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Many researchers are working on growing human organs in a lab with 3D printing due to the lack of availability of organs for transplant. There has been much success in 3D printing heart valves and single parts of organs. The new technique that has been created is called Sacrificial Writing into Functional Tissue (SWIFT), which prints vessels within a pre-existing live cell matrix. This innovation is the first that gives these organs viability and function by providing a vascular network for oxygen delivery. This method has created cardiac tissue that beats over a seven-day period. Ultimately, this innovation will lead to engineering a fully functional 3D printed human heart.

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September 6, 2019

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This video explains Sacrificial Writing into Functional Tissue (SWIFT).

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20 people die waiting for an organ transplant every day in the US, but lab-grown organs so far lack the cellular density and functions required to make them viable replacements. The new SWIFT method from the Wyss Institute and Harvard SEAS solves those problems by 3D printing vascular channel networks directly into living tissue constructs, enabling the creation of larger tissues approximating the size and function of organs.

Credit: Wyss Institute at Harvard University

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This is a photo of a hollow vascular channel printed using the SWIFT method.

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This is a mini-heart that kept beating for 7 days based off this new innovation.

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Harvard University Wyss Institute for Biologically Inspired Engineering and Harvard University and John A. Paulson School of Engineering and Applied Sciences (SEAS) Researchers: Mark A. Skylar- Scott, Sebastien G. M. Uzel, Lucy L. Nam, John H. Ahrens, Ryan L. Truby, Sarita Damaraju and Jennifer A. Lewis

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1. SWIFT focuses on single vessels rather than entire organs

2. Cost/ time/ resources

3. Longevity- 7-day period currently

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I believe this is a fantastic advancement in the world of 3D printing of organs. I have worked as a Cardiacthoracic and Transplant ICU nurse for the past 3 years. I have seen many patients receive heart transplants, I have seen many patients wait for months, and I’ve even seen many die while waiting for transplant. This further innovation in 3D printing provides the vascular network needed for a heart to beat and have blood supply ultimately leading to creation in the future of a completely 3D printed heart that beats and pumps just like a human heart. This could change wait times for transplant, how sick patients are going into transplant, and devices we use to support patients pre-transplant may no longer even be needed if hearts and other organs are readily available because of 3D printing. Because of this advancement many lives will be saved.

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Essop, A., Jackson, B., Vialva, T., Lo, A., & Petch, M. (2019, September 9). Harvard researchers develop SWIFT method to 3D print organ building blocks. Retrieved from https://3dprintingindustry.com/news/harvard-researchers-develop-swift-method-to-3d-print-organ-building-blocks-161358

Hastings, C. (2019, September 9). Sacrificial ink writing technique for 3D printed organs. Retrieved from https://www.medgadget.com/2019/09/sacrificial-ink-writing-technique-for-3d-printed-organs.html

Skylar-Scott, M. A., Uzel, S. G. M., Nam, L. L., Ahrens, J. H., Truby, R. L., Damaraju, S., & Lewis, J. A. (2019). Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels. Science Advances, 5(9). doi: 10.1126/sciadv.aaw2459

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1. Advances the field of organ engineering

2. Provides 3D organs with vascular network/effective oxygen delivery

3. Possible solution to lack of organ availability for transplant

4. Reduces transplant wait time

5. If patients own stem cells used to create organ this could decrease chances of rejection or even possibly not having to take anti-rejection medications

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iPSC culture

Personal Genome Project 1 (PGP1) iPSCs or, for all EB-derived cardiac OBB protocols, BJFF iPSCs (provided by S. Jain at Washington University) were cultured on Matrigel-coated plates (Corning Life Sciences) and maintained in mTeSR1 medium (STEMCELL Technologies). Once colonies began to merge (60 to 80% confluency), cells were rinsed with phosphate-buffered saline (PBS) without calcium or magnesium and passaged by adding an enzyme-free passaging solution (ReLeSR, STEMCELL Technologies) at 1 ml/10 cm ² of tissue culture area. The ReLeSR was immediately aspirated away, and the flask was incubated for 7 min at 37°C, 5% CO ₂. To lift off colonies, fresh mTeSR1 (1 ml/10 cm ²) was gently added to the flask using a serological pipette and slowly pipetted back up once to break up the colonies. The cells were then added to a freshly prepared Matrigel-coated flask, performing a split between 1:8 and 1:3, and mTeSR1 was added to a total volume of 2 ml/10 cm ² of tissue culture area. Cells were maintained at 37°C/5% CO ₂ in an incubator.

Microwell array fabrication

Microwell array plates were used to generate large numbers (>10 ⁵) of uniform EBs via forced aggregation. The microwells, in the form of inverted pyramids with 400-μm base width, were arranged in a six-well plate format and manufactured via polydimethylsiloxane (PDMS) molding. The following process was used to facilitate demolding and ensure casting compatibility between polymers. Briefly, SYLGARD 184 (Dow Corning) was mixed 10:1 with its cross-linker and poured into a commercially available microwell plate, degassed in a vacuum chamber, and cured at 80°C for 2 hours to generate a negative cast of the microwells. Next, Ecoflex 00-30 (Smooth-On Inc.) was used to create a positive mold by following a similar method of pouring, degassing, and curing as for the SYLGARD 184. After removing the cured Ecoflex, a two-part polyurethane (Smooth-On Inc.) was poured onto the Ecoflex positive to create a polyurethane-negative mold. Next, ~80 g of SYLGARD 184 was mixed with 8 g of its cross-linker in a planetary mixer, poured into a polyurethane negative mold, degassed for 10 min in a vacuum, and allowed to cure at room temperature for 24 to 48 hours. The cured SYLGARD 184 was then placed in the oven at 80°C for >2 hours to ensure complete cross-linking before removal from the polyurethane mold.

