Biotech

Small spheroids for head and neck cartilage tissue engineering

Time Stamp: December 29, 2024 7:00 PM
Source Node: 842409

Optimization of spheroid size and cell density

An important consideration for spheroid culture is the choice of optimal cell numbers. The goal is to use the minimum number of cells necessary to produce stable aggregates of a sufficient size for easy handling and analysis, without requiring unnecessary 2D cell expansion. To estimate the minimal cell density for producing viable aggregates with stable shape and sufficient size, we prepared nasal CC and CPC spheroids with cell numbers ranging from 0.5 to 10 × 10⁴ per single construct and cultured them in the ChDif medium. Nasal spheroids tend to be relatively small compared to auricular aggregates³⁵, which determined our choice for nasal CC and CPC in this experiment.

Spheroids formed with cell numbers as low as 0.5 × 10⁴ and increased proportionally in size with higher cell densities (Fig. 1A and B, Figure S1A). In most spheroids, a reduction of the initial size (shrinking) was observable already by culture day seven and progressed until day 21. Typically, size reduction was more pronounced in larger spheroids, and was observable in CPC spheroids from both donors, as well as CC constructs from donor 1 (Fig. 1A and B, Figure S1A). The area of CPC spheroids was reduced by 3-fold for medium-sized aggregates prepared from 2.5 × 10⁴ cells and by more than 4-fold for larger spheroids containing 5 × 10⁴ and 10 × 10⁴ cells (Fig. 1C and D). In contrast, small CPC spheroids containing 1 × 10⁴ cells shrunk by < 2-fold, while the smallest CPC spheroids prepared from 0.5 × 10⁴ cells remained constant or even slightly increased in size (Fig. 1C and D). Due to the strong shrinkage of larger spheroids, the differences between different spheroid diameters were reduced by culture day 21, constituting 243.5 ± 7.2 μm for the smallest, 264.4 ± 29.3 μm for medium (2.5 × 10⁴) and 417.5 ± 55.2 μm for the largest CPC spheroids (Fig. 1C).

Unlike CPCs, CC spheroids showed contrasting behavior between the two donors. Although both donors formed similar-sized spheroids per given cell density (Fig. 1A and B, Figure S1A) aggregates from donor 1 shrank similarly to their CPC counterparts (Fig. 1), while those from donor 2 increased slightly in size irrespective of the cell density (Figure S1A, Fig. 1C and D). The shrinkage pattern in donor 1 CC spheroids resembled that of the CPC aggregates but with an even more pronounced reduction of the initial size than observed with CPCs. Namely, the size of larger spheroids (5 and 10 × 10⁴) was reduced by more than 5-fold, while a 3-fold reduction from the initial size was seen in smaller spheroids with 2.5 × 10⁴ cells, and less than 2-fold reduction was observed in the smallest spheroid with 1 and 0.5 × 10⁴ cells (Fig. 1D).

As expected, auricular CCs and CPCs formed larger spheroids than nasal cells. Spheroids from both types of auricular cells exhibited growth in culture with no tendency to shrink, forming substantially large constructs by day 21 even with a cell density as low as 1 × 10⁴ (Figure S1B).

In summary, consistent shrinkage-growth patterns were observed among medium-sized (2.5 × 10⁴) and large nasal spheroids (5 and 10 × 10⁴), while lower cell numbers (0.5 and 1 × 10⁴) formed small spheroids with very little size change over 21 days in culture, probably indicative of low cell-cell interaction and matrix remodeling within the aggregates. Furthermore, aggregates formed with 2.5 × 10⁴ cells were sufficiently large and exhibited comparable viability to their larger counterparts. Given these factors, 2.5 × 10⁴ cells per spheroid were assumed as an optimal minimal cell number for nasal spheroids. For consistency, auricular spheroids were also prepared with a similar cell density throughout the study.

Finally, we also assessed whether spheroids at this density could fuse into larger constructs when co-cultured. We repeatedly observed a complete fusion of constructs with 2.5 × 10⁴ cells, which typically took up to 12 days to complete (Figure S2).

Comparison of MM and spheroid culture

To investigate if alternative scaffold-free culture methods could effectively generate cell aggregates with the established minimal cell density of 2.5 × 10⁴, we conducted micromass (MM) cultures using nasal CCs with comparable cell numbers. Alternatively, we prepared MM cultures with a high cell density of 30 × 10⁴ cells, approximating the average numbers reported in previous studies for MM culture^21,22,23. Both types of scaffold-free cultures were cultivated in ChDif or CDM supplemented with TGF-β1 (Table 1).

