3D printing of cellular constructs

The high-resolution fabrication of cellular constructs was achieved by using a 3D printer previously employed to print aqueous droplets surrounded by lipid monolayers in oil8. Printer modifications allowed mammalian cells loaded inside the droplets to be patterned in a hydrogel-based bioink (Figs 1 and 2, Methods). The printed cellular constructs were then encapsulated in a thin layer of gel to allow transfer into aqueous medium (Fig. 3 and Supplementary Fig. 1). Human embryonic kidney (HEK-293T) cell derivatives (Supplementary Methods) were selected to evaluate the printing method, while oMSCs were used to investigate the differentiation capacity of cells in printed constructs.

Figure 1 3D printing of cellular constructs. (a) Schematic of cell printing. The dispensing nozzle ejects cell-containing bioink droplets into a lipid-containing oil. The droplets are positioned by the programmed movement of the oil container. The droplets cohere through the formation of droplet interface lipid bilayers. (b) A confocal fluorescence micrograph showing droplet interface bilayers (stained yellow) within a cell-free printed construct (11 × 14 × 7 droplets). The bilayers were visualised by adding sulforhodamine-101 (~10 μM) to the print solution. (Scale bar = 100 μm). (c) Histogram showing the mean HEK-293T cell density in printed droplets under oil as a function of the cell density in the bioink. The cell density was calculated as the mean number of cells per droplet (n = 25) divided by the mean droplet volume. Error bars represent the compound error of droplet size variance and cell per droplet variance. (d) A bright-field micrograph of a patterned cell junction, containing two cell types, printed as successive layers of 1 nL droplets (d = 130 μm) ejected from two glass nozzles (d = ~150 μm). (e) A confocal fluorescence micrograph of a printed HEK-293T cellular construct (11 × 14 × 2 droplets) under oil. Live/dead cell staining was performed with calcein-AM (CAM, green) and propidium iodide (PI, red), respectively. Visible are approximately 700 cells at 4 × 107 cells mL−1 with a viability of 85% (determined by manual cell counting). (Scale bar = 150 μm). (f) A high magnification, confocal fluorescence micrograph of a live/dead assay performed on an HEK-293T cellular construct (7 × 8 × 4 droplets) printed at a starting concentration of 1.5 × 107 cells mL−1, with a mean occupancy of 38 cells per droplet equivalent to 3 × 107 cells mL−1. Visible are some of the droplet boundaries. (Scale bar = 75 μm). Full size image

Figure 2 High-resolution patterning of two cell types. (a–c, e,f) Confocal fluorescence micrographs of printed cellular constructs in oil, immediately after printing. HEK-293 cells stained with Deep Red (DR) or Red CMPTX (RC) CellTracker™ dyes were false-coloured blue and yellow, respectively. (a) A Y-shaped structure within a square construct (8 × 9 × 4 droplets), with a mean feature width of 180 μm. (Scale bar = 200 μm). (b) A cruciform pattern of HEK-293 cells within a square construct (10 × 12 × 5 droplets). (Scale bar = 250 μm). (c) A high magnification image of the patterned HEK-293 cells in (b). (Scale bar = 100 μm). (d) A 3D model of a cuboidal cellular construct with an interface between two HEK populations (HEK 1, yellow; and HEK 2, cyan) at a diagonal in the x-z plane. (e,f) Partial cross-sections at fixed vertical positions (45 and 192 µm respectively) of a cellular construct (21 × 24 × 7 droplets) printed based according to the model in (d), showing both HEK populations. (Scale bars = 250 μm). (g–j) Side-on images of lamellar constructs, comprising CellTracker™ stained HEK-293 cells before and after phase transfer. The lower, DR-stained HEK-293 cell layers (yellow) were 3 droplets thick, while, the upper, RC-stained HEK-293 cell layers (blue) were 4 droplets thick (g,h) or 3 droplets thick (i,j). Images were recorded: (g) at day 0, in oil, immediately after printing; (h) immediately after transfer to culture medium; (i) on day 3 of culture, in medium and; (j) on day 5 of culture, in medium. (Scale bar = 250 μm). Full size image