EB formation

To prepare the microwell arrays for cell culture, the arrays were immersed in isopropyl alcohol for 1 hour and then immersed in water and autoclaved for 1 hour. Immediately before preparing the cells, the autoclaved water was drained from the wells, and then, they were placed into a sterile one-well tray (Omnitray, Falcon). Next, 1.5 ml of AggreWell Rinsing Solution (ARS; STEMCELL Technologies) was added per well before centrifuging the wells at 2000 g for 5 min to remove trapped air bubbles from the wells. The ARS was aspirated, and the wells were rinsed twice with 2 ml of Dulbecco's modified Eagle's medium (DMEM). Last, the DMEM was replaced with 3 ml of EB culture medium (EBCM) supplemented with 10 μM ROCK inhibitor (ROCKi; Y27635; EMD Millipore), and the microwell arrays were maintained at room temperature. To prepare EBCM, mTeSR1 was mixed 1:50 with a polyvinyl alcohol (PVA) stock solution to generate a solution of 4 mg/ml. Penicillin-streptomycin was added to 100 U/ml, and the solution was sterile-filtered and stored at 4°C for up to 2 weeks. The PVA stock solution was prepared by adding 20 g of PVA (30 to 70 kDa; Sigma-Aldrich) to 80 ml of deionized water while stirring at room temperature. Under continuous stirring, the solution temperature was increased to 80°C to fully dissolve PVA in water, after which the PVA solution was stored at 4°C until ready for use.

To form EBs, PGP1 iPSCs were grown to 60 to 80% confluency in Matrigel-coated 225-cm ² T225 flasks (Falcon). One T225 of PGP1 cells was typically used to seed 24 wells (four six-well plates), each containing ~4200 microwells. To lift off the iPSCs from the substrate, the cells were first rinsed in PBS without calcium or magnesium and incubated in Accutase (Innovative Cell Technologies) for 15 min at 37°C to generate a mostly single-cell suspension. The cells were added to prewarmed DMEM/F12 and Hepes and centrifuged at 220 g for 5 min. The supernatant was removed, and the cells were resuspended in EBCM and10 μM ROCKi. Cells were counted using a LIVE/DEAD imaging system, and 1 ml of the cell suspension is seeded into each well at a cell density of approximately 500 cells per microwell. The well plates were then centrifuged at 100 g for 3 min to compact the cells. After 24 hours, the cell media was replaced with 4 ml of fresh EBCM to remove the ROCKi. The well plates then underwent two half-media changes (2 ml of EBCM is replaced with fresh media) per day. Care was taken during media changes with media added and removed slowly as to not disturb the EBs from their individual microwells. After 4 days of culture, a total of eight six-well microwell array plates could generate a sufficient volume of EBs to render ~1 ml of OBB slurry.

Cerebral organoids

To generate cerebral organoids, PGP1 iPSCs were grown to ~80% confluency and, on day 0, were lifted from Matrigel-coated T225 flasks by incubation with gentle cell dissociation reagent for 12 min. The flasks were rinsed with DMEM/F12 containing Hepes and centrifuged for 5 min at 220 g. Next, the cells were resuspended in EBCM and 10 μM ROCKi and seeded into microwell arrays, prepared as described above, at a density of approximately 500 cells per microwell, and compacted by centrifugation at 100 g for 3 min. The media was replaced on day 1 with fresh EBCM without ROCKi. On day 2, the EBs were measured to be approximately 200 μm in diameter, and the media was replaced with neural induction medium (NIM) composed of DMEM/F12 with GlutaMAX, 1:1000 of heparin (10 mg/ml; Sigma-Aldrich), 1:100 N-2 supplement (Thermo Fisher Scientific), and 1× minimum essential medium nonessential amino acids (MEM-NEAA) (Thermo Fisher Scientific) with 1:2000 of 10 mM SB431542 in dimethyl sulfoxide (DMSO) (SB, EMD Millipore) and 1:2000 of 0.2 mM LDN 193189 in DMSO (LDN, EMD Millipore). Aliquots of SB and LDN were stored at −20°C and added to the other media components immediately before adding NIM to cells. On day 3, the OBBs were removed from the microwells by pipetting up and down with DMEM/F12 containing 15 mM Hepes buffer, and the aggregates were transferred to a 50-ml centrifuge tube. The OBBs were allowed to settle under gravity, the supernatant was aspirated, and then, the OBBs were resuspended in 6 ml of NIM per two microwell arrays that were harvested. Next, they were seeded into untreated T25 flasks, and 6 ml of NIM was mixed with the OBBs per flask. After harvesting, an additional 5 ml of NIM was pipetted into each flask. The flasks were transferred to an orbital shaker (VWR Shaker, 1000 Standard) that was set to 60 rpm and placed in an incubator. The media was replaced on days 5 and 7 by placing the flasks on a custom-built sedimentation rack that held the flask at 45° such that cells settle into a single corner of the T25 flask. Once settled, the supernatant was aspirated and replaced with 6 ml of fresh NIM. On day 8, the medium was replaced with neural differentiation medium (NDM) composed of a 1:1 mix of DMEM/F12 with Neurobasal medium (Thermo Fisher Scientific), 1:200 N-2 supplement (Thermo Fisher Scientific), 1:100 B27 without vitamin A (Thermo Fisher Scientific), 1:4000 insulin solution (Sigma-Aldrich), 1:100 GlutaMAX, and 1:200 MEM-NEAA. A 1000× solution of β-mercaptoethanol (1:1000; Thermo Fisher Scientific) was added to NDM immediately before adding the media to the OBBs. Thereafter, the media was replaced every other day, and the cerebral organoids were harvested for SWIFT vascularization on day 21.