In contrast to spheroids, the aggregation of cells in MM cultures was often delayed or even ineffective. The aggregates were only visible on the third day in MM culture with TGF-β1, while spherical self-assembly was visible already at the start of the culture on low-attachment plates (Fig. 2A, Figure S3A). The viability of cells in both MM and spheroid cultures in TGF-β1 medium was visibly compromised, as indicated by the increased intensity in the SYTOX nuclear stain (Fig. 2C). Although the cell density of the MM culture did not seem to affect the viability of cell aggregates in the TGF-β1 medium, it did affect the shape of the aggregate, which occasionally deviated from a spherical structure and acquired an elongated form (Figure S3).

The behavior of both cell aggregate types was different in ChDif as compared to the culture with TGF-β1 supplementation. Namely, a complete lack of cell aggregation was observed in MM cultured in ChDif medium at a density of 2.5 × 10⁴ and only delayed cell aggregation with a formation of non-spherical, spread-out cellular structure was observed at a higher density of 30 × 10⁴ (Fig. 2B). In some donors, no MM aggregate formation was observed even at higher cell density in ChDif (Figure S3B). In contrast, spheroids from all donors formed effectively in the ChDif medium on the ULA surfaces. Furthermore, unlike spheroids, an outgrowth of cells attaching to the plastic was usually observed in large MM structures, which disrupted the sphericity of the aggregate structure (Fig. 2B).

The IHC analysis revealed no collagen II staining in MM cultured in TGF-β1 medium at any cell density, whereas moderate positive staining for collagen II was detected at the edges of spheroids (Fig. 2A). A limited presence of GAGs was only observed in spheroids and, to a lesser extent in MM with 30 × 10⁴ cells in TGF-β1 CDM, as represented by an inhomogeneous and weak Alcian Blue stain.

Consistently with the lack of effective cell aggregation, MM cultures of any cell density also did not show positive collagen II staining after 21 days in the ChDif medium. In contrast, the collagen II-positive area was observed centrally in spheroids cultured in ChDif on ULA plates (Fig. 2B). Alcian Blue staining was also more pronounced in ChDif cultures, particularly in spheroids and partially formed MM aggregates with 30 × 10⁴ cells. Collagen I staining was positive in all 3D aggregates, regardless of the culture condition, but was especially high in low-density MM with 2.5 × 10⁴ cells (Fig. 2A).

Overall, the formation of spherical cell aggregates was more efficient on ULA plates as compared to the MM method, which could not facilitate effective cell aggregation at a lower cell density. Additionally, chondrogenic differentiation was observed in nasal spheroids cultured in ChDif medium, but not in nasal MM, as indicated by the production of GAGs and collagen II.

The influence of cytokines on HNC spheroid size, growth dynamics, and viability

The composition of the culture medium is crucial for the successful in vitro maintenance of cartilage spheroids. To evaluate the maturation and growth of HNC spheroids under varying conditions, we supplemented CDM for spheroid culture with different cytokines that are commonly used in chondrogenic cultures (Table 1). ChDif medium was used as a reference throughout the study, as a standard commercially available medium for chondrogenic differentiation.

CDM composition impacted spheroid characteristics, such as size, and viability. Differences in the response to cytokines were observed between auricular and nasal cells, and to some degree, between CCs and CPCs from the same tissues. In all conditions, auricular cells generated overall larger spheroid constructs as compared to the nasal counterparts (Figs. 3A–D and 4). Non-viable cell staining of spheroids from both tissues varied among donors. In particular, auricular donor 1 displayed pronouncedly high SYTOX staining selectively for CC spheroids (Figure S4A), while nasal donor 2 showed poor viability of spheroids prepared from both CC and CPCs (Figure S4B). Nevertheless, the influence of medium composition on viability was discernable with culture time, whereby the donors with poor viability pronouncedly reduced the SYTOX staining in favorable conditions (e.g. ChDif, or IGF-1-containing media), reflecting the tendencies observed in donors with good viability (Figure S4). As a general trend, CPCs from both tissues displayed lower staining for non-viable cells. While similarities were observed between the response of nasal and auricular spheroids concerning size and growth dynamics, the extent of visible cytokine influence differed among the two tissues. The details on the characteristics of different HNC spheroids are described below separately per each tested condition.

All spheroids showed good viability in the ChDif medium relative to other CDM conditions (Fig. 3). Lower staining for non-viable cells was observed in auricular spheroids, particularly in the early culture period (Figure S 4A). The tendency of decrease in SYTOX signal with culture time was observed in spheroids from both tissues, except for aurCPC donor 1, which showed a sudden drastic increase in the SYTOX staining at days 14 and 21 (Figure S4A, Fig. 3E). Irrespective of the viability, auricular spheroids showed a gradual increase in size (Figs. 3A and B and 4A). By day 21 in the ChDif medium, the diameter constituted on average 969 ± 65.7 μm for aurCC spheroids and 1057 ± 143.3 μm for aurCPC spheroids.