Figure 3 Phase transfer and culture of printed constructs containing HEK-293T cells. (a) Gel encapsulation of a printed construct and phase transfer. The printed cellular construct was gelled by standing at 4 °C for 20 to 25 min and the lipid in the oil was removed by washes with silicone oil AR20 at room temperature. The construct was then coated with a thin layer of cell-free bioink, which was gelled by standing at 4 °C for 20 to 25 min. The gelled construct was then transferred into the upper phase of an oil-culture medium two-phase system. The construct fell through the oil into the culture medium. (b) Image of a z-stack 3D reconstruction of live/dead-stained HEK-293T cells printed as a cuboid construct (7 × 8 × 4 droplets) immediately after printing under oil. The printed droplets had a mean density of 2.9 × 107 cells mL−1 with a viability of 96%. (Scale bar = 200 μm). (c) Image of a z-stack 3D reconstruction of live/dead-stained printed HEK-293T cells after gel encapsulation and transfer to culture medium. (Scale bar = 200 μm). (d) Graph showing HEK-293T cell viability (including standard error of the mean) of five printed constructs at day 0 after transfer to culture medium. Viabilities were determined by using automated object counting, values of which were used either unmodified or resolved with respect to mean cell size. (e) Image of a z-stack 3D reconstruction of immunocytochemistry performed on a construct in culture medium at day 7: cell nuclei (DAPI, blue); cytoplasm of live cells (CAM, green) and; mitotic marker (phospho-histone H3 ICC, PH3, white). (Scale bar = 200 μm). Full size image

HEK-293T cells or oMSCs were harvested and dispersed in a sterile bioink, which was kept at 37 °C prior to printing. The bioink contained serum-free defined cell culture medium, ultra-low-gelling-temperature (ULGT) agarose, fluorenylmethyloxycarbonyl (Fmoc) protected dipeptide gelators48 and routinely, type I collagen (Methods). The bioink was also compatible with other extracellular matrix proteins, and alternatively could be supplemented with fibronectin and laminin. In typical experiments, the cell-laden bioink (5 μL) was loaded into the glass bioprinter nozzle (Fig. 1a and d), which was subsequently immersed in a sterile blend (35:65 v:v) of undecane and silicone oil AR20 containing diphytanoyl phosphatidylcholine (DPhPC, Methods). The oil mixture had been optimized to allow aqueous droplets to descend by gravity within the oil phase, and to prevent freezing during the temperature-induced (4 °C) gelation performed in the gel encapsulation process (Supplementary Fig. 2). A programmed piezo-actuated impulse was used to eject cell-containing droplets (volume = 1 nL) from the nozzle into the oil (Fig. 1d). As the droplets sank (Fig. 1a), they acquired a DPhPC monolayer8, which allowed the subsequent formation of droplet-droplet interface bilayers (DIBs)49. Bilayer formation (Fig. 1b) conserved the print resolution by initially confining cells to limited volumes within the growing structure. Because the constructs were assembled in oil, the droplets packed in a hexagonal array. By contrast, when droplets are printed in air they flatten and the print resolution is reduced in the horizontal plane39. The constructs could be printed with high concentrations of cells without significant coalescence or loss of structural fidelity, which was attributed to the incorporation of Fmoc-dipeptides and the omission of fetal bovine serum (FBS) from the bioink (Supplementary Figs 3–5). To stabilise the constructs during gel encapsulation and phase transfer, the bioink was supplemented with agarose (1.0 to 1.2 w/v %), allowing the printed pattern to be preserved within solidified hydrogel (Supplementary Fig. 6). The presence of Fmoc-dipeptides also aided pattern retention by increasing the interfacial adhesion between printed droplets, and thereby producing a more cohesive structure (Supplementary Fig. 7).

Hundreds of nanolitre droplets were printed to give millimetre-scale constructs (Fig. 1e). Over the course of five minutes, structures with approximate dimensions of 1 × 1 × 0.5 mm could be printed. This was the typical size of printed constructs, but larger structures, up to nine times this volume were also fabricated (Supplementary Fig. 8). The printed droplets, had an average occupancy of between 1 and 38 cells depending on the bioink cell density (Supplementary Fig. 9). This resulted in a printed cell density in the range of 0.1 × 107 to 3 × 107 cells mL−1 (Fig. 1c), similar to the cell densities found in physiological tissues50,51,52. Typically, bioinks with starting concentrations of 1.0 × 107 to 1.5 × 107 cells mL−1 were employed as they achieved the highest printed cell densities (3 × 107 cells mL−1), which arose due to sedimentation of the cells within the printer nozzle (Fig. 1f and Supplementary Fig. 9). This sedimentation enabled the printed cell density to be systematically varied by changing the initial cell density of the bioink (Supplementary Fig. 9). The small volume of bioink required for printing and the concentration of the cells by sedimentation makes our approach particularly appealing for the fabrication of tissues with scarce or high-value cells.

