Aggregation volume change and viability of LF spheroids over time
To evaluate the growth trend during the aggregation of LF cells, both 2D in vitro expansion and ULA 96-well plates for 3D cultures at different cell seeding numbers were examined (Figure 1). Figure 1a shows the cell morphology of minced LF tissues placed in a petri dish maintained over three weeks adjacent to the tissue (white dotted line) or away from the tissue (black dotted line). The LF spheroid formation process was observed using different cell numbers (100, 1000 and 5000) continuously from day 0 to day 14 (Figure 1b). Based on the bright-field microscopy images, we found that the cells began to aggregate after 6 hours (D0) of cell seeding. One hundred cells quickly formed a complete spheroid on day 1, and 1000 cells also smoothly aggregated on day 2. While the aggregation of 5000 cells was slower than that of 100 cells and 1000 cells, it still formed compact spheroids on day 4. During the 14-day period, the spheroids grown under the three different cell number conditions all became round in shape. The volume of the 1000-cell and 5000-cell spheroids was quantified on days 4, 7 and 14 (Figure 1c). As a control group, we used cells obtained from patients with herniated intervertebral disc instead of LFH. According to the quantitative results, the size of the 100 cells gradually increased over time (≈2-4Í10-3 mm3 at day 14). Interestingly, while the size of the 1000 cells did not significantly change during the culture time (≈5Í10-3 mm3 at day 14), the size of the 5000-cell spheroids consistently decreased as the culture time increased (≈15Í10-3 mm3 at day 14). The growth trends of the LFH and control (non-LFH patient sample) groups were similar, with no distinctive differences observed.
Figure 1. In vitro 2D LF cell expansion, aggregation processes and volume change in LF spheroids over time. (a) Minced LF tissues placed in a petri dish maintained over three weeks showing the cell morphology adjacent to the tissue (white dotted line) or away from the tissue (black dotted line). The scale bar is 100 μm. (b) Different cell numbers (100, 1000 and 5000 cells) were seeded in a well. Continuous observation for 14 days. The scale bar is 100 μm. (b) Quantification of LF spheroid volume on days 4, 7, 10 and 14. Four different patient samples and different cell numbers were quantified (n = 3).
To confirm that the ULA 96-well platform is a stable method for generating LF spheroids, we evaluated the cell viability within the spheroids (Figure 2). The cells were cultured for 14 consecutive days and assessed on day 7 and day 14. The results showed that regardless of the cell number in the spheroids, most cells exhibited green fluorescence. Few scattered dead cells were detected with the ethidium homodimer (red) staining and could be observed in the center of the spheroids. Therefore, most LF cells in the spheroids presented good viability through day 14.
Figure 2. Live and dead cell staining of LF spheroids in spheroids with different cell concentrations. Spheroids were cultured for 14 days. Live and dead staining on days 7 and 14. BF represents a bright-field image. Live cells exhibit intense green fluorescence. Dead cells exhibit red fluorescence. Hoechst was used to stain the nuclei.
Investigation of LF spheroid long-term culture for 28 days
To investigate whether LF cells are suitable for long-term culture, we tested all three concentrations of 100, 1000 and 5000 cells and cultured the cells for 28 days. The bright-field images showed that the spheroids still maintained compact aggregation (Figure 3a). The live/dead staining results showed that most cells maintained good viability at all concentrations in the spheroids. Furthermore, the quantitative results (Figure 3b) indicated that the cell size in the 5000-cell culture exhibited a decreasing trend over 28 days. The cell size in the 1000-cell culture remained relatively constant throughout the long-term culture. As previously described, the cells in the 100-cell culture continued to grow in size. Over the long-term observation, we found that these cells reached the same size as the cells in the 1000-cell culture on day 21 (≈4.3Í10-3 mm3) and surpassed the size of the cells in the 1000-cell culture by day 28 (≈6.1Í10-3 mm3). Similarly, while the spheroids of 5000 cells were the largest in size among all cells under the three density conditions on day 4 (≈15Í10-3 mm3), the 5000-cell spheroids gradually reduced their size to slightly pass the size of the 100-cell spheroids over 28 days (≈8.0Í10-3 mm3). Considering the lack of a significant indication of cell death based on the different cell densities using the qualitative live/dead fluorescence recording, the 1000-cell spheroids were selected as a representative group to quantitatively determine the viability of the spheroids under long-term maintenance. The PI fluorescent DNA-binding dye often used for apoptosis and cell cycle analyses was used. The highlighted sub G1 region showed that a minute amount of DNA fragmentation could be found in both the 2D and spheroids on different days, while the final time point of the flowcytometric analysis was performed on day 26 due to sample availability (Figure 3c). The quantification of cell apoptosis showed that less than 5% of the cell population was apoptotic under the conditions evaluated. These results demonstrate how spheroids containing different cell numbers gradually approach the same size. In addition, we cultured the LF cells for 28 days to ensure that the cells could be maintained in long-term culture.
