Three-dimensional bio-impedance analysis
Bio-impedance analysis of HUCLs was carried out to compare two different cell culture media (DMEM/F12 and K-SFM) at three different cell densities (30,000, 60,000 and 100,000 cells per well) as shown in Figure 1 (A – F). HUCLs formed a mature confluent barrier as indicated by a plateau in the impedance (Z) represented as log normalized values on the y-axis in the 3D model. As such, HUCLs grown in DMEM/F12 media at all three seeding densities (A – C) formed a mature confluent barrier faster than similar cells grown in K-SFM media (D – F). Furthermore, three-dimensional representations of normalized impedance across HUCLs as a function of both time and log frequency showed DMEM/F12 at a density of 60,000 was most optimal for barrier maturation (B). Likewise, at a 60,000-seeding density, the logarithmic growth curve reached a plateau (time to confluency) after 6 hours with DMEM/F12 (B) compared to >14 hours with K-SFM (E). Thus, indicating that HUCLs grown in the supplemented media more efficiently form an epithelial barrier than cells similarly grown in unsupplemented media.
Next, we aimed to dissect the influence of DMEM/F12 and K-SFM media on the two components of impedance: pure resistance (R) and capacitance (C). When cells are challenged with an AC, both R and C are created, resulting in the overall impedance, Z. To determine which frequency to use in subsequent evaluations, frequency dependence spectra of these parameters were measured as shown in Figure 2. The frequency dependence of variables Z, R, and C for cells grown in DMEM/F12 at the three cell densities at T = 15 hours are shown in panels A – C, respectively. Panels D – F display the same information for HUCLs grown in K-SFM at T = 15 hours. As shown in Figure 2, the impedance spectrum showed a characteristic frequency of 32 kHz, providing the greatest possible range for group comparison of cells grown in DMEM/F12 (A) and K-SFM media (D). On the other hand, we observed that 4000 Hz produces the maximum resistance in both DMEM/F12 and K-SFM media (Fig. 2B & E, respectively). Further, capacitance ratios displayed that optimal cell spreading was achieved at 64 kHz for both DMEM/F12 (Fig. 2C) and K-SFM media (Fig. 2F). However, K-SFM showed overlap between the three cell densities with greater standard deviations than DMEM/F12, thus indicating potential suboptimal conditions for HUCLs growing in the K-SFM media.
Impedance (capacitive reactance) measurements, as shown in Figure 3A – E, calculated at a high frequency provide information as to when the cell monolayer is in place and confluent. This is reflected by the plateau in the impedance when measured at 32 kHz. Cells grown in DMEM/F12 reached the plateau phase at 15 hours for the 30,000-seeding density, and at 4 – 6 hours for both 60,000- and 100,000-seeding densities. Whereas HUCLs seeded at the same densities but grown in K-SFM did not display a distinct plateau phase, indicative of poor HUCL spreading. This trend is further illustrated in both total (Fig. 3D) and endpoint (Fig. 3E) impedance measurements generated at 32 kHz; impedance values for HUCLs grown in DMEM/F12 were significantly higher when compared to K-SFM media. These impedance measurements indicate that the HUCLs grown in the DMEM/F12 are able to form and maintain a strong and confluent monolayer. Thus, indicating that investigation of HUCL barrier formation should be carried out using supplemented DMEM/F12 media in place of the classically used K-SFM media.
Resistance measurements taken at a low frequency provides insight into the barrier formation and function. Figure 4A – C show resistance measurements generated at 4000 Hz from HUCLs seeded at the three different cell densities and grown in the two different culture media. Barrier formation is indicated by the plateau phase in each resistance profile. HUCLs grown in supplemented DMEM/F12 reached the highest resistance (9,000 Ω) at 60,000 (B) and 100,000 (C) seeding densities. However, HUCLs cultured in K-SFM failed to reach a distinct “plateau phase” with a maximum resistance of ~ 6,000 Ω. The total and endpoint resistance values shown in Figure 4D and E, respectively, were generated out to a maximum of 16 hours as determined by the barrier formation plateaus for both groups of media. Total resistance values were significantly higher in DMEM/F12 versus K-SFM at seeding densities of 60,000 and 100,000 cells. Furthermore, endpoint resistance measurements showed that all three cell densities grown in DMEM/F12 were significantly higher compared to K-SFM cells, indicating the formation of tighter and stronger epithelial cell barriers when grown in DMEM/F12. Collectively, these results indicate that the optimal growth, barrier formation, and sustaining conditions for HUCLs are best carried out in the DMEM/F12 media. Without the supplementation, as indicated by HUCLs grown in K-SFM, “mature” barriers are not formed, thus providing further evidence for the use of supplemented DMEM/F12 media when studying corneal epithelial function in vitro.