Cardiac spheroids and EB-derived aggregates

To create the cardiac spheroids used for Fig. 1, D and E and fig. S2, we adapted a previous protocol by Lian et al. (). Briefly, PGP1 iPSCs were grown to 80 to 95% confluency on T225 plates in mTeSR1 medium. Cardiac differentiation media was prepared by combining 500 ml of RPMI 1640 (Thermo Fisher Scientific) with either 10 ml of B27 without insulin (to form cardiomyocyte differentiation medium, CDM) or 10 ml of B27 (to form cardiomyocyte maturation medium, CMM). On day 0, differentiation was initiated by switching the mTeSR1 media to CDM and 6 μM CHIR99021 for 24 hours. The following media changes were then made: day 1, CDM; day 3, CDM and 2 μM iWR1 (BioGems; CDMI); days 5 and 7, CDM; day 9 (or by day of beating), CMM; days 11 and 13, RPMI without glucose supplemented with 2% B27 (to purify cardiomyocytes); and day 15, CMM. In a separate T225 flask, primary ventricular normal human cardiac fibroblasts (Lonza) were cultured in stromal cell growth medium (Lonza). Next, cardiomyocytes, at day 17, and fibroblasts were passaged by rinsing with PBS and then incubated with TrypLE for 9 min (for cardiomyocytes) or 5 min (for fibroblasts) at 37°C. Next, the TrypLE was rinsed using 37°C DMEM, and the cells were pelleted into separate 15-ml tubes via centrifugation at 220 g for 5 min. Both cell types were resuspended in cardiac spheroid medium (CSM), comprising DMEM with high glucose (American Type Culture Collection) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/ml). The cells were counted and combined at 70% cardiomyocytes and 30% fibroblasts and then seeded into molded 800-μm-diameter PDMS microwells at a cell density of 2000 cells per microwell. The microwell arrays were centrifuged at 100 g for 3 min to form OBBs from the cells. CSM was half-changed daily, and the cardiac spheroids were observed to be beating by 2 to 4 days after aggregation, at which point they were harvested for SWIFT vascularization.

To demonstrate the versatility of this process, we also generated cardiac OBBs according to a protocol adapted from () and (). BJFF iPSCs were formed into EBs on day −4, as described above. Two T225s (at ~80% confluency) were used to seed four six-well plates of microwells. On day −3, to remove ROCKi, the media was replaced with 4 ml of EBCM. On day −2, EBs from two wells were harvested, combined, and allowed to settle under gravity. The supernatant was aspirated, and the EBs from two wells were resuspended in 12 ml of EBCM and transferred to a single untreated T25 flask. Thus, EBs from four six-well plates (i.e., 24 wells in total) were harvested and placed into 12 T25 flasks. These flasks were incubated on an orbital shaker with a rotational speed of 60 rpm. To maintain and differentiate the cells in the T25 flasks, the following media changes were performed: day −1, 12 ml of EBCM; days 0 and 1, 10 ml of CDM and 5 μM CHIR99021 (BioGems) (CDMC); day 2, 10 ml of CDM; days 3 and 4, 10 ml of CDMI; and day >5, 10 ml of CDM daily. On days 0, 2, and 5, a rinse was performed after aspiration, using 4 ml of the medium that the cells were being changed into, to increase the removal of growth factors or small molecules from the previous medium. After the cells resettled, the rinsing medium was removed before the new culture medium was added. Beating was typically initially observed by day 7, and almost all cardiac OBBs were visibly beating by day 9. SWIFT printing was performed using cardiac OBBs between days 9 and 11, after which the cells were maintained in CMM.

ECM preparation

An ECM prepolymer gel was prepared as follows: A transglutaminase (TG) working solution was first prepared by adding a microbial tissue TG formulation (20 mg/ml; Moo Gloo TI) to cell culture medium (mTeSR1 for EBs, NIM for cerebral organoids) and sterile-filtering the solution via a 0.2-μm syringe filter. For cardiac OBBs, TG was not added to the ECM. Next, the volume ( x milliliters) of high-concentration rat tail collagen I (Corning) required to create a collagen concentration (4 mg/ml) in the final ECM solution (total volume, y milliliters) was calculated. The ECM prepolymer gel solution composed of (0.825 y − 1.135 x) ml of mTeSR1, 0.125 y ml of TG working solution (if applicable), x/10 ml of 10× PBS, y/100 ml of 250 mM CaCl ₂, ~ x/40 ml of 1 N NaOH, x milliliters of high-concentration rat tail collagen type I, and 0.04 y ml of Matrigel was added, in order, on ice. For example, to generate 8 ml of ECM prepolymer gel using a solution of collagen I (8.90 mg/ml), the following was added in order: 2.56 ml of mTeSR1, 1 ml of mTeSR1 containing TG (20 mg/ml), 0.36 ml of 10× PBS, 0.080 ml of CaCl ₂, 0.090 ml of 1 N NaOH, 3.596 ml of collagen I, and 0.320 ml of Matrigel. The volume of NaOH was adjusted, as necessary, to attain a final pH of ~7.4. To prevent premature gelation, the pipette was precooled using ice-cold PBS before pipetting the collagen and Matrigel. After addition and mixing of all components, the gel was centrifuged at 2000 g for 3 min at 2°C to remove air bubbles. The ECM prepolymer gel was stored on ice and used within ~1 hour.

