CD44 expression trends of mesenchymal stem-derived cell, cancer cell and fibroblast spheroids on chitosan-coated surfaces

Preparation of coated cultured plates

Chitosan (Sigma-Aldrich, St. Louis, MO, USA; C3646) with more than 75% deacetylated was used, which was extracted from shrimp shells. Chitosan was coated on tissue culture polystyrene (TCPS; Corning Inc., Corning, NY, USA) using methods described in a previous study [36]. One percent (w/w) chitosan dissolved in 0.5 M acetic acid was coated to plates that were about 10 μL/cm² in size. The chitosan-coated plates were dried in an oven at 60°C and electric-neutralized by 0.5 N NaOH. Next, the plates were exposed under ultraviolet light for sterilization.

Attenuated total reflection-Fourier transform infrared (ATR-FTIR)

Before the plates were analyzed under the ATR-FTIR spectroscopy (Bio-Rad, Hercules, CA, USA; UMA 600), they were washed by water twice and air-dried in laminar flow. Absorbances were evaluated from 4000 cm⁻¹ to 650 cm⁻¹ with a spectral resolution of 4 cm⁻¹. The absorbances of the materials and background were evaluated using 64 scans for each sample.

Cell culture

The 3A6 (human mesenchymal stem-like cell line), SW620 (human colon cancer cell line), and Hs68 cells (human newborn foreskin fibroblast line) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Biological Industries, Cromwell, CT, USA), 100 U/mL penicillin G, 100 μg/mL streptomycin, and 250 μg/mL amphotericin B (Biological Industries).

The 3A6 cell line was a gift from Professor Hung, Shih-Chieh (Institute of Clinical Medicine, National Yang-Ming University, Taiwan), 3A6 cells were isolated from human bone marrow, and have been confirmed to have the basic characteristics of mesenchymal stem cells [38]. The cell seeding densities were about 1.5×10⁴/cm² on TCPS and chitosan-coated surfaces.

Immunofluorescence

Cell spheroids were fixed in 4% paraformaldehyde and incubated with mouse anti-CD44 primary antibody (Invitrogen, Carlsbad, CA, USA; 33-6700). FITC conjugated goat anti-mouse IgG antibody (Millipore, Billerica, MA, USA; 12-506) was used as the secondary antibody. The cells were mounted and observed using a confocal fluorescence microscope (LEICA Microsystems, Wetzlar, Germany; SP5).

Flow cytometry

Monolayer and spheroid cells were harvested by trypsinization for 10 min and pipetted to produce single cell suspensions. To determine cell surface antigen expression, the samples were incubated with the CD44-APC (BD Biosciences Pharmingen, San Diego, CA, USA; 559942) antibody. Cells were analyzed on FACSCalibur (BD Bioscience Pharmingen) and positive cells were determined by the higher fluorescent proportion of the population, which was compared with the isotype controls.

Hypoxia and stravation conditions

For hypoxia conditions, cells were cultured in a gas mixture composed of 94% N₂, 5% CO₂, and 1% O₂, which was maintained using an incubator with two air sensors (Baker Ruskinn, South Wales, UK; Invivo2). For stravation conditions, cells were cultured in 1% FBS medium instead.

Preparation of a chitosan-solution-added medium

Preparation of a chitosan-solution-added cultured system followed the previous study [39]. Chitosan was dissolved in 0.5 M acetic acid and mixed with the medium to achieve 0.3 mg chitosan per mL medium. The without-chitosan groups were prepared in the same manner as the chitosan-containing medium without adding chitosan.

Statistical analysis

All measurements are presented as mean±standard deviation. Statistical significance was evaluated using at least three independent samples; comparing groups were performed using the Student’s t-test. Statistically significant values were defined as p<0.05 and p<0.01. These data were generated from several cultures that were generated in different situations and analyzed with TCPS groups as the reference every time.

Chitosan-coated surface

The chemical composition of the chitosan-coated surface was analyzed by ATR-FTIR. Figure 1 shows the ATR-FTIR spectra of TCPS, chitosan, and the chitosan-coated surface before and after the immersion into the medium. In contrast with spectra of TCPS and chitosan, there are significant peaks at 895 (saccharide structure), 1030 and 1060 (C–O stretching vibrations), 1150 (C–O–C in glycosidic linkage), 1580 (N–H bending vibration), 1650 (C=O stretching vibration), and 3000–3600 (N–H and O–H stretching vibration) cm⁻¹ in spectra of chitosan and chitosan-coated surfaces. Moreover, after 3, 5 and 7 days of immersion into the medium, the specific chitosan IR spectra did not significantly change on chitosan-coated TCPS. These results assured that chitosan was successfully coated on TCPS to form a thin film and was stable in the culture medium environment.