As with the resistance measurements described above, the growth characteristics of HUCLs were observed in the real-time formation of confluent cell layers and measured as capacitance (Fig. 5A – E). Cells grown in the supplemented DMEM/F12 media displayed more efficient cell spreading at all seeding densities compared to cells grown in K-SFM. At the 30,000 seeding cell density (A), cells grown in DMEM/F12 reached a confluent monolayer between 12 and 14 hours. HUCLs seeded at 60,000 and 100,000 cells grown in the DMEM/F12 formed a confluent monolayer between 2 – 4 hours (B & C). Whereas HUCLs at either 30,000 or 60,000 seeding densities grown in K-SFM exhibited much less efficient cell spreading. Cells grown in K-SFM were able to establish a confluent layer; however, it took much longer at 15 hours. To further illustrate the differences between DMEM/F12 and K-SFM in the formation of a confluent layer, total and endpoint capacitance measurements are also shown. Total capacitance (Fig. 5D) was significantly lower at 60,000 and 100,000 cell seeding densities for DMEM/F12 compared to K-SFM. As shown in Figure 5E, endpoint capacitance for all three seeding densities was significantly decreased in DMEM/F12, as well. Because of the inverse relationship between capacitance and cell spreading, it is indicated that the DMEM/F12 media better supports the growth, spreading and formation of a confluent cellular layer compared to the classically used K-SFM growing conditions.
Mathematical modeling of the R data - Rb, α and Cm
The ECIS software has the ability to model the impedance into parameters that distinguish between cell-cell (Rb) and cell-matrix (α) adhesions, as well as membrane capacitance (Cm). Rb is the resistivity of cell-cell contacts to the current flow. α is measures the impedance contributions arising from the cell–electrode junctions. Therefore, the contribution of Rb, α, and Cm to the observed changes in previous experiments was calculated by fitting a mathematical model developed by Giaever and Keese7. Rb, α, and Cm values were measured from HUCLs at the 60,000 cell seeding density grown in DMEM/F12 compared to K-SFM media and are presented in Figure 6A–F.
The constructed parameter α, indicating the strength of interaction between the cells with the basal substrate, is higher in cells grown in DMEM/F12 compared to K-SFM throughout the entire time course (Fig. 6A). These results combined with total and endpoint α measurements (Fig. 6D), which are also significantly higher for HUCLs grown in DMEM/F12 compared to K-SFM, indicate that cells grown in the DMEM/F12 media create stronger cellular attachments to the basal substrate. These data may also contribute to the overall differences seen in the resistance values between HUCLs grown in DMEM/F12 versus K-SFM.
Furthermore, Rb values, which is indicative of paracellular barrier strength, were higher in HUCLs cultured in DMEM/F12 media when compared to HUCLs grown in K-SFM media (Fig. 6B). This observed increase in barrier function is further demonstrated by corresponding total and endpoint Rb values (Fig. 6E), where HUCLs grown in DMEM/F12 displayed significantly higher Rb values than K-SFM media. In addition to the α value, the fact that HUCLs grown in DMEM/F12 displayed higher Rb values compared to the cells grown in K-SFM indicates the stronger cell-cell interactions are also playing an underlying role in the overall differences observed in resistance.
Cm, or the capacitance of the cell membrane, is indicative of temporal alterations in membrane thickness and composition, as shown in Figure 6C. Additionally, Cm measurements are used to determine if variations in capacitance are only due to changes in electrode coverage or are a function of microvariations in the apical membrane structures. Total Cm is not presented since confluent monolayers are required to model this parameter, which did not consistently occur at earlier timepoints for cells grown in K-SFM. As a result, only end-point Cm is shown, which is significantly lower in HUCLs grown in DMEM/F12 compared to K-SFM (Fig. 6F). Therefore, the interpretation from the data is that the differences in Cm are due to differences in electrode coverage and not membrane structure.