Twenty people die every day waiting for an organ transplant in the United States, and while more than 30,000 transplants are now performed annually, there are over 113,000 patients currently on organ waitlists.
Artificially grown human organs are seen by many as the “holy grail” for resolving this organ shortage, and advances in 3D printing have led to a boom in using that technique to build living tissue constructs in the shape of human organs.
However, all 3D-printed human tissues to date lack the cellular density and organ-level functions required for them to be used in organ repair and replacement.
Living embryoid bodies surround a hollow vascular channel printed using the SWIFT method. Credit: Wyss Institute at Harvard University
Now, a new technique called SWIFT (sacrificial writing into functional tissue) created by researchers from Harvard’s Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Sciences (SEAS), overcomes that major hurdle by 3D printing vascular channels into living matrices composed of stem-cell-derived organ building blocks (OBBs), yielding viable, organ-specific tissues with high cell density and function. The research is reported in Science Advances.
“This is an entirely new paradigm for tissue fabrication,” said co-first author Mark Skylar-Scott, Ph.D., a Research Associate at the Wyss Institute. “Rather than trying to 3D-print an entire organ’s worth of cells, SWIFT focuses on only printing the vessels necessary to support a living tissue construct that contains large quantities of OBBs, which may ultimately be used therapeutically to repair and replace human organs with lab-grown versions containing patients’ own cells.”
SWIFT involves a two-step process that begins with forming hundreds of thousands of stem-cell-derived aggregates into a dense, living matrix of OBBs that contains about 200 million cells per milliliter. Next, a vascular network through which oxygen and other nutrients can be delivered to the cells is embedded within the matrix by writing and removing a sacrificial ink. “Forming a dense matrix from these OBBs kills two birds with one stone: not only does it achieve a high cellular density akin to that of human organs, but the matrix’s viscosity also enables printing of a pervasive network of perfusable channels within it to mimic the blood vessels that support human organs,” said co-first author Sébastien Uzel, Ph.D., a Research Associate at the Wyss Institute and SEAS.
The cellular aggregates used in the SWIFT method are derived from adult induced pluripotent stem cells, which are mixed with a tailored extracellular matrix (ECM) solution to make a living matrix that is compacted via centrifugation. At cold temperatures (0-4 °C), the dense matrix has the consistency of mayonnaise – soft enough to manipulate without damaging the cells, but thick enough to hold its shape – making it the perfect medium for sacrificial 3D printing. In this technique, a thin nozzle moves through this matrix depositing a strand of gelatin “ink” that pushes cells out of the way without damaging them.
When the cold matrix is heated to 37 °C, it stiffens to become more solid (like an omelet being cooked) while the gelatin ink melts and can be washed out, leaving behind a network of channels embedded within the tissue construct that can be perfused with oxygenated media to nourish the cells. The researchers were able to vary the diameter of the channels from 400 micrometers to 1 millimeter, and seamlessly connected them to form branching vascular networks within the tissues.
Organ-specific tissues that were printed with embedded vascular channels using SWIFT and perfused in this manner remained viable, while tissues grown without these channels experienced cell death in their cores within 12 hours. To see whether the tissues displayed organ-specific functions, the team printed, evacuated, and perfused a branching channel architecture into a matrix consisting of heart-derived cells and flowed media through the channels for over a week. During that time, the cardiac OBBs fused together to form a more solid cardiac tissue whose contractions became more synchronous and over 20 times stronger, mimicking key features of a human heart.
“Our SWIFT biomanufacturing method is highly effective at creating organ-specific tissues at scale from OBBs ranging from aggregates of primary cells to stem-cell-derived organoids,” said corresponding author Jennifer Lewis, Sc.D., who is a Core Faculty Member at the Wyss Institute as well as the Hansjörg Wyss Professor of Biologically Inspired Engineering at SEAS. “By integrating recent advances from stem-cell researchers with the bioprinting methods developed by my lab, we believe SWIFT will greatly advance the field of organ engineering around the world.”
Collaborations are underway with Wyss Institute faculty members Chris Chen, M.D., Ph.D. at Boston University and Sangeeta Bhatia, M.D., Ph.D., at MIT to implant these tissues into animal models and explore their host integration, as part of the 3D Organ Engineering Initiative co-led by Lewis and Chris Chen.