Live/dead cell staining performed on the day of fabrication (Fig. 1e and f) showed that the HEK-293T cell viability was 88 ± 5% (\(\bar{x}\) ± s.d., n = 5) after printing. Similar viabilities were observed for non-printed HEK-293T cells cultured in blocks of gelled bioink and HEK-293T cells ejected as droplets in air directly into culture medium (Supplementary Fig. 10). Therefore, neither the bioink components nor piezo-induced stress was a primary cause of cell death. Indeed, the small fraction of dead cells that was observed was attributed to the periods spent away from ideal culture conditions (2 to 4 h total, during printing and processing) and to extended illumination during imaging by microscopy53 (Supplementary Fig. 11). Viability was increased to 90% or greater by returning printed constructs to the incubator between processing steps, minimising pH fluctuations during imaging by incorporating 20 mM HEPES buffer and imaging cellular constructs only once (Supplementary Methods).

Fabrication of patterned cellular constructs

In addition to constructs printed from one type of cell, constructs containing two types of cell were printed in various high-resolution patterns (Fig. 2). These materials contained either separately labelled populations of the same cell line (e.g. HEK-293T stained with either Deep Red or Red CMPTX CellTracker™ dyes) or different cell types (osteoblasts and chondrocytes, see below). For example, HEK-293T cells were patterned as a Y-shaped junction within a cuboid (Fig. 2a), demonstrating the ability to fabricate features at a resolution of 1 to 2 droplet diameters (feature width 180 ± 60 μm, \(\bar{w}\) ± s.d., 14 measurements). HEK-293/YFP and HEK-293/CFP cells were patterned into structures (Fig. 2b and c), which incorporated a cross motif (feature width 425 ± 73 μm, \(\bar{w}\) ± s.d., 12 measurements) within a cuboid, with discrete cell types in the cruciform and corner segments. Constructs containing more complex 3D patterns of cells were also fabricated. For example, a 3D junction between HEK-293T and HEK-293/CFP cells was printed within a cuboidal structure (Fig. 2d, e and f, and Supplementary Figs 12 and 13). Here, the interface between the two cell populations was at a diagonal in the vertical (x-z) plane (Supplementary Figures 12 and 13) with a measured angle of 21 ± 8° with respect to the print surface (\(\bar{x}\) ± s.d., 12 measurements, Supplementary Methods). Additionally, a high-resolution interdigitated interface between the two cell populations was achieved by patterning cell-laden droplets in an interdigitated fashion in the x-y plane (Supplementary Figure 12).

Finally, lamellar structures (Fig. 2b and e–h) were fabricated from two types of stained HEK-293 cells. Each lamellar construct had a final thickness of ~400 μm and comprised two layers of cells, with each layer 3 to 4 droplets thick. In this instance, droplet printing allowed precise and repeatable fabrication of lamellar structures with well-defined external dimensions (1.1 × 1.1 × 0.4 mm) and internal layer thicknesses (lower layer: 160 ± 26 μm and upper layer: 200 ± 17 μm, \(\bar{x}\) ± s.d., n = 3 constructs). The ability to print spatially organised structures with predefined lineages was also possible. oMSC-derived osteoblasts and primary chondrocytes were suspended in separate bioinks and printed in a layered, 3D geometry as a model of a 3D osteochondral interface (Supplementary Fig. 14).