Sacrificial ink preparation

A gelatin stock solution was used to prepare the sacrificial ink for SWIFT. The 15% (w/w) gelatin stock solution was made by slowly adding 45 g of gelatin into 255 ml of PBS without calcium or magnesium at 85°C while stirring. A separate gelatin ink was created for printing tissue molds used in the perfusion chambers following the same protocol temperature, except that a temperature of 70°C was used. In each case, the samples were held at temperature and stirred for 12 hours to fully dissolve the gelatin, after which their pH was adjusted, while stirring, to ~7.5 using 2 N NaOH. Each hot gelatin stock solution was sterile-filtered, aliquotted, and stored at 4°C for up to 6 months.

Cell density measurements

The EBs were harvested from the microwell arrays once they grew 200 to 250 μm in diameter (typically 2 to 4 days in microwell culture) by vigorous pipetting up and down in the wells with a P1000 pipette tip. The EB-containing media was transferred into a 50-ml tube, one tube per six wells. The wells were further rinsed with an excess of DMEM/F12 and Hepes using a 10-ml serological pipette to remove most (>90%) of the EBs. The media, with suspended EBs, was added to 50-ml centrifuge tubes, and the EBs were allowed to settle by incubating on ice for 10 min. All further cell handling was performed on ice to prevent gelation of the ECM. The supernatant was aspirated, and the EBs were collected into a single 50-ml tube. Next, 50 ml of fresh DMEM/F12 and Hepes was added to further wash away single cells. The EBs were then allowed to settle for another 10 min, and then, the supernatant was aspirated, and the EBs were resuspended up to 15 ml using DMEM/F12 and Hepes and transferred into a 15-ml tube and allowed to settle for 10 min. Next, to compact the EBs, the 15-ml tube was centrifuged at 100 g for 3 min at 2°C, the supernatant was aspirated, and the total volume of the EB matrix was estimated. To replace the DMEM/F12 and Hepes in the extracellular space with ECM, the cells were rinsed in the ECM solution at a 1:3 (EB matrix:ECM solution) volume ratio by pipetting with a P1000 pipette and then centrifuged at 100 g, and the supernatant was aspirated. Last, the consolidated EB-ECM solution was resuspended in an equal volume of ECM solution, forming a dense EB-ECM slurry that could be handled and pipetted in subsequent steps. To estimate the cell density of the OBB matrix, the EB-ECM slurry was centrifuged at 150 g for 3 min in an Eppendorf tube, and the OBB matrix volume was estimated visually by pipetting known volumes of water into a second Eppendorf tube to match the height of the pellet in the first tube. Next, the EBs were dissociated into single cells by incubating in Gentle Cell Dissociation Reagent for 15 min and triturating with a P1000 pipette, and the total cell number was counted. The cell density of the OBB matrix was then estimated by the ratio of cell number to OBB matrix volume.

Rheological measurements

Rheological measurements were carried out by first compacting the prepared EB, ECM, and EB-ECM slurries (mean diameter of EBs, ~200 μm) into 3-cm ³ syringes (Nordson EFD) and then extruding them directly onto the rheometer plate of a TA-instruments Discovery HR-3 stress-controlled rheometer. The plate, with a diameter of 25 mm, was precooled to 2°C. Both the plate and disc were coated with sandpaper to prevent wall slip, and the gap height was brought to 1 mm. Any excess slurry was trimmed with a spatula before these measurements. First, oscillatory frequency sweeps were performed from 0.1 to 10 rad/s with an applied shear stress of 0.5 Pa. Next, oscillatory amplitude sweeps were performed from 0.01 to 100 Pa at a frequency of 0.5 Hz. Apparent viscosities were determined by flow sweeps performed from shear rates of 0.01 to 5/s. The EB matrix yield stress was estimated by fitting a Herschel-Bulkley curve to the increasing shear-rate sweep. Next, to measure the gelation of the ECM upon raising the temperature, the storage and loss moduli were probed with an oscillatory shear stress of 5 Pa and a frequency of 0.5 Hz at 2°C before raising the temperature to 37°C and lowering the gap height to 900 μm to account for EB matrix shrinkage after gelation. To measure gelatin ink rheology, the sacrificial ink was prepared in a 0.5-cm ³ glass syringe (Hamilton) and extruded through a 1.5-inch straight metal nozzle with an inner diameter of 250 μm (Nordson EFD). The printed gelatin was collected and transferred onto the rheometer, which was set up as described above.