Figure 3. Investigation of LF spheroid long-term culture for 28 days. Morphology of LF spheroids with different cell numbers cultured for 28 days. (a) Bright-field image, live/dead cell staining, and Hoechst staining. (b) The growth trends of 100, 1000 and 5000 cells over 28 days; the linear regression of each curve is indicated as color dotted lines. (c) Cell death analysis in 2D and 1000-cell spheroids performed by flow cytometry. (4) Quantification of the percentage of cell death in 2D and 1000-cell spheroids on days 7, 14 and 26 (data of day 28 are not available).
Identification of the phenotypes of LF cells in 2D culture and 3D culture using immunofluorescence staining
LFs are fibroblast-like cells composed mainly of elastin fibers and collagen. Immunofluorescence staining was used to identify the cell phenotype of human lumbar LF cells isolated from surgical specimens obtained from patients and the cultured cells (Figure 4). Both the 2D monolayer cells and 3D spheroids with different numbers of cells after 7 days of culture were compared. On the 2D substrate, the cells uniformly expressed fibronectin, collagen I and collagen III. Weak fluorescence of elastin was also observed in the 2D-cultured cells. The 3D spheroids expressed fibronectin and elastin, and their structure could not be clearly recognized because of the compact cell arrangement as previously mentioned. In addition, collagen I and collagen III exhibited strong fluorescence that was evenly distributed throughout the spheroid, indicating that the cultured 3D spheroid model maintained the typical phenotype after 7 days of culture. Similar results were observed in the 1000-cell spheroids cultured until day 14 (Figure S3).
Figure 4. Identification of the phenotype of cultured LF cells using immunofluorescence staining. Immunofluorescence staining of fibronectin, elastin, collagen I and collagen III in 2D-cultured cells and 3D spheroids (100, 1000 and 5000 cells) was performed.
Protein expression in LF cells
Based on the stable growth trend of the spheroids of 1000 cells in terms of both morphology and volume and the results of the cell cycle (supplement) and phenotypic expression analyses, we further investigated protein expression in spheroids of 1000 cells cultured for 7 days in comparison with the 2D cells (Figure 5). The protein expression of fibroblast-related markers in the spheroids on days 4 and 7 was determined by Western blotting and compared with that in the 2D-cultured cells (Figure 5a). The Western blot analysis revealed that collagen I and collagen III did not significantly change over time (Figure 5a-c). In addition, there was no significant difference in the expression of collagen I or III between the 2D and 3D groups. We also measured the protein expression of elastin and fibronectin (Figure 5d-f). The results showed that elastin expression in the 3D spheroids was lower than that in the 2D culture after 4 days of culture. However, elastin protein expression increased in the 3D group after 7 days. Fibronectin protein expression on day 7 in the 3D-cultured cells was significantly (p value < 0.05) higher than that in the 2D-cultured cells.
To further understand the components of the cell changes under different culture conditions, a Western blot analysis of ECM protein expression in the 2D-cultured cells and spheroids was performed. Normal LF tissue is composed of elastin fibers and collagen in a 2:1 ratio, while a reduction in the elastin-to-collagen ratio is apparent in LFH during aging and degeneration. Both the 2D-cultured cells and 3D spheroids expressed collagen type I and III, and the collagen expression levels did not significantly differ between the two groups. Elastin expression significantly differed between the 3D spheroids and 2D-cultured cells at 4 days of culture. While elastin expression increased in the spheroids on day 7, no significant difference was observed in the 2D-cultured cells between days 4 and 7.
Figure 5. Protein expression in LF cells in 2D culture and 3D culture. One thousand spheroid cells were cultured for 7 days, and collagen I, collagen III, elastin and fibronectin were detected on days 4 and 7. (a) The expression of collagen I and III was determined by Western blotting. (b)(c) Quantitative evaluation of (a). (d) The expression of fibronectin and elastin was determined by Western blotting. (e)(f) Quantitative evaluation of (d). The data represent the mean ± SD. “*” denotes statistical significance with a p value < 0.05 among the experimental groups, whereas “**” denotes statistical significance with a p value < 0.01. Equal protein loading was verified by GAPDH.
Histological comparison of spheroids and human LF tissue
To examine the cell structure and elastin distribution of tissue, LF cells and tissue were stained with H&E. In the normal LF tissue (Figure 6a), a large area presented pink staining, indicating the presence of rich elastic fibers in the tissue. In addition, the elastic fibers and cells were organized in parallel. However, in the LFH tissue (Figure 6b), several nuclei could be observed, and the elastic fibers in the LFH tissue were fragmented and disorganized compared with those in the normal tissue. The H&E staining results of the 2D-cultured cells (Figure 6c) showed that the cells were randomly distributed on the slides, with an obvious polarized and extended morphology. Due to the difficulty of frozen tissue sectioning, the 5000-cell spheroids were selected and cultured for 4 days (Figure 6d). In these spheroids, several nuclei in a compact structure and random aggregation were observed. In contrast to the cells in the 2D culture, the 3D-cultured cells tended to accumulate tightly in the spheroid.
Figure 6. Histological comparison of spheroids and human tissue. (a) Normal LF tissue with abundant normal elastin fibers organized in parallel. (b) LFH tissue with disorganized and fragmented elastic fibers. (c) 2D LF cells were randomly distributed on the slides. (d) Frozen sections of 3D spheroids. Many nuclei could be observed and exhibited compact, nondirectional aggregation. Scale bars = 200 μm