Fig. 1:

ATR-FTIR spectra of TCPS, chitosan membrane, and chitosan-coated surfaces before and after the immersion into the medium within the 7 days.

Morphologies and diameters of multicellular spheroids

Figure 2 shows 3A6, SW620, and Hs68 cells successfully attached and proliferated on TCPS at day 3 after seeding. Both 3A6 and Hs68 cells exhibited the spindle morphology. SW620 exhibited the typical epithelial cell shape with round morphology.

Fig. 2:

Optical images of cells on TCPS. (a) Mesenchymal-derived stem cells 3A6. (b) Cancer cells SW620 (c) Fibroblasts Hs68. Scale bar=200 μm.

Figure 3 shows morphologies of 3A6, SW620, and Hs68 cells cultured on a chitosan-coated surface for 3, 5, and 7 days. Consistent with previous studies [27], [36], [40], almost all cells could suspend and form aggregates spontaneously. This indicates that the cell–cell distance of traditional cultures on TCPS was obviously greater than that of a suspension culture on chitosan-coated surfaces. The average spheroid size is shown in Table 1. The size of Hs68 cell spheroid decreased with culture time, while SW620 and 3A6 spheroids had the largest size on the fifth day. In these spheroids, SW620 could aggregate the largest spheroids on the fifth day.

Fig. 3:

Optical images of cell spheroids on chitosan-coated surfaces during a 7-day cultured period. Scale bar=100 μm.

CD44 expressions of cell spheroids

CD44 proteins participate in many cellular processes, which include the regulation of growth, survival, differentiation, and motility [41], [42], [43], [44], [45]. Especially, CD44 protein is the common surface marker of MSCs and cancer cells [1], [2], [3], [4], [8], [9], [10], [11], so we assessed the surface antigen CD44 expression of MSCs and cancer cells by confocal microscopy after the cells were cultured on chitosan-coated surfaces for 3, 5, and 7 days. As shown in Fig. 4, overall multicellular spheroids were stained with the CD44 antibody. Thus, the flow cytometry was further used to quantitatively analyze the expression of CD44 in monolayer cells on TCPS and spheroid-derived cells on chitosan-coated surfaces.

Fig. 4:

Confocal immunofluorescence images of CD44 expressions of cell spheroids on chitosan-coated surfaces during a 7-day cultured period. The center images are the xy planes, and the images below and right are xz and yz planes. Scale bar=75 μm.

Fibroblasts were included in this study because they are well differentiated and stable. As shown in Fig. 5, there was no significant change in the CD44 expression of Hs68 cells, regardless of substrates or culture period. For 3A6 and SW620 cells, they exhibited the different tendencies for the CD44 expression. After having the cells cultured on chitosan-coated surfaces for 3 days, the CD44 expressions of 3A6 and SW620 cells in the spheroids were less and more than that on TCPS, respectively. On further culture, the CD44 expression of 3A6 and SW620 cells in the spheroids increased and decreased with culture time, respectively. On the 7^th day, the CD44 expressions of 3A6 and SW620 cells in the spheroids have been more and less than that on TCPS, respectively. Thus, the CD44 expression of 3A6 and SW620 cells in the spheroids were dynamic and totally different.

Fig. 5:

CD44 expressions of multicellular spheroids on chitosan-coated surfaces during a 7-day cultured period. The positive ratios of CD44 expression were determined by flow cytometry. n=3, ∗p<0.05, ∗∗p<0.01.

CD44 expressions of cells in different cultured environments

Subsequently, various culture environments were established to investigate how chitosan-coated surfaces affected the CD44 expressions of cell spheroids. Major differences between cells cultured on TCPS and chitosan-coated surface were considered in this study, such as cell–cell distance, hypoxia/starvation, and the effect of chitosan itself.

When cells were cultured on TCPS, the cells grew in a 2D environment and the cell–cell distance was obviously greater than that of cells being aggregated into multicellular spheroids. To simulate compact cell contact within cellular spheroids, we increased the seeding density for cells cultured on TCPS to decrease the cell–cell distance. Figure 6a shows the CD44 expression of 3A6 cells decreased with increasing seeding density (p<0.05 and p<0.01), while in the cases of SW620 and Hs68 cells, CD44 expression remained stationary. The differentiation of stem cells has been reported to be greatly regulated by their niche [46]. If mesenchymal stem cells can aggregate closer and communicate well with each other, it would greatly improve differentiation capability [31], [34]. Therefore, the 3D spheroid structure with high aggregation degree provided 3A6 cells with the tendency to differentiate so the CD44 expression of 3A6 cells significantly decreased at high seeding density after 3 days of culture.