Silicone mold and perfusion chamber fabrication

Direct ink writing was used to manufacture customized silicone molds for printing and perfusing bulk tissues. First, a printable silicone ink was prepared by combining a 10:1 mass ratio of SE1700 base:curing agent (Dow Corning) and then adding 1% (w/w) black silicone pigment (Silc-Pig, Smooth-On). The components were then mixed using a speed mixer for 3 min at 2000 rpm (THINKY Inc.). Using a spatula, freshly mixed silicone ink was loaded into a 30-cm ³ syringe (Nordson EFD) and centrifuged at >3000 g to remove entrapped air. A tapered nozzle with a 0.41-mm inner diameter tip (Nordson EFD) was attached to the outlet of the syringe, and the syringe was affixed to a custom 3D printer (). Ink was dispensed from the syringe by means of pressure generated by an Ultimus V pressure controller (Nordson EFD) onto an underlying glass substrate. The tissue molds are composed of silicone walls and small channels for tube-insertion sites, i.e., inlets and outlets. After printing, each mold was transferred to 80°C for >2 hours to cure. After curing, fresh silicone ink was extruded onto the top layer of the mold (either manually or using the 3D printer), and a second glass slide was added to the top, such that the silicone gasket was sandwiched between two glass slides. For the mold used in Fig. 3, clear silicone was printed and cured onto the second glass slide before assembly, such that the assembled gasket had a slit in the middle of the top side for nozzle insertion. The mold was transferred to 80°C to cure the silicone. Stainless steel tubes serving as inlets and outlets with an inner diameter of 0.83 mm were inserted into the inlet/outlet channels of the molds used for perfusion, and Versilon silicone tubing, with an inner diameter of ¹/ ₃₂ inches, was fitted to the external ends of the tubes and crimped with a hose clamp to seal the inlets and outlets. For the custom perfusion mold in Fig. 3, the stainless-steel tubes had a small ~1-mm notch cut into one end to facilitate a continuous connection from the printed vascular channels and the tube. Molds were autoclaved and stored at room temperature before use.

Alternately, polycarbonate chambers and front and back plates were machined to produce custom perfusion devices (fig. S7). The use of glass windows allowed for tissue visualization, and watertight seals were formed between the windows and chamber walls via o-rings. Metal tubes serving as fluidic inlets and outlets were press-fit on the side and bottom of the chamber. The perfusion chambers were autoclaved and stored at room temperature before use. Before printing vascular channels inside the OBB-based matrices, an anchoring structure was printed using a stereolithography 3D printer (Perfactory Aureus, Envisiontec. Inc), using E-Shell 300 clear resin, postcured under ultraviolet (UV) illumination, and positioned within the chamber. This structure had a pair of anchoring prongs for embedding inside the tissue, which served to hold the tissue in place during perfusion, while allowing for media perfusion around the outer surfaces of the tissue. Next, a tissue template (which ultimately forms the shape of the tissue) was stereolithographically printed, postcured under UV illumination, plasma-treated, and dipped into a 10% (w/w) aqueous solution of Pluronic F-127 (Sigma) and allowed to air-dry. This Pluronic F127-coated template was inserted into the chamber and was fitted over the anchoring prongs. Next, 5% (w/v) molten gelatin, prepared by adding PBS to a stock solution of 15% (w/v) gelatin, was poured into the chamber and injected into the side metal tubes and allowed to gel around the template at 4°C for 15 min. The template was then removed by pulling straight up, leaving a tissue mold with a custom tissue geometry and anchoring prongs protruding at its top. An o-ring was placed around the tissue inlet tube located at the bottom of the mold after which the printer was aligned to the chamber. To form a tissue-anchoring plug around the inlet, a solution of high-concentration ECM (hcECM) was prepared similar to the ECM described above, but with collagen (7.5 mg/ml) and 2% (w/v) TG [from a stock of TG (160 mg/ml) in medium]. This material was dispensed at the base of the tissue mold and around the O-ring before gelling at 22°C for 15 min. Last, 5% (w/v) molten gelatin was injected into the bottom metal pin before filling the mold with an uncompacted mix of cell aggregate and ECM solution for SWIFT printing.

Cardiac mold design and fabrication

A 3D computer-aided design (CAD) model of a normal 17-year-old heart was downloaded from the NIH 3D Print Exchange in the form of a stereolithography (.stl) file. The model was first rotated to orient the apex downward, and a section of the myocardium was digitally truncated using Tinkercad (Autodesk). The geometry was then alternatively smoothed and simplified in MeshLab to eliminate the noise and asperities of the model surface. Tinkercad was next used to generate positive and negative models of the desired geometry and to digitally carve out a wedge in the section of the anterior wall and the septum, in the vicinity of the LAD coronary artery, which subsequently served as a reservoir for the cardiac OBB matrix. These models were built at a 1:2 scale using an Objet30 3D printer (Stratasys) to serve as masters for the casting of the final heart visualization model and cardiac tissue receptacle. This three-step process consisted of the casting of a transparent block of PDMS (SYLGARD 184 at 10:1 base:curing agent) off of the outer surface of the sectioned myocardium master, the casting of the actual truncated myocardium using a lightly colored PDMS (using red and white Silc-Pig in SYLGARD 184), and the assembly of those two parts with PDMS acting as glue and filling the ventricles. A 3D printed wedge plug was used during the curing step to ensure that the cardiac tissue wedge reservoir remained open. The print path was generated by extracting the LAD and one of its diagonal branches from the original CAD model. The stereolithography file was sliced, and a MATLAB routine was used to skeletonize the vascular channels and convert it into GCode. Because of the low resolution of the CAD model, only a portion of the LAD and a single diagonal could be extracted from patient data; extra septal and diagonal branches were manually added to the coronary vasculature print path, using geometries similar to those found in vivo.

Sacrificial writing into functional tissues

A sacrificial gelatin ink was prepared using the procedure described above to enable embedded 3D printing of vascular networks. The gelatin stock solution (solubilized at 85°C, as described above) was melted at 80°C for <30 min and then diluted in PBS to generate a 5% (w/v) solution. Next, red food coloring was added at 2% (v/v) to enable visualization of printed ink. The freshly prepared sacrificial gelatin ink was loaded into a 0.5-cm ³ glass syringe and subsequently cooled to 4°C for 15 min to induce gelation. The glass syringe was then loaded onto a custom-built syringe pump that was mounted onto the 3D printer. The syringe pump was operated by an Arduino microcontroller and a stepper-motor driver. The syringe barrel was housed in a water-cooled custom-built temperature controller to control the stiffness of the gelatin ink by maintaining a temperature of approximately 20°C. A metal nozzle, with a length of 1.5 inches and an inner diameter of 0.25 mm (Nordson EFD), was fitted to the bottom of the syringe. Before printing, the syringe was incubated in its temperature-controlled housing for >15 min.