Transfer of cellular constructs to medium for culture

To transfer the 3D printed cellular constructs from oil to culture medium without loss of pattern fidelity, a stabilization process was developed (Fig. 3). The constructs were cooled from ambient temperature and kept at 4 °C for 20 to 25 min to trigger gelation of the agarose in the bioink (Supplementary Fig. 1). After cooling, the constructs were encapsulated in additional ULGT-agarose to confer further stability during the phase transfer procedure (Methods). To mediate gel coating, the lipid of the print oil was first diluted to ~15 μM, i.e. ~1% of its original concentration, by repeated silicone oil (AR20) washes at room temperature (Methods). Afterwards, a droplet (0.2 to 0.4 µL) of the bioink (containing agarose, but without cells) was pipetted onto the external surface of the construct and solidified by standing at 4 °C for 20 to 25 min to produce a robust veneer (Fig. 3a and Supplementary Fig. 1). The calculated thickness of a veneer formed from the added bioink was 45 to 81 μm, assuming that it spreads evenly over the printed construct (Supplementary Methods). Confocal micrographs showed that the average veneer thickness at the vertical surface of the construct was 32 ± 9 μm (n = 3 constructs, Supplementary Methods). For phase transfer54, a coated cellular construct was loaded into a truncated pipette tip (20 μL) and transferred into the upper phase of an oil-above-culture-medium two-phase system. The cell construct fell into the aqueous phase by gravity sedimentation, shedding the bulk oil phase. The gelation process had a negligible impact on the organization of the printed structures as demonstrated for the printed lamellar constructs (Fig. 2e,f). The external dimensions were unchanged and the discrete cell layers were retained after phase transfer. The transfer process was performed successfully with more than 100 cuboidal cellular constructs (Supplementary Fig. 15).

The ability to deliver live/dead stain to cellular constructs in culture medium demonstrated the penetration of small dye molecules into the structures (Fig. 3c), which indicated that the lipid bilayers between adjacent droplets had been disrupted as a result of the gelation and oil washing steps. However, the structural role of the bilayers was no longer required at this point and their breakdown enabled the ingress of nutrients necessary for cell growth and the maintenance of physiological osmolarity.

After transfer to culture medium, the printed constructs were cultured and assayed for cell viability and proliferation over one week (Supplementary Fig. 16) with a change of culture medium every 2 to 3 d. Live/dead assays performed on the HEK-293T constructs revealed cell viabilities in excess of 80% immediately after phase transfer (Fig. 3d) and rising to >95% on days 3 and 7 (n = 4, Supplementary Table 4). DAPI nuclear staining showed that the cell population within the HEK-293T constructs exhibited cell division over the seven-day period. The average number of visible DAPI stained cells was observed to increase from 3,400 ± 1,600 cells (\(\bar{x}\) ± s.d., n = 4) on day 3 to 11,600 ± 5,100 cells (\(\bar{x}\) ± s.d., n = 4) on day 7 (Supplementary Fig. 17). Immunocytochemical (ICC) staining (Methods) showed dividing HEK-293T cells throughout the entire printed structures at day 3 and 7 of culture (Fig. 3e and Supplementary Figs 18 and 19), with 2 to 8% of the cells (n = 8) displaying phospho-histone H3 signals, characteristic of the G2/M transition (Supplementary Fig. 17). Furthermore, after 7 d, HEK-293T constructs had evolved from structures with individually distinguishable cells compartmentalised within printed droplets (Fig. 3b) into structures dense with multiple cell aggregates up to 140 μm in width (Fig. 3e), indicating that cells had proliferated and outgrown the confines of the printed droplets. Similar behaviour was also found in lamellar cellular constructs (Fig. 2e–h), which retained printed patterns of discrete cell layers after 5 d of growth.

