Assessment of liquid media requirements for storing and evaluating respiratory cilia motility

Storage media and the general maintenance of cilia motility

Except for samples stored in saline, cell counting revealed mostly minor differences in the number of cells with motile cilia when stored in all media at all time points, with or without FBS (Fig. 2).

Storage in saline, both at 4 °C or room temperature resulted in a dramatic decrease in the number of cells with motile cilia, which became significantly decreased after only 1 h of storage (−63.7 ± 28% decrease at 4 °C, −42.4 ± 31.4% decrease at room temperature). This decrease continued up to 6 h of storage after which only random isolated cells with motile cilia could be found (if searched for). Storage in 4 °C saline was mildly better than storage at room temperature after 12 h with only a −64.0 ± 24.2% decrease at 4 °C vs a −92.1 ± 10.7% decrease at room temperature (Fig. 2) (Movie S2).

Other cell counting results of note include a small but significant elevation from baseline of cell number when stored at room temperate in HBSS + FBS, which became significantly elevated after 2 h storage (+17.3 ± 12%), and remained slightly elevated up to 12 h (+16.3 ± 14.6%). Conversely the 12-hour samples stored in 4 °C HBSS w/o FBS was slightly but significantly reduced (−20.8 ± 17%) (Fig. 2).

The ability of L15 to maintain the number of cells with motile cilia per observed field of view appeared to be significantly impacted by the absence of FBS. Samples stored in L15 w/o FBS for ≥ 2 h displayed significant reductions in cell number following storage at either 4 °C (−16.4 ± 23%) or room temperature (−23.8 ± 31.5). These reductions remained constant up to 12 h storage (−21.9 ± 14.6% at 4 °C; -23.7 ± 13.9% at room temperature) (Fig. 2).

RPMI w/o FBS showed a single significant anomalous elevation following 2 h storage at room temperature (+24.5 ± 12.1%), while 4 °C storage for 12 h caused small significant decreases in cell numbers when stored in M199 + FBS (−18.7 ± 33.2%), and DPBS/DMEM w/o FBS (−30.1 ± 19.9% / −20.5 ± 19.8%) (Fig. 2).

Storage media and the maintenance of cilia beat frequency (CBF)

A range of different changes in CBF were observed for samples stored in the different media (Fig. 3).

Short term storage in saline (≤ 2 h) resulted in significant reductions in CBF for samples stored at both 4 °C (-43.0 ± 13.5%) and room temperature (38.8 ± 39.1%). This was followed by CBF in 4 °C saline samples returning to baseline levels after 12 h (3.8 ± 35.6%) while CBF in room temperature saline samples dropped by −84.5 ± 18.2% (Fig. 3). It should be highlighted that the CBF results for saline need to be taken in context with the cell count numbers shown in the previous figure (Fig. 2), cell numbers were significantly reduced after short term storage in saline and the CBF results were obtained from the very sparsely remaining ciliated cells that in many cases were hard to find.

One general trend observed for just about all media tested was that room temperature storage of ciliated samples resulted in significant elevations in CBF from baseline values, and this appeared generally more prevalent in media containing FBS (Fig. 3). As seen in DPBS + FBS (+52.5 ± 21.7% after 2 h); HBSS w/o FBS (+63.6 ± 22.0% after 12 h) and HBSS + FBS (+31.4 ± 8.8% after 6 h); RPMI w/o FBS (+45.6 ± 21.3% after 2 h) and RPMI + FBS (+34.7 ± 29.0% after 6 h); M199 + FBS (+28.7 ± 15.3% after 2 h); DMEM + FBS (+26.4 ± 21.1% after 6 h); MEM + FBS (+27.5 ± 15.3% after 6 h); and L15 + FBS (+23.7 ± 11.1% after 2 h).

For just about all media, 4 °C storage was markedly more capable in maintaining tissue CBF at baseline values for all timepoints examined (Fig. 3). One exception being DMEM w/o FBS, where 4 °C storage resulted in a significant elevation in CBF after 6 h storage (+24.7 ± 27.5%). The ability of L15 to maintain CBF in ciliated samples also appeared to rely on the presence of FBS. Samples stored in L15 w/o FBS at both 4 °C and room temperature all showed significant reductions in CBF starting after 1 h storage (−22.6 ± 17.5% at 4 °C; −20.8 ± 20.3% at room temperature) and remaining depressed up to 12 h storage (−41.7 ± 13.3% at 4 °C; −25.7 ± 14.7% at room temperature) (Fig. 3).

Storage media and the maintenance of cilia generated flow (velocity)

Cilia generated flow velocity was significantly impaired for samples stored in saline (Fig. 4), which was significantly decreased after just 30 mins room temperature storage (−23.9 ± 26.4%) and after 60 min 4 °C storage (−53.1 ± 18.4%). Flow velocity remained significantly decreased at all later timepoints for saline stored samples. As flow velocity was determined by tracking microspheres contained within the media, and not if cilia were generating linear fluid flow across the ciliated epithelial surface, it should be noted that the low velocity values measured from saline samples at later timepoints represented random bead movement by Brownian motion rather than cilia mediated flow (Movie S2) (