The OBB-ECM matrix was loaded into the silicone molds or perfusion chambers, and the following steps were performed rapidly to ensure that it remained cold and that the ECM did not undergo gelation before SWIFT was completed. First, for cardiac tissue fabrication, the volume of cardiac OBBs was estimated by compacting via centrifugation at 100 g. Next, the ECM solution was mixed with 10 ⁷ HNDF cells/ml of cardiac OBBs before resuspending the cardiac OBBs in the ECM:HNDF cell mixture to generate a slurry. The freshly prepared OBB slurry was pipetted into the molds, and the molds and their housing were centrifuged at 150 g at 2°C for 3 min to compact the aggregates, forming an OBB-based matrix, containing ~2.4 × 10 ⁸ ± 0.4 × 10 ⁸ cells/ml and 6 × 10 ⁵ ± 2 × 10 ⁵ HDNF cells/ml. Most of the supernatant ECM was removed leaving ~1 mm of the ECM solution above the top of the compacted bed of OBBs. To keep the matrix cool during the long-duration print in Fig. 3 (C and D), the mold was transferred onto the front of a custom-built water-cooled metal plate, comprising a metal plate, backed by an acrylic sheet that had milled channels through which ice-cold water was pumped. Both the metal plate and the acrylic sheet were cut to enable back illumination to facilitate visualization of the printing process. The mold was held on the water-cooled plate by a pair of clamps and was maintained in its housing in a vertical orientation by pressing it against two metal right-triangle prisms.

The desired vascular network was then printed within the OBB-based matrix at a print speed ranging from 0.5 to 4 mm/s. The extrusion rate of the syringe pump was set to define a certain diameter filament. The vascular geometry was designed in a custom-built MATLAB script that generated branched cubic splines, automatically assigned a printing order to prevent the nozzle from translating through previously printed features, and rendered each curvilinear line as piecewise linear segments in GCode. To vary the filament (and, hence, channel) diameter, the print speed was changed while maintaining a constant ink deposition. After each line segment, ink extrusion is stopped, and to avoid agitating the printed patterns, the nozzle was withdrawn vertically above the top of the tissue, translated to its new x and y coordinates, and reinserted to the correct new z coordinate by moving down vertically.

Design of hierarchical vascular networks

When templating a complex hierarchical vascular network ( Fig. 3, C and D), the channel diameters were computed to ensure an equivalent shear stress within each segment of the network, independent of their diameter. Assuming Poiseuille flow, we determined the channel radius R and the flow Q, along with the pressure P at each node by solving the following set of equations (in MATLAB). On the basis of mass conservation, the sum of flow coming to a node is zero

For the vascular network depicted in Fig. 3 (C and D), the ratio between the largest vessels (inlet and outlet) and the smallest one was found to be 1.3. To print these channels at a constant deposition rate, the nozzle velocity was modulated proportionally to the inverse of the square of the diameter of each channel (or to its cross-sectional area).

Perfusion of SWIFT tissue constructs

After SWIFT printing of cardiac tissues in machined perfusion chambers, the tissue was capped with hcECM to provide mechanical stability required to maintain patency for long-term culture. The mold was then transferred to a 37°C, 5% CO ₂ incubator for 45 min to complete gelation of the OBB-ECM matrix, and to melt the printed sacrificial gelatin filaments and gelatin that surrounds the tissue. After incubation, silicone molds were sealed via an excess of autoclaved SE1700 silicone base (without cross-linker) into the gap at the top of the mold, while machined perfusion chambers were sealed by screwing on the polycarbonate lid. The sealed perfusion devices were then transferred to a custom-made tissue perfusion housing that held the tissue vertically and adjacent to a media reservoir, containing mTeSR1 media with PVA (4 mg/ml; for EB perfusion) or CMM (for cardiac tissue perfusion) with 1% antibiotic-antimycotic (Thermo Fisher Scientific). Next, the tissue was maintained at 37°C, where the sacrificial gelatin ink is molten, and a peristaltic pump (Ismatec Inc.) was used to introduce fluid media that removes the gelatin and leaves behind open channels. For the cardiac tissue perfusion, after evacuating the printed gelatin at a vascular flow rate of 0.01 ml/min for 5 min, the inlet for the external perfusion was opened, and the molten gelatin that surrounds the cardiac tissue was evacuated at a high perfusion rate (0.8 ml/min) for 5 min. To ensure the removal of all gelatin, this process was repeated but with a vascular perfusion rate of 0.02 ml/min. Over the course of ~1 hour, the perfusion rate was then increased gradually to its final perfusion rate. Perfusion rates used are 250 μl/min for Fig. 3 (C and D), 100 μl/min for EB-tissue perfusion for viability measurements, and 500 μl/min for cardiac tissue perfusion. For specific experiments, the media reservoir was hyperoxygenated by means of bubbling a mixture of sterile-filtered 95% O ₂/5% CO ₂ (Airgas Inc.) through the media at a volumetric flow rate of ~10 ml/min.