“The ability to support living human tissues with vascular channels is a huge step toward the goal of creating functional human organs outside of the body,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS, the Vascular Biology Program at Boston Children’s Hospital, and Professor of Bioengineering at SEAS. “We continue to be impressed by the achievements in Jennifer’s lab including this research, which ultimately has the potential to dramatically improve both organ engineering and the lifespans of patients whose own organs are failing,”
Additional authors of the paper include John Ahrens, a current graduate student at the Wyss Institute at Harvard University and Harvard SEAS, as well as former Wyss Institute and Harvard SEAS members Lucy Nam, Ryan Truby, Ph.D., and Sarita Damaraju. This research was supported by the Office of Naval Research Vannevar Bush Faculty Fellowship, the National Institutes of Health, GETTYLAB, and the Wyss Institute for Biologically Inspired Engineering at Harvard University.
The ability to construct whole organs for therapeutic use remains a daunting challenge, requiring billions of cells to be rapidly organized into functional microarchitected units that are supplied with nutrients via pervasive vascular channels (1). Without a readily perfusable circulatory network, engineered human tissues are limited to several hundred micrometers in thickness (2–10).
This constraint arises due to the delay between implantation and anastomosis with host vasculature, which necessitates a reliance on the diffusive transport of oxygen and nutrients to maintain cell viability (2, 8, 9).
Although avascular tissue grafts may provide a measurable improvement in organ function upon implantation (3, 8, 9), the de novo biomanufacturing of three-dimensional (3D) grafts and, ultimately, full-scale organs will inevitably require a perfusable vascular network. While 3D vascularized tissues (~1 cm thick) have recently been fabricated via multimaterial 3D bioprinting (11, 12) and stereolithography (13), they lack the requisite cellular density and microstructural complexity needed to achieve physiologically relevant levels of function.
Engineered tissues composed of individual cells suspended in extracellular matrices (ECMs), i.e., so-called cells in gels, typically contain at least one to two orders of magnitude lower cell density than those observed in vivo (1, 14).
Recent advances in the self-assembly of human embryonic stem cells and induced pluripotent stem cells (iPSCs) have led to the development of organoids that have several characteristics akin in their in vivo organ counterparts (15–17).
Most organoid protocols [e.g., cerebral (16), kidney (17–19), and cardiac (20) organoids] begin by generating embryoid bodies (EBs) composed of many iPSCs placed into microwells and cultured under static conditions, whose differentiation can be directed into “mini organs” of interest.
We posit that these organoids may serve as ideal organ building blocks (OBBs) for biomanufacturing patient- and organ-specific tissues with the desired cellular density, composition, microarchitecture, and function provided that a perfusable network of vascular channels can be introduced within these living matrices.
Embedded 3D printing offers one viable strategy for achieving this goal. Lewis et al. first demonstrated this method by writing a viscoelastic, sacrificial (or fugitive) ink within acellular hydrogel (21) and silicone (22) matrices.
After printing, these matrices were cured, and the sacrificial ink was removed, leaving behind a 3D network of interconnected channels. Building on this advance, other researchers developed synthetic (23) and biopolymer (24, 25) matrices that exhibit a self-healing, viscoplastic response that simplifies the patterning of complex 3D architectures. However, to date, these methods have only been used to construct acellular (23, 24) or sparsely cellular (25) matrices.
Here, we report a biomanufacturing method that relies on sacrificial writing into functional tissue (SWIFT) composed of a living OBB matrix to generate organ-specific tissues with high cell density, maturation, and desired functionality (Fig. 1).
First, we create hundreds of thousands of iPSC-derived OBBs in the form of EBs, organoids, or multicellular spheroids. Next, these OBBs are placed into a mold and compacted via centrifugation to form a living OBB matrix. We then rapidly pattern a sacrificial ink within this matrix via embedded 3D printing, which upon removal yields perfusable channels in the form of single or branching conduits. Last, we demonstrate that these bulk vascularized tissues function and mature when perfused over long durations.