Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Component isolation (Figure 1 and Figure 2)
Before utilizing the core explants and cell populations in assembloid co-culture, these components are to be checked under the microscope (Figure 1). Core explants should have a uniform diameter (100-200 µm) and no visible kinks or wrinkles. Endothelial cells should present an elongated shape in contact with other cells, which they do not when seeded at a too-low density because of a low initial yield from the isolation. In this case, the endothelial cells assume a more roundish shape with cytoskeletal extensions and proliferate markedly slower. Split them 1:5 after 7-10 days. Tendon fibroblasts isolated from the Achilles tendons assume a more roundish morphology compared to their human counterparts within 1-2 passages (10-14 days each) when they were split 1:6. Macrophages are much smaller than fibroblasts or endothelial cells and do not proliferate after the isolation. Depending on the batch, their shape can vary from pyramidal to round.

The phenotypes of the cellular components were verified with flow cytometry. A conjugated CD31 antibody was used as a marker for endothelial cells (Figure 2A). Setting the fluorescence threshold based on an unstained control sample (grey), 90.1% of passage 1 (P1) and 48.7% of passage 2 (P2) endothelial cells were identified as CD31-positive. A genetically modified mouse line co-expressing the tendon fibroblast marker Scleraxis alongside a green fluorescent protein (ScxGFP) and a conjugated CD146 antibody was used to characterize the tendon fibroblasts (Figure 2B)^35,60. After one passage (P1), 37.3% of the fibroblasts were ScxGFP⁺CD146^–, 0.2% were ScxGFP⁺CD146⁺, 4.3% were ScxGFP^–CD146⁺, and 58% were ScxGFP^–CD146^–. After two passages (P2), the percentage of ScxGFP⁺CD146^– cells decreased to 27.6%, the percentage of ScxGFP⁺CD146⁺ cells increased to 6.9%, the percentage of ScxGFP^–CD146⁺ cells increased to 10.6%, and the percentage of ScxGFP^–CD146^– cells decreased to 54.9%. To identify and characterize the macrophages, a F4/80 antibody was used in combination with a CD86 and a CD206 antibody (Figure 2C). After isolation and culture, 96.4% of the bone marrow-derived cells were F4/80-positive. Among these F4/80-positive cells, 8.6% were CD206⁺CD86^–, 23.6% were CD206⁺CD86⁺, 28.3% were CD206^–CD86⁺, and 39.4% were CD206^–CD86^–. Collagen crosslinking speed may vary from batch to batch and is to be tested before starting experiments.

Assembloid appearance (Figure 3)
In lesion-like culture conditions (36 °C, 20% O₂), the core explant remained mechanically stretchable, did not change in appearance, and continued to be visually distinguishable and physically separatable from the surrounding hydrogel over at least 21 days (Figure 3A,B). The surrounding hydrogel was compacted over time, with the compaction speed depending on the cell population seeded into it. Achilles tendon-derived fibroblasts contracted their surrounding hydrogel the fastest and did so radially when in a hydrogel cast around a core explant and in all directions when not (Figure 3B,C). Initially, cell-free hydrogels placed around a core explant compacted, as well. This contraction was likely caused by migrating cells from the core explant, indicating a dynamic cross-compartmental interface. As cell-free hydrogels without an embedded core explant did not compact detectably, the contribution of water loss-induced shrinkage appears to be negligible (Figure 3B and Supplementary File 6).

A lack of hydrogel compaction can, therefore, be used to detect mistakes in the assembloid assembly (i.e., low cell concentrations) and should be checked before continuing with more expensive readout methods. While establishing this method, common mistakes reducing the cell concentration included dying cells in the extrinsic hydrogel because they were left for too long in the relatively harsh crosslinking solution (high pH, low temperature) and drying core explants because the time between medium aspiration and hydrogel injection was too long, or because the core explant was clamped too high to be embedded in the collagen.