Fig. 6:

CD44 expressions of cells cultured with different environments on TCPS. Cells were cultured (a) with different seeding densities. (b) with hypoxia and starvation. (c) with and without chitosan solution-added medium. The positive ratios of CD44 expression were determined by flow cytometry at 3-day culture. n=3, ∗p<0.05, ∗∗p<0.1.

In addition to reducing the cell–cell distance, the dense spheroid structure developed hypoxia and starvation at distances beyond the diffusion capacity of oxygen and nutrients [47]. Recently, low oxygen levels have been proposed to extensively affect cell behaviors [48]. Thus, the concentrations of oxygen and serum were decreased for cells cultured on TCPS to simulate the microenvironment of multicellular spheroids. As shown in Fig. 6b, the CD44 expression of 3A6 cells increased when the concentrations of oxygen (p<0.05) and serum were reduced on TCPS. However, the CD44 expressions of SW620 cells significantly decreased with low oxygen and nutrient levels (p<0.01). Therefore, a spheroid structure with a high aggregation degree provided hypoxia and a starvation environment for 3A6 and SW620 cells to exhibit opposite CD44 expressions, especially for cells within spheroids after a longer culture period.

It is likely that the effects of chitosan-coated surface on the CD44 expression of 3A6 and SW620 cells stem from its interaction with cells, or specific proteins and then to interact with cells for desired functions. Actually, chitosan is able to interact with many proteins, which is important in mediating cell and tissue responses [39], [49]. Therefore, chitosan was directly added into the cultured medium for cells cultured on TCPS. Obviously, Fig. 6c shows the CD44 expression of SW620 cells increased when cells were cultured in chitosan-added medium (p<0.01), suggesting chitosan played an important role in inducing the higher CD44 expression of SW620 cells on a chitosan-coated surface after 3 days of culture. On the other hand, the expression of CD44 for 3A6 cells did not significantly change in the presence of chitosan. Therefore, the reason for the lower CD44 expression of 3A6 cells on chitosan-coated surface after 3 days of culture may be the compact cell contact within the cellular spheroids (Fig. 6a).

For organism, there are various kinds of cells with varied morphologies and functions in the body that cooperate to maintain life. In this study, it has been confirmed that fibroblasts were relatively stable when cultured on different substrates and environments. However, MSCs and cancer cells were easily affected by environment factors to exhibit the different tendencies of protein expressions. These results indicated that responses of different kinds of cells should be taken into consideration simultaneously when biomaterials were implanted into the body for further applications. Therefore, we investigated and compared the effect of chitosan on CD44 expressions of 3A6, SW620, and Hs68 cells simultaneously in this study. The advantage of chitosan is that it is capable of establishing a suspension culture model to develop multicellular spheroids, which mimics in vivo 3D environment. Notably, CD44 expression on 3A6 and SW620 cells was dynamic and diverse so it is not suitable for observing the interaction between biomaterials and cells in vitro at a specified cultured time. In other words, the evaluation of biomaterials should be more cautious. Furthermore, many factors such as cell–cell distance, hypoxia/starvation, and the effect of chitosan itself in a 2D environment was designed to figure out how to regulate CD44 expression of 3A6 and SW620 cells in 3D environment. Interestingly, the complex responses of 3A6 and SW620 cells in multicellular spheroids could be delineated by the interactions between cell and cell, cell and chitosan, as well as cell and the microenvironment. Based on these data, the cell behaviors were regulated not only by the chitosan properties but also by many other factors. This indicates that more factors and aspects should be well considered when analyzing the interaction between the biomaterials and cells, which would help us to use biomaterials and regulate cells precisely.

Finally, upon seeding cell spheroids back to TCPS, Fig. 7 shows that cells could attach to TCPS and migrate out from the spheroids. This suggested cells were could be cultured and regulated for further tissue engineering applications.

Fig. 7:

Optical images of reseeding cells on TCPS. Cells cultured on chitosan-coated surfaces for 3 days and reseeded to TCPS for 3 days. (a) 3A6. (b) SW620. (c) Hs68. Scale bar=200 μm.