In contrast, nasal spheroids, which were substantially smaller than their auricular counterparts (Fig. 3C and D), shrank further with culture time (Fig. 4B). Here, CPC spheroids were smaller than CCs and the difference in size became even more apparent at later culture time points, with CPC aggregates shrinking more in comparison to CC spheroids (Fig. 4B). By day 21, nasCC spheroids measured 427 ± 38.9 μm and nasCPC constructs reached the average diameter of 325 ± 15.7 μm. Importantly, in contrast to the experiments with the cell density (Figure S 1 A), all nasCC spheroids cultured in ChDif in this experiment exhibited shrinking behavior without an exception.

The two growth factors of the TGF-β family had very comparable effects on the size and viability of different spheroids. For both TGF-β1 and TGF-β3, auricular and nasal spheroids alike showed relatively high SYTOX staining (Fig. 3E), which reduced only slightly with culture time (Figure S 4) and even increased in the case of aurCPCs (Figure S 4A). Auricular spheroids cultured in a media with TGF-β1/3 were distinctively small (Fig. 3A and B) and showed no change in size during the culture period, irrespective of changes in viability (Fig. 4A). Nasal CCs and CPCs showed some shrinkage in TGF-β media but the size decrease was more moderate as compared to nasal spheroids cultured in ChDif (Fig. 4B).

CTGF is a downstream effector of TGF-β³⁶ and stimulates the expression of IGF-1 in chondrocytes³⁷. Furthermore, it is known to bind and synergize with both IGF-1 and TGF-β in different physiological and pathologic conditions³⁷. In auricular samples, CTGF produced spheroids with intermediate characteristics in terms of viability and size as compared to those grown in TGF-β1/3 and all other CDM. Namely, CTGF spheroids were slightly larger (Figs. 3A and B and 4A) and showed somewhat lower SYTOX staining (Fig. 3E) than TGF-β1/3 aggregates but were smaller and less viable as compared to other CDM conditions. CTGF-grown nasal spheroids overall resembled those cultured with TGF-β1/3 in terms of size and shrinkage patterns (Figs. 3C and D and 4B) but showed higher SYTOX signal compared to TGF-β1/3 counterparts (Fig. 3E).

IGF-1 is an anabolic cytokine, which is produced by chondrocytes and, similarly to the CTGF plays an important role in chondrocyte proliferation, GAG synthesis, and maintenance of phenotype^37,38. In terms of spheroid size, supplementation of medium with IGF-1 yielded very similar spheroids to those cultured in IGF-1 and CTGF combination.

A strikingly pronounced effect of IGF-1 was observed on the size of auricular spheroids cultured with IGF-1 alone or in combination with CTGF (Fig. 3A and B). As with ChDif, the distinctively large size of auricular spheroids in IGF-1-containing CDM was apparent already at culture day 3 and progressively increased with culture time (Fig. 4A). Non-viable cell staining was also comparable to that of auricular spheroids in ChDif typically displaying low MFI values for SYTOX (Fig. 3E).

Nasal spheroids cultured with IGF-1-containing media were not as large as auricular aggregates but were nevertheless the largest among spheroids in all conditions compared (Fig. 3C and D). Furthermore, with IGF-1 only negligible shrinkage was observed after culture day 3 in nasal CPC aggregates, while no shrinkage and even slight growth was observed in nasal CC spheroids, which were on average larger than their CPC counterparts (Fig. 4B). Stable viability of both nasal CC and CPC spheroids was observed in IGF-1-containing media (Fig. 3E), with a low SYTOX signal in donor 1 and a reducing SYTOX signal in donor 2 (Figure S4B).

The influence of cytokines on mitochondrial ATP

ATP is the primary source of cellular energy. It is commonly used as an indirect indicator of metabolic activity, overall cell health, and viability. To confirm the presence of viable, metabolically active cells within HNC spheroids at culture day 21, the aggregates were labeled with a mitochondrial ATP-binding fluorescent dye. Positive staining of ATP was observed in all spheroids, confirming the presence of viable cells within aggregates (Fig. 3A-D). However, the intensity of the mitochondrial ATP signal across various conditions was not always reflective of SYTOX non-viable cell staining. Namely, ChDif cultures had the greatest mitochondrial ATP signal in both auricular and nasal (Fig. 5A) spheroids, including spheroids with high SYTOX staining (e.g. AurCPC, donor 1). In contrast, spheroids in IGF-1-containing media showed the weakest staining signal of mitochondrial ATP (Fig. 5A).