Confocal fluorescence microscopy: Viability and morphology analysis (Figure 3)
Once removed from the clamps with scissors (Figure 3B), assembloids can be fixed, stained, and imaged with a confocal microscope as a whole without sectioning. Here, core // endothelial cell, core // macrophage, and core // fibroblast assembloids were stained with DAPI (NucBlue) and Ethidium Homodimer (EthD-1) to analyze the viability and DAPI and F-actin to analyze morphology and cell spreading in the 3D collagen hydrogel (Figure 3D). The viability of core // endothelial cell assembloids (Figure 3E) was quantified and found to be generally lower after assembloid assembly than previously reported for core // macrophage and core // fibroblast assembloids⁸⁴. However, the viability remained stable during assembloid culture until at least day 7.

Mechanically induced microdamage and measurement of mechanical properties (Figure 4)
The screws and pins attached to the clamp holders allow the fixation of clamped assembloids to uniaxial stretching devices. The custom-made stretching device used here is equipped with a 10 N load cell and has been described in previous publications (Figure 4A)²². All samples were pre-conditioned with five stretch cycles to 1% strain prior to the measurements.

Recording the full stress-strain curve of core explants or assembloids (Figure 4B) would allow quantification of the linear elastic modulus (α), the maximum stress (β), and the maximum strain (у). However, it also irreversibly damages the core explant or the assembloid, which makes it impossible to assess the longitudinal development of the maximum stress (β) and the maximum strain (у) for the same samples (Figure 4B). Here, the linear elastic modulus was used as a measure for the sample's ability to withstand forces, as this measurement requires stretching the sample to only 2% strain, which has been shown previously to not cause permanent reductions in the linear elastic modulus¹⁸. In particular, core // endothelial cell assembloids were exposed to the clamping procedure to 2% strain (approximately the end of the linear elastic region) or 6% strain (approximately the maximum strain). The resulting microdamage was assessed by measuring the linear elastic modulus before and after the procedure (Figure 4C).

In line with previously conducted experiments exploiting mono-cultured core explants, core // endothelial cell assembloids retained their linear elastic modulus for at least 14 days when cultured in quasi-homeostatic niche conditions (29 °C, 3% O₂) and exposed to strains no higher than 2%^18,21. Regarding mechanical baseline stimulation, the static stretch applied through the clamps seemed to sufficiently mimic native strain levels experienced by tendon core units in vivo to prevent catabolic processes generally associated with matrix unloading⁸⁷. Indeed, the progressive and statistically significant decline of the linear elastic modulus observed in core // endothelial cell assembloids exposed to 6% strain could be attributed to the matrix unloading stemming from mechanically-induced matrix microdamage.

When performing these experiments, it is important to prevent the drying of the assembloid. Here, they were encased in autoclaved and wetted paper, but other methods could also be viable depending on their compatibility with the stretching device used. As the friction between the metal clamps and the core explant is limited, add small pieces of paper between the metal and the core explant during clamping to prevent slippage and closely monitor the stretching process to detect and exclude slipped core explants and assembloids.

Compartment-specific transcriptome and assembloid-specific secretome analysis (Figure 5 and Figure 6)
In the first set of core mono-culture experiments presented here, the stability of core gene expression after explant isolation was assessed to decouple isolation from experimental effects (Figure 5A). Although higher replicate numbers are necessary for precise conclusions, the expression of Vegfa and Mmps increased strongly in freshly isolated core explants within hours after the explant isolation when cultured in lesion-like niche conditions (37 °C, 20% O₂).

Neovascularization is a central hallmark of tendon disease and repair that could, in part, be driven by endothelial cells activated by pro-angiogenic factors (i.e., vascular endothelial growth factor, Vegfa) secreted by the tendon core under hypoxia⁸⁸. Examining the first step of this potential crosstalk (Figure 5B), the expression of both Vegfa and the hypoxia marker carbonic anhydrase 9 (Ca9) was found to be increased statistically significant in explants mono-cultured under low oxygen tension (3% O₂) in contrast to those mono-cultured under high oxygen tension (20% O₂). Meanwhile, the lower oxygen tension did not seem to cause changes in the expression of tendon fibroblast markers such as Scleraxis (Scx) and collagen-1 (Col1a1). Together, these results identify core-resident cells as plausible contributors to pro-angiogenic signaling in a hypoxic niche.

