework that effectively integrates different
modes of control modalities as discussed in Sec. III remains a challenge. 3D multicellular simu-
lation tools such as Morpheus127 and others87,128 integrate gene regulation, cell signaling, and
biomechanics and may serve as a useful basis for M-CELS design tools. However, missing are
effective abstractions that support M-CELS 3D organ and multicellular machine design objec-
tives, akin to gene circuit design tools.129–132
operation. Synthetic biology at the single-cell level has had many successes,56,57 but it has also
had to accept that progress has been slower than once hoped.135 One of the many challenges is
the non-intuitive behavior of molecular circuits: complex topologies, non-linear relationships,
and feedback loops often make the dynamics of a circuit impossible to predict without the aid
of computer simulations—these unexpected behaviors may be considered as examples of the
emergence, discussed above. Much of this work still revolves around trying to understand the
dynamics of simple circuits that are already constructed rather than trying to design de novo cir-
cuits, but this ambitious challenge of engineering new circuits is also gaining ground.34,130–132
Switching from single-celled circuits to multicellular systems brings a level of complexity
for computer modeling. Feedback loops now exist at more scales than the purely molecular.
Although the active dynamics of cells are known to be largely controlled by their molecular
states, it is now equally clear that macroscopic events directly feed back to control molecular
events. For example, tissue growth may push a group of morphogen-secreting cells away from
their target cells, thus changing their expression profiles as a direct consequence. If the behavior
of feedback loops in single-celled gene circuits is hard to predict, the complexity of these
multi-cellular and multi-scale feedbacks is dramatically more challenging. That is, the degree of
complex and subtle emergence is even higher than for molecular circuits alone. Understanding
and predicting them will not be possible without good computer models, so a clear expectation
for the future is that multi-scale numerical simulations must become a key goal for M-CELS.
A wide variety of modeling formalisms exist to tackle multi-cellular systems. At one end
of the spectrum are continuum approaches such as Finite Element Modeling (FEM), which
approximate a tissue as a continuous material, and they are optimal for questions involving
physical mechanics.136 At the other end of the spectrum, Cellular Potts Models (CPMs) employ
TABLE III. New technologies needed for the development and manufacture of M-CELS.
Imaging High resolution, high content imaging of large, multi-cellular structures, label-free methods,
and 4D imaging
Computational analysis Multi-scale modeling, agent-based methods, and data-driven modeling
Bioprinting Simultaneous printing of the matrix and multiple cell types with single cell resolution
Scaffold design Artificial and natural biomaterials with controllable chemistry and mechanical stiffness
Microfluidics Systems that facilitate spatiotemporal control of micro-environmental properties and co-
differentiation processes
Biofabrication Providing appropriate cell-matrix stimuli for organoid growth and stability and new
manufacturing methods that leverage intrinsic self-assembly
Optogenetics To facilitate the capability for spatiotemporal patterning of the function in growing M-CELS
Robotics Methods to handle high-volume production of organoids and other M-CELS for industrial
applications
multi-scale nature and we hope to control dynamic macroscopic processes through “molecular
programs,” it will probably take a significant phase of further basic research before computer
modeling aids us in “forward” rational design of these systems. Indeed, for the foreseeable
future, computer modeling research will probably focus on obtaining satisfactory models of
existing multicellular systems, and this will largely depend on “reverse-engineering” from large
quantitative datasets. Organoids may prove a particularly tractable system to work on in the
IX. BIOMANUFACTURING
The biomanufacture of M-CELS poses significant challenges to existing bioprocesses.148
M-CELS present unique needs based on their inherent complexity and reliance on emergent
behavior. The overarching need—developing effective methods to reliably and robustly produce
M-CELS at a desirable scale—highlights a number of challenges and opportunities.
The development of M-CELS relies upon the integrated formation of cellular structures.
Such structures can be achieved through either self- or guided-assembly of multiple cell types
into functional units (see Sec. I). As we consider methods for manufacturing these systems, it
will be important to understand whether such complexity can be reduced or segmented in a
modular fashion to create M-CELS subunits, followed by assembly into desired M-CELS in a
controllable or bio-foundry manner. It is expected that efforts to better understand the phenom-
ena associated with emergence will help elucidate where such processes may be simplified or
deconstructed and where they cannot. The goal of such efforts is to enable the creation of a
“bio-assembly line” to achieve industrial scale production of desired M-CELS.