Cell viability assays

For EB viability measurements, the tissues were removed from flow after 12 hours of perfusion. Next, the tissues and their molds were first immersed in a large bath of ice-cold PBS. The perfusion chambers were opened by unscrewing the front panel and removing the glass window. The tissues were then sectioned using a pair of microdissection scissors and transferred via aspiration into a 35-mm petri dish containing a cut silicone gasket to house the tissue for imaging. Next, the PBS was changed to mTeSR1 and PVA medium (4 mg/ml) by repeatedly flushing ~50% of the volume at a time with fresh mTeSR1. An equal volume of 2× concentrate LIVE/DEAD solution [calcein (1 μl/ml), ethidium homodimer-1 (EthD-1, 4 μl/ml), and Hoechst solution (0.5 μl/ml)] was then added, and the tissue was incubated at 37°C for 30 min. After incubation, LIVE/DEAD images were acquired on a confocal microscope (ZEISS) using a 5× ApoFluor objective. A cell number-normalized viability score was calculated as the ratio of the area of a thresholded calcein-AM viability signal to the area of a thresholded Hoescht signal. To obtain cell viability data for cardiac tissues, the same process was used. However, the tissues were incubated on ice immediately before imaging to suppress beating artifacts during imaging.

Lumen endothelialization

Before endothelialization, an EB-based tissue featuring a SWIFT-printed single vessel was perfused with DMEM:F12 supplemented with 1% (v/v) of Matrigel for 20 min to enhance endothelial cell adhesion. HUVECs, cultured in EGM2 medium, were harvested from their flasks by incubating for 5 min in 0.05% trypsin/EDTA. The cells were centrifuged at 220 g in DMEM with 10% FBS, strained, and resuspended at a density of 10 ⁷ cells/ml in EGM2. The HUVEC suspension was perfused through the lumen at a flow rate of 40 μl/min until they could be seen exiting the tissue. The flow was then stopped, and the tissue gasket was laid flat for 10 min in the incubator to allow cells to settle at the surface of the lumen. This process was repeated three times, each time laying the gasket flat at a different angle such that the four quadrants of the lumen were coating with HUVECs. Tissues were then returned to being perfused with 1:1 mTSeR1:EGM2 medium at a flow rate of 40 μl/min for 24 hours before being fixed for analysis.

Calcium imaging

A concentrated solution of Rhod-2 (Thermo Fisher Scientific) was prepared by dissolving 50 μg of the calcium indicator in 100 μl of DMSO. The solution was then diluted 1:250 in Tyrode's solution (Alfa Aesar). Blebbistatin (Sigma-Aldrich) was added at a final concentration of 5 μM (from a 5 mM stock solution). Cardiac OBB tissues were first rinsed with Tyrode's solution (Alfa Aesar) and incubated with the calcium indicator solution for 30 min. Next, the tissues were rinsed once with a blebbistatin-containing Tyrode's solution. The samples were subjected to a 555-nm light illumination, and the wide-field signal was captured by a scientific complementary metal-oxide semiconductor camera (sCMOS, Andor technology). Data were acquired using Micro-Manager open source software. During imaging, the plate containing the cardiac tissues was placed on top of a heated pad (Stoelting).

Electrical stimulation

To electrically stimulate the SWIFT cardiac tissues within the perfusion chamber, the back panel was replaced with a plate featuring two platinum wires (0.25 mm in diameter) looped into rectangles (14 mm by 5 mm) and such that both platinum wires were located 15 mm apart when mounted onto the perfusion chamber. A monophasic signal with a pulse of 2 ms was generated by a custom-made Arduino-based controller at a frequency of 1 or 2 Hz and an amplitude of 6 V/cm. For point stimulation of cardiac OBB tissue in fig. S12 and movie S6, the electrodes were composed of the same platinum wires but with extremities placed at a distance of 750 μm from one another and were positioned near the tissue to stimulate with a manual micromanipulator. The voltage was set to 20 V to trigger pacing of the tissue.

Pharmacological assays

On day 8, calcium was supplemented to the culture medium to a final concentration of 2 mM. Cardiac tissues were perfused for 20 min before videos were acquired. Then, isoproterenol (Sigma-Aldrich) was administered intraluminally for 20 min to cardiac tissues at a concentration of 10 μM before they were imaged again. On separate sets of experiments, 1-heptanol (Sigma-Aldrich) was added to the medium on day 11 at a concentration of 1 mM, and cardiac tissues were perfused intraluminally for 20 min, after which each tissue was imaged. Beating patterns were acquired by tracking the anchoring prongs of the cardiac tissue using the software Tracker ( physlets.org/tracker).

Cryosectioning, immunostaining, and flow cytometry

Tissues were cryosectioned by first fixing for 30 min in 4% formaldehyde and then rinsed three times in PBS containing 0.05% Tween 20 detergent (PBST). The fixed tissue was incubated overnight at 4°C in PBS containing 30% sucrose (sucrose solution) and then transferred into a 1:1 solution of Optimal Cutting Temperature compound:sucrose (OCT:sucrose) for 1 hour and 30 min. Next, the tissue was gently placed into a cryostat tissue mold, which was subsequently filled with 100% OCT solution and frozen on a cryostat Peltier cooler. The tissue was sectioned using 40- to 60-μm slices and transferred onto a Superfrost Plus slide (VWR Inc.). Sections were stored at −20°C before immunostaining.