Next, the activation of endothelial cells by pro-angiogenic core signaling was assessed in core // endothelial cell assembloid co-culture under high (20% O₂) and low (3% O₂) oxygen tension. Fortunately, the modular composition of assembloids allows compartment-specific transcriptome analysis after culture by physically separating the core explant from the extrinsic collagen hydrogel (Figure 6A). In the core explant (Figure 6D), Vegfa expression was again confirmed to increase under low oxygen tension, although the effect on other hypoxic markers such as Fgf2 was less clear and requires higher replicate numbers for precise conclusions. In addition, the expression of pro-inflammatory markers such as Tnf-α and markers for extracellular matrix degradation such as Mmp3 were decreased in the core under low oxygen tension. In the extrinsic hydrogel initially seeded with endothelial cells (Figure 6E), the presence of an alive core explant (aC) decreased Vegfa expression under low oxygen tension, but not under high oxygen tension. In addition, the presence of a devitalized (dC) core explant under low oxygen tension did not decrease Vegfa expression either. Under low oxygen tension, Tnf-α expression in the extrinsic hydrogel was comparable around aC/dC but increased under high oxygen tension around alive core explants. Fgf2 expression was decreased in all conditions compared to the extrinsic endothelial cell-laden hydrogel cultured around a devitalized core explant under high oxygen tension but most under low oxygen tension. Mmp3 expression was highest around alive core explants under high oxygen tension and lowest around devitalized core explants under low oxygen tension. Overall, the co-cultured endothelial cells seem responsive to both the active core explant, which is capable of initiating crosstalk and variations in oxygen levels. A more comprehensive transcriptome analysis would facilitate the elucidation of their respective contributions.

The modularity of the assembloid system allows the integration of genetically modified cells containing fluorescent reporter genes. Here, endothelial cells isolated from Pdgfb-iCreER mG mice⁸⁹ were seeded into the hydrogel compartment. These cells co-express the endothelial cell marker platelet-derived growth factor subunit b (Pdgfb) alongside the enhanced green fluorescent protein (EGFP), which makes Pdgfb-expressing endothelial cells appear green under the microscopy (Figure 6C). Using this method, the presence of Pdgfb-expressing endothelial cells was confirmed to be maintained over 7 days in culture (37 °C) and appeared to be independent of oxygen tension (20% O₂ compared to 3% O₂).

To analyze the secretome of assembloids, the culture medium used respectively for core // cell-free and core // fibroblast, core // macrophage, or core // endothelial cell co-culture was replaced with its serum-free counterpart three days before aspirating and freezing the supernatant now enriched with the secretome (Figure 6A). This enrichment time was sufficient to detect cytokines such as vascular endothelial growth factor (VEGF) with an MSD assay, as shown here for core explants and core // fibroblast assembloids cultured in lesion-like niche conditions (Figure 6B).

Important considerations when analyzing the secretomes and transcriptomes of core explants and assembloids concern the usage of proper controls. Freshly isolated core explants have limited value, as especially their expression of Vegfa and Mmps increases strongly within hours after the isolation (Figure 5A). Time-matched explants surrounded by an initially cell-free hydrogel are more suitable as controls for the core compartment gene expression. For the extrinsic hydrogel, cell-laden hydrogels cultured without a core explant are inferior controls compared to cell-laden hydrogels cultured around devitalized core explants (Supplementary File 7), mainly because they compact into roundish shapes instead of elongated hydrogels which greatly changes cell morphology (Figure 3A).

Figure 1: Assembloid component isolation and assembly to model in vivo crosstalk. Tendon core explants were extracted from mouse tails, cut, and clamped. Mouse leg muscles (i.e., quadriceps femoris (QF), gastrocnemius (G), and tibialis anterior (TA)) were digested to isolate endothelial cells that were then cultured on tissue culture plastic. The Achilles tendons (AT) were digested as well to isolate tendon fibroblasts, which were then cultured on tissue culture plastic. The bone marrow from the tibia and the femur was flushed out of the bones. Then, the isolated monocytes were cultured on tissue culture plastic and differentiated into naïve macrophages. The light microscopy images (10x) depict the appearance of core explants, endothelial cells, tendon fibroblasts, and macrophages immediately before their integration into assembloids. During the assembly, the cells cultured on plastic were put in suspension and then seeded into a collagen-1 solution (1.6 mg/mL). Then, the cell-hydrogel mixture was cast around the clamped core explant and polymerized for 50 min at 37 °C before adding culture medium. Culture conditions were controlled via the clamps (mechanical tension) and the incubator settings (oxygen concentration, temperature). Please click here to view a larger version of this figure.