Similarly to insulin, IGF-1 is implicated in glucose transport and oxidation in vitro^38,39. We, therefore, hypothesized that IGF-1 could have affected the level of mitochondrial ATP through its influence on metabolic activity in the individual cells, with subsequent changes in the ATP staining signal.

To test the influence of ATP on individual cells, we cultured all cell types in 2D in either ChDif medium or CDM supplemented with IGF-1 and measured the ATP signal using a luminescence-based assay. Furthermore, the 2D cultures were also stained using the ATP-Red Live Cell Dye, to ensure that our staining of mitochondrial ATP was reflective of the intracellular ATP levels. ATP measurements using a luminescence-based assay were performed immediately after cell attachment at an early time point of 4 h to exclude the increase of ATP due to cell proliferation. In contrast to the spheroid cultures, the measurements of the luminescence-based assay showed a significant increase in ATP levels in all IGF-1 samples, which was particularly pronounced in the aurCCs and even more so in aurCPCs (Figure S5).

To evaluate the dynamic changes in the ATP levels in IGF-1 CDM or ChDif monolayer cultures, we monitored the cells with ATP-binding fluorescent dye until maximal cell confluence. The ATP levels showed a steady increase in all conditions and cell types (Fig. 5B, Figure S6). Consistent with the luminescence measurements, the strongest fluorescence was detected in aurCPCs followed by aurCCs (Fig. 5B). Significantly higher ATP staining was observed in both auricular and nasal CCs and CPCs cultured in IGF-1 CDM as compared to ChDif after 48 h and 72 h (Fig. 5B). As the cells reached confluence at later time points, the overall intensity of fluorescence staining in both conditions began approximating each other. Nevertheless, the differences between the cells cultured in ChDif and IGF-1 CDM were discernable. In particular, ChDif cultures reached a higher density of cells and were confluent by 144 h and over confluent by 240 h (Figure S6), whereas CCs and CPCs in IGF-1 CDM proliferated less, reaching confluence by 240 h, and showed a more granular, defined ATP staining pattern than the cells in ChDif.

Overall, while low ATP staining was observed in spheroids grown in IGF-1-containing CDM, monolayer cultures showed that IGF-1 increases ATP staining in individual cells.

Assessment of cell viability by TUNEL assay

TUNEL assay has been widely used to detect cell-death-associated DNA fragmentation and can be combined with IHC in formalin-fixed paraffin-embedded (FFPE) tissues²¹. Because the conventional imaging method has limitations in evaluating viability, such as the inability to visualize individual cells, and due to the impact of culture medium composition on ATP viability staining as described above, we conducted the TUNEL assay on FFPE auricular spheroids to validate the viability monitoring of live samples in our study ( as shown Fig. 3). Overall, the distribution of TUNEL-labeled nuclei in the spheroid sections was relatively similar to the SYTOX labeling pattern. Similarly, the ATP label corresponded well with the distribution of total cell nuclei within the spheroid (Figure S7).

The influence of cytokines on spheroid morphology and ECM

To assess the quality of chondrogenic cultures, we performed IHC staining of spheroids grown for 21 days in CDM with various cytokine supplementations (Table 1). The spheroids were stained for key cartilage ECM constituents, such as collagen II, aggrecan, and elastin. Furthermore, staining for collagen I was performed to evaluate the tendency for fibrocartilage formation. Positive staining for collagen I was observed in all spheroids regardless of CDM conditions of cell type (Figure S8).

ChDif produced spheroids of moderate cellularity and density of ECM arrangement (Fig. 6A, Figure S9A). All cartilage ECM components tested were positive in all spheroids cultured in ChDif, showing a good chondrogenic capacity of the medium (Fig. 6G). Little to no structural patterns or characteristic distribution of ECM was observed.

TGF-β1 and TGF-β3 produced spheroids with a compact ECM structure, densely populated with cells, and often showed a cellular and ECM arrangement pattern. Particularly, in spheroids cultured with TGF-β1/3, a dense cell layer with a parallel arrangement was often observed surrounding an intermediate layer of cells with a more perpendicular, less orderly pattern and a loose necrotic core in the center (Fig. 6B and C, Figure S9B and C, Figure S10). This structure was most clearly pronounced in nasal CC spheroids (Figure S10).