Development of printed stem cells

Having successfully printed and cultured constructs of HEK-293T cells, a robust mammalian cell line, the methodology was applied to mesenchymal stem cells (Fig. 4), which have therapeutic potential in regenerative medicine17. The viability of printed oMSCs immediately after printing (Fig. 4c) was 91 ± 4% (\(\bar{x}\) ± s.d., n = 5), which was similar to the high post-print viabilities observed for HEK-293T cells (Fig. 3d). The ability of the printed oMSCs to differentiate was investigated by introducing TGF-β3, a chondrogenic growth factor, into the culture medium (Methods, Supplementary Fig. 20). ICC performed at days 3 and 7 revealed expression of the early chondrogenic transcription factor SOX-9 in oMSC constructs exposed to TGF-β3 (Fig. 4d,e and Supplementary Fig. 21), but not in untreated network controls (Supplementary Fig. 22). SOX-9 immunofluorescence was present in all cells in treated constructs. Tissue-wide expression of SOX-9 protein was also observed in a positive control of engineered cartilage, formed over 35 d using oMSCs seeded within a polyglycolic acid scaffold (Supplementary Fig. 22). Digital polymerase chain reaction (dPCR) analysis was used to quantify SOX-9 mRNA, normalised to the endogenous expression of β-actin mRNA, in printed constructs (n = 22) taken from four oMSC sources (Fig. 4f and Supplementary Figure 23). After 7 d, dPCR showed upregulation of SOX-9 mRNA for the treated printed constructs only. The upregulation of SOX-9 mRNA expression was lower for the treated printed constructs (0.61 ± 0.15, \(\bar{x}\) ± s.d., n = 22) than for treated pellet cultures (1.17 ± 0.15, \(\bar{x}\) ± s.d., n = 24), the gold standard for chondrogenesis55 (Fig. 4f). This was unsurprising given that the reduced intercellular space in cell pellets is commonly used to maximise the juxtacrine signalling (e.g. via N-cadherin, N-CAM) required for optimal chondrogenesis. Significantly, within 3 d, the oMSCs in the printed constructs treated with chondrogenic factors underwent spontaneous re-organisation to form spherical aggregates, 93% of which were between 20 and 60 μm in diameter (Fig. 4b, Supplementary Figs 20 and 24). This process resembled cellular condensation, a critical stage preceding chondrogenesis56 and was also observed in the untreated cellular constructs, however, the distribution of aggregate sizes was different (Supplementary Fig. 24). Cell aggregates within treated constructs remained a similar size over 10 d during early stage chondrogenesis, whereas untreated constructs contained fewer aggregates, with the majority of the cells present within larger aggregates that were >100 μm wide (Supplementary Figs 20 and 24). Advantageously, our approach distributes cells homogenously throughout the construct, which is in contrast to the inhomogeneous cell distributions often observed after seeding scaffolds for conventional tissue engineering57.

Figure 4 Growth and differentiation of printed oMSCs. (a,b) Image of a z-stack 3D reconstruction of live/dead-stained printed oMSCs: (a) immediately after printing and; (b) after 10 days in culture with the TGF-β3 supplement. (Scale bars = 250 μm). (c) Graph of oMSC viabilities (including standard error of the mean) for five printed constructs immediately after transfer to culture medium. Viabilities were determined by using automated object counting, values of which were used either unmodified or resolved with respect to mean cell size. (d) Confocal fluorescence micrograph of immunocytochemistry performed on a printed oMSC construct after 3 days of culture with a TGF-β3 supplement: SOX-9 (orange); nuclei (DAPI, blue); cytoplasm of live cells (CAM, calcein-AM, green). (Scale bar = 50 μm). (e) High-magnification micrograph of immunohistochemistry performed on a printed oMSC construct after 35 days of culture with TGF-β3 supplement; type II collagen (diaminobenzidine tetrahydrochloride (DAB), brown); nuclei (hematoxylin QS, blue). (Scale bar = 25 μm). (f) Digital PCR measurements of SOX-9 mRNA expression in printed oMSC constructs (n = 22) and oMSC pellet cultures (n = 24) after 7 days in chondrogenic medium with or without supplementation of TGF-β3. Each printed and pellet sample was replicated 4 to 6 times from four oMSCs sources, each extracted from a different sheep. SOX-9 expression was normalised to an endogenous β-actin control. Error bars represent standard deviations. Differences were tested by using a paired t-test, with two-tailed p values < 0.05 considered significant. Full size image

Following the preliminary chondrogenesis results, the ability to engineer tissues with properties of hyaline cartilage from printed oMSCs was investigated over a five-week period. Printed oMSC constructs (n = 13) were cultured for 35 days in a differentiation medium containing ascorbic acid, dexamethasone, TGF-β3 and insulin, a typical protocol for stem cell cartilage engineering58. During culture, the majority of printed oMSCs condensed into ~60-µm-wide aggregates, with each construct comprising 20–30 aggregates evenly distributed across the 1 × 1 × 0.5 mm printed structure (Supplementary Fig. 25). Immunoperoxidase staining of the printed constructs revealed the presence of secreted type II collagen, a key component of the extracellular matrix (ECM) of hyaline cartilage (Fig. 4e and Supplementary Fig. 25). Interestingly, the printed constructs exhibited negligible expression of type I collagen, a major component in fibrocartilage, but unwanted in engineered hyaline cartilage. These results demonstrate that printed cellular constructs can be maintained in long-term culture (35 days) without loss of structural fidelity and that the component cells can undergo differentiation and matrix secretion to produce tissue-like structures.