Figure 2: Characterization of cellular assembloid components. (A) Representative flow cytometric analysis of muscle-derived endothelial cells after one passage (P1, top row) and two passages (P2, bottom row). The counts for unstained (grey) and CD31-stained (green) cells were normalized to modal. The percentages are given for the CD31-stained group. (B) Representative flow cytometric analysis of Achilles tendon-derived fibroblasts after one passage (P1, top row) and two passages (P2, bottom row). The axes report fluorescence intensities of unstained cells (grey) and cells both expressing ScxGFP and stained with CD146 antibodies (rainbow colors). (C) Representative flow cytometric analysis of bone marrow-derived macrophages after culture. In the top row, the counts for unstained (grey) and F4/80-stained (green) cells were normalized to modal. The percentages are given for the F4/80-stained group. The graph in the bottom row reports fluorescence intensities of unstained cells (grey) and the F4/80⁺ subset of cells stained with CD206 antibodies and CD86 antibodies (rainbow colors). Please click here to view a larger version of this figure.

Figure 3: Assembloid imaging and appearance. (A) Representative photographs taken at day 0 (d0) and day 21 (d21) of culture (37 °C, 20% O₂) show a multi-dimensional contraction of a hydrogel containing extrinsic fibroblasts without an embedded core explant and strong radial compaction of a hydrogel containing extrinsic fibroblasts around a core explant. (B) Representative photographs taken at day 21 (d21) of culture (37 °C, 20% O₂) show differences in compaction speed between cell-free hydrogels, cell-free hydrogels cast around a core explant, and tendon fibroblast-laden hydrogels cast around a core explant. (C) The representative light microscopy images (10x) taken at day 0 (d0) and day 21 (d21) of culture (37 °C, 20% O₂) indicate longitudinal changes in the presence of cell populations and the compaction speed of the collagen hydrogel (HG) around the core explant (E) in core // cell-free and core // fibroblast assembloid co-culture. The schematic representation depicts the differences in hydrogel compaction between core // cell-free assembloid and core // fibroblast assembloid co-culture. (D) Representative confocal microscopy images taken at day 7 (d7) of core // endothelial cell, core // macrophage, and core // fibroblast assembloid co-culture (37 °C, 20 % O₂). Images in the left row depict assembloids with cell nuclei stained in blue (DAPI) and dead cells stained in pink (Ethidium homodimer-1). The other two rows depict assembloids with cell nuclei stained in blue (DAPI) and actin filaments in green (F-actin). (E) Boxplots depicting the quantified viability of core // endothelial cell assembloids at day 1 (d1) and day 7 (d7) of co-culture. N = 5. The upper and lower hinges correspond to the first and third quartiles (25^th and 75^th percentiles) and the middle one to the median. Whiskers extend from the upper/lower hinge to the largest/smallest value no further than 1.5 times the interquartile range. P-values: ^n.s.p > 0.05. Please click here to view a larger version of this figure.

Figure 4: Mechanical stimulation of assembloids and measurement of assembloid mechanical properties. (A) Graphical depiction of the custom-made stretching device comprising the clamp holder platforms, a force sensor, and a stepper motor. The photographic image shows an assembloid mounted to the stretching device with clamps. The lid of a 15 mL plastic tube (Ø: 17 mm) used for scale. (B) Graph depicting representative stress/strain curves for core explants (light blue) and assembloids (light red). The linear elastic modulus (α), maximum stress (β), and maximum strain (у) can be extracted from the data to mechanically characterize the core explant or assembloid. (C) Graph showing the linear elastic modulus (Emod) of core // endothelial cell assembloids co-cultured (29 °C, 3% O₂) over a 14-day time course after being clamped (solid line), clamped and stretched to 2% L₀ strain (dotted line), or clamped and stretched to 6% L₀ strain (dashed line) at the start of the experiment. N = 5. The data points were normalized to the initial modulus linear elastic modulus before the stretching and are all displayed as mean (±sem). P-values: *p < 0.05, **p < 0.01. Please click here to view a larger version of this figure.