For immunostaining, the tissue cryosections were first permeabilized in a 0.125% solution of Triton X containing 0.5% bovine serum albumin (BSA) or 2% donkey serum (DS) for 10 min, rinsed three times in PBST, and blocked for at least 30 min in a solution of PBS containing 3% BSA or 2% DS. Next, primary antibodies [Oct 4 (1:200; sc-5279, Santa Cruz Biotechnology), Sox 2 (1:40; AF2018, R&D Systems), Tuj1 (1:1000; MAB1195, R&D Systems), collagen I (1:500; ab90395, Abcam), cTnT (1:200; ab45932, Abcam), and sarcomeric α-actinin (1:200; ab9465, Abcam)] were added in 3% BSA or 2% DS in PBS and incubated overnight at 4°C. On the following day, the primary antibody was removed by rinsing three times in PBST, and Alexa Fluor Plus-conjugated secondary antibodies were added at 1:400 in 3% BSA or 2% DS in PBS for 45 min. The secondary antibodies were removed by rinsing three times in PBST. The nuclei were then labeled using a 1:5000 dilution of 4′,6-diamidino-2-phenylindole in PBS for 3 min, followed by rinsing twice with PBST. The sections were imaged using a Zeiss confocal microscope.

To carry out flow cytometry, after measuring its volume (as described above for EB density estimation), the cardiac OBB matrix was rinsed thoroughly with cold DMEM to remove ECM. The remaining cardiac OBBs were dissociated by incubation for 30 min at 37°C in a papain solution [papain (25 U/ml) in Earl's balanced salt solution containing l-cystein and EDTA, per the manufacturer's instructions (Worthington, Inc.)]. The cells were rinsed in fresh CDM, triturated via repeated pipetting with a P1000 pipette tip, and counted using a hemocytometer. The cells were then rinsed twice in PBS containing 3% BSA, fixed by incubating with CytoFix (BD Biosciences) for 15 min at 4°C and then rinsed twice via centrifugation and addition of PBS containing 3% BSA. For flow cytometry, 1 × 10 ⁶ cells were resuspended in perm-wash solution (BD Biosciences) and incubated for 15 min at room temperature before rinsing in fresh perm-wash solution. Next, samples were blocked for 15 min at room temperature by incubation in Pharmingen solution (BD Bioscience). The cells were then incubated for 1 hour in labeled primary antibodies (Vimentin PE, 562337, BD Pharmingen; cTnT Alexa Fluor 647, 565744, BD Pharmingen; 1:10 dilution in Pharmingen solution). After incubation, cells were washed twice in perm-wash and analyzed using an LSR II Analyzer (BD Biosciences). Gating of cell populations was performed as shown in fig. S10.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw2459/DC1

Supplementary Materials and Methods

Fig. S1. OBB displacement during SWIFT.

Fig. S2. Luminal channels formed after sacrificial ink printing and removal.

Fig. S3. Rheological characterization of tissue matrix constituents.

Fig. S4. Tissue viability assessment.

Fig. S5. Branching vascular network embedded within an EB-based tissue matrix via SWIFT.

Fig. S6. Embedded 3D printing of OBBs within different living matrices.

Fig. S7. Schematic illustrations of perfusion apparatus for SWIFT biomanufacturing and tissue maintenance.

Fig. S8. Viability of perfused and nonperfused EB-based tissues.

Fig. S9. Custom-made perfusion chip for large-scale viability assay.

Fig. S10. Flow cytometry data for cardiac OBBs.

Fig. S11. Cardiac tissue composition.

Fig. S12. Calcium imaging of cardiac tissue matrix.

Fig. S13. Characterization of cardiac tissue produced by SWIFT.

Fig. S14. Characterization of perfused cardiac tissue.

Movie S1. Nozzle translation during embedded printing within a tissue matrix composed of OBBs.

Movie S2. Biomanufacturing of a helical vascular feature within a tissue matrix composed of OBBs.

Movie S3. Biomanufacturing of a branched vascular network within a tissue matrix composed of OBBs.

Movie S4. Endothelialization of a vascular channel within a tissue matrix composed of OBBs.

Movie S5. OBBs composed of beating cardiac organoids.

Movie S6. Calcium imaging of cardiac OBB matrix.

Movie S7. Cardiac tissues with perfusable vascular channels via SWIFT.

Movie S8. Contractile activity of a bulk cardiac tissue (T-shaped construct) produced by SWIFT biomanufacturing.

View Abstract

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Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels

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Engineering organ-specific tissues for therapeutic applications is a grand challenge, requiring the fabrication and maintenance of densely cellular constructs composed of ~108 cells/ml. Organ building blocks (OBBs) composed of patient-specific-induced pluripotent stem cell-derived organoids offer a pathway to achieving tissues with the requisite cellular density, microarchitecture, and function.

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My top 5 strengths are responsibility, discipline, relator, restorative, and belief. Based off my strengths, I have created a team of different strengths so we can all work together to further this innovation into a complete 3D organ. This innovation would not currently be adopted by a facility for use with patients, but could be used by a facility to further advancement in a 3D functional organ. I would choose Faith, Daisy, Ivan, Sheila, and Andrew.

Faith is futuristic. She is inspired by the future which is important as this advancement is just one step in creating an entirely 3D printed and functional organ. She can inspire the team and researchers to look to the future.

Daisy is a developer. As a team we will need to work together and with the researchers to develop an entire organ based off this most recent innovation. She recognizes the potential in this advancement and can help improve based off this new technique.

Ivan has intellection. He will be able to guide the team in intellectual conversations and possibly has new ideas on how to advance this technology.

Sheila is strategic. She is able to create alternative ways to proceed with the creation of an organ and when faced with challenges will be able to work with the other team members to create a new plan.

Andrew is an activator. Andrew will make things happen and help continue to progress this innovation into something more.

Together we will work as a team to create an organ and save lives!