Biofabrication of subunits and their further assembly into M-CELS requires manufacturing
technologies across multiple disciplines (Table I) and technologies (Table III). Simple 2D and
3D cell culture methods can be augmented or replaced by newer technologies such as 3D bio-
printing,149–153 and automated bioreactor systems are key to enabling the generation of
compartmentalized M-CELS structures in an organized manner (Fig. 3). The continuing devel-
opment of effective 3D bioprinting technologies may enable the effective implementation of
this approach in M-CELS biomanufacture. Although the resolution that is currently achievable
with 3D bioprinting may not yet permit direct implementation on the length scales (10’s of
microns) required for M-CELS formation, they can help, at least, spatially position cells in a
manner that will facilitate further self-assembly and may prove to be effective tools for auto-
mating and standardizing key steps in M-CELS biomanufacturing.
Technologies that enable both scale up—relying on novel culture platforms with
increased capacity (e.g., bioreactors) and scale out—increasing the number of culture systems
040901-15 Kamm et al. APL Bioeng. 2, 040901 (2018)
FIG. 3. Manufacture of M-CELS. (a) Design and selection of cellular and substrate components. (b) Product manufacture
requiring a variety of manufacturing methods, many being unique to biological systems. (c) The manufactured product. (d)
supply-chains that are dependent upon reliable preservation. Cryo- or other preservation tech-
nologies of M-CELS pre-products or sub-assemblies will streamline and simplify the
manufacturing process. The ability to preserve completed M-CELS could enable building
“finished-goods” inventories and shipping to end-users.
X. ETHICAL CONSIDERATIONS
A fundamental question for researchers working in synthetic biology, emergent behavior,
and living systems is whether they are “creating life.” Some researchers in synthetic biology
believe microorganisms are “just” machines, and that the creation of new such machines
shouldn’t be considered differently.149 Others may argue that researchers need to be cognizant
of the degree to which projects might be perceived as creating life and what special obligations
might exist in this creative process. The language of “creating life” raises fundamental ques-
tions, including religious issues, for some observers who ask whether there should be natural
limits beyond which we should not be trespassing. While many of these issues have been raised
in the context of synthetic biology, they grow in importance when, for example, we become
able to produce a functioning brain organoid that can collect, process, and act based on infor-
mation gained from the environment. In addition, the concept of pain and sentience150,151 would
need to be addressed in the development of M-CELS that could be perceived as replication of
living entities. As many of the future M-CELS could by pass the natural developmental pro-
cess, the use of the 14 day rule150,152 employed to provide an ethical framework around embryo
research would have to be modified.153 We are also cognizant that some communities, in vari-
ous parts of the world, may be more sensitive to these concerns than others.
both accountability and responsibility. Collaboration between researchers would provide a sys-
tem for checks and balances, with sharing of information between labs, while acknowledging
the tension such sharing would have with pathways for preserving research/publication rights.
M-CELS researchers, in close consultation with bioethicists, should develop a shared ethics
framework and consider creation of a shared governance structure.
It is unclear to what degree researchers and trainees who work on M-CELS are considering
or fostering discussion of these questions surrounding “creating” or “re-creating life” or control-
ling emergent behavior in their daily work. Those involved in research may benefit from an
intentional structure for identifying underlying, competing values and ethical principles in their
work and providing a forum for discussion, especially for trainees, about how scientific values
and personal values or beliefs coincide. The development of such a frame work and an ethics
code of conduct for M-CELS research is an imperative task for the research community to
develop. Being able to articulate the ethics underlying one’s work is so important in communi-
cating within the scientific community, with funders and policy makers and with the public at
large.
ACKNOWLEDGMENTS
We are grateful for support for the Workshop in Engineered Living Systems provided by the
NSF, UIUC, and MIT. This framework for research in this field at MIT, UIUC, and Georgia Tech
was funded by a National Science Foundation (NSF) Science and Technology Center on Emergent
Behavior of Integrated Cellular Systems (EBICS) (Grant No. CBET0939511), with additional
support from the NIH/NCI (U01 CA202177). We also wish to thank Janet Sinn-Hanlon,
DesignGroup@VetMed, University of Illinois, for creating the illustrations and Dr. Umer Hassan at
UIUC for reviewing this manuscript. M.L. gratefully acknowledges the support via the Allen
Discovery Center award from The Paul G. Allen Frontiers Group (12171) and the Templeton World
Charity Foundation (TWCF0089/AB55).
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