Concerning chondrogenic differentiation, the presence of cartilage ECM was confirmed in all spheroids cultured in CDM with TGF-β3. In contrast, TGF-β1 induced the production of all tested ECM components in all spheroids, except for auricular aggregates from one of the two donors tested in our study, showing no detectable collagen type II in either aurCC or aurCPC in CDM with TGF-β1 (Fig. 6G, Figure S9B and C, Figure S10). All auricular spheroids grown in TGF-β1/3 showed positive aggrecan staining in both donors. In contrast, nasCPC spheroids from one donor displayed only scarce positive aggrecan staining in TGF-β1/3. Similarly scarce aggrecan staining was also seen in nasCC spheroids from the same donor (Fig. 6G, Figure S9B and C).

While CTGF produced relatively small spheroids with similar size and viability dynamics as TGF-β1/3, structurally the culture groups differed substantially. In contrast to TGF-β1/3, spheroids cultured in CTGF CDM had a looser structure, with randomly arranged cells surrounding an often amorphous and hypocellular core with scarce ECM content (Fig. 6D, Figure S9D, Figure S10).

NasCPC spheroids were an exception and were more compact than all other spheroid types cultured with CTGF, albeit lacking the characteristic outer layer of cells, as seen with TGF-β1/3 (Figure S 10). Remarkably, in one out of the two donors tested, CTGF led to no detectable aggrecan expression in nasCPCs and only very scarce aggrecan production in all other spheroids (Fig. 6D and G, Figure S 9D). Collagen II was detected in all spheroids grown in CTGF CDM, albeit only scarcely in aurCPC spheroids from one of the donors (Fig. 6D and G, Figure S9D). Elastin staining was positive and abundantly distributed throughout the spheroids (Fig. 6D and G, Figure S9D).

Spheroid structure and ECM content in spheroids cultured with IGF-1 were somewhat comparable to those of CTGF-grown aggregates. IGF-1 spheroids showed an even more hypocellular, loose ECM, with a pronounced sponge-like, aerated texture not only in the spheroid core but often throughout the aggregate structure (Fig. 6E, Figure S9E, Figure S10). In contrast to CTGF, and somewhat similar to TGF-β1/3, spheroids cultured with IGF-1 often displayed some discernable zonality particularly in CC aggregates, displaying a distinctive outer layer with a parallel arrangement of cells and ECM (Fig. 6E, Figure S9E, Figure S10). In contrast to TGF-β spheroids, however, here the outer layer was looser than the adjacent intermediate zone, which showed a perpendicular arrangement of cells and ECM and was more densely structured than the outer layer and the spheroid core. As with CTGF, nasCPCs had a relatively compact structure throughout the aggregate as compared to all other spheroid types in the same condition.

Further similarity with CTGF-grown spheroids was the predominantly low presence of aggrecan in one of the donors in the study. In these samples, IGF-1 culture resulted in scarce positive staining in aurCC and nasCPC, moderate staining in aurCPC, and no detectable staining in nasCC spheroids (Fig. 6G, Figure S9E). As with CTGF, collagen II and abundant elastin were found in all aggregates cultured with IGF-1 (Fig. 6E and G, Figure S9E).

A combination of the two cytokines produced large spheroids that showed morphological resemblance to both IGF-1 and CTGF-grown aggregates. Here, however, the abundant presence of aggrecan, elastin, as well as collagen I was detected throughout all spheroid types (Fig. 6F and G, Figure S9F, Figure S8). Similarly, strong collagen II staining was detected in auricular and nasal CC, nasCPC, and somewhat less so, in aurCPC spheroids (Fig. 6F, Figure S9F).

In summary, spheroids cultured in different CDMs showed distinctions in terms of architecture, as well as staining for cartilage ECM proteins. Albeit at varying degrees, the production of all cartilage ECM molecules was stimulated by ChDif, TGF-β3, and a combination of IGF-1 + CTGF in all constructs (Fig. 6G), with particularly superior immunoreactivity of all ECM components visualized by IHC in ChDif and IGF-1 + CTGF spheroids. Notably, however, the latter conditions also stimulated high collagen I production (Figure S8). In other conditions, some cartilage ECM components were absent or scarcely present in either one of the two donors tested in the study, such as collagen II in auricular spheroids cultured with TGF-β1, or aggrecan in nasCPC spheroids cultured with CTGF and nasCC spheroids grown in CDM with IGF-1.

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Source: https://www.nature.com/articles/s41598-024-83847-w

Small spheroids for head and neck cartilage tissue engineering

Optimization of spheroid size and cell density

Comparison of MM and spheroid culture

The influence of cytokines on HNC spheroid size, growth dynamics, and viability

The influence of cytokines on mitochondrial ATP

Assessment of cell viability by TUNEL assay

The influence of cytokines on spheroid morphology and ECM

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