Figure 5: Changes in the core transcriptome after isolation and culture under different niche conditions. (A) Scatterplot depicting the fold changes in Vegfa, Mmp13, and Mmp3 gene expression in mono-cultured (37 °C, 20% O₂) murine core explants 2 h, 4 h, 6 h, and 8 h after isolating them from the tail. The fold changes at the respective time points were normalized to the gene expression 2 hours after the isolation. N = 2. (B) Boxplots depicting the fold changes in Ca9, Vegfa, Scx, and Col1a1 gene expression in core explants mono-cultured under low oxygen tension (3% O₂) normalized and compared to those mono-cultured under high oxygen tension (20% O₂). N = 5-6. The upper and lower hinges of the boxplots correspond to the first and third quartiles (25^th and 75^th percentiles) and the middle one to the median. Whiskers extend from the upper/lower hinge to the largest/smallest value no further than 1.5 times the interquartile range. Datapoints used for normalization are depicted as black dots and individual datapoints as red dots. P-values: **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.

Figure 6: Assembloid-specific secretome and compartment-specific transcriptome analysis. (A) Representative photograph showing the assembloid at day 7 (d7), when secretome and transcriptome samples were taken, and depiction of the underlying workflow. (B) VEGF concentration (pg/mL) in the supernatant of core // cell-free and core // fibroblast assembloids after 7 days of co-culture (37 °C, 20% O₂) depicted as boxplots. N = 6. (C) Representative confocal microscopy images of core // endothelial cell assembloids after 7 days of co-culture (37 °C) under high oxygen tension (20% O₂) and low oxygen tension (3% O₂). Cell nuclei are stained in blue (DAPI), and the embedded endothelial cells co-express enhanced green fluorescent protein (EGFP) alongside the endothelial cell marker platelet-derived growth factor subunit b (Pdgfb). The dotted line indicates the compartmental interface between the core explant (E) and the endothelial cell-laden hydrogel (HG). (D) Scatterplot depicting the fold changes in Vegfa, Tnf-α, Fgf2, and Mmp3 gene expression in the core compartment from core // endothelial cell assembloids co-cultured under low oxygen tension (3% O₂) normalized and compared to those cultured under high oxygen tension (20% O₂). N = 2. (E) Scatterplot depicting the fold changes in Vegfa, Tnf-α, Fgf2, and Mmp3 gene expression in the extrinsic compartment of core // endothelial cell assembloids with an alive core (aC) or a devitalized core (dC) co-cultured under high oxygen tension (20% O₂) and low oxygen tension (3% O₂). The fold changes in the respective conditions were normalized to the extrinsic compartment of a core // endothelial cell assembloid with a devitalized core (dC) co-cultured under high oxygen tension (20% O₂). N = 3-4. In B, the upper and lower hinges of the boxplots correspond to the first and third quartiles (25^th and 75^th percentiles) and the middle one to the median. Whiskers extend from the upper / lower hinge to the largest / smallest value no further than 1.5 times the interquartile range. Outliers are depicted as black dots. P-values: *p < 0.05. In D and E, the datapoints used for normalization are depicted as black dots, and individual datapoints are depicted as red dots. Please click here to view a larger version of this figure.

Table 1: Input requirements for tendon disease and injury model systems. A list of primary tendon disease triggers and secondary drivers matched to a selection of input parameters whose tractability is central for modeling tendon disease and injury. Please click here to download this Table.

Table 2: Output requirements for tendon disease and injury model systems. A selection of tendon disease hallmarks matched to a selection of output parameters whose quantifiability is central for the interpretation of tendon disease and injury model behavior. Please click here to download this Table.

Supplementary File 1: .stl file for the clamp holders, the mounting station, and the chamber molds. Please click here to download this File.

Supplementary File 2: Plan of right clamp holder. Please click here to download this File.

Supplementary File 3: Plan of left clamp holder. Please click here to download this File.

Supplementary File 4: Plan of the mounting platform Please click here to download this File.

Supplementary File 5: Plan of metal clamps. Please click here to download this File.

Supplementary File 6: Image showing cell-free hydrogel shrinkage. Please click here to download this File.

Supplementary File 7: Image showing a devitalized core explant. Please click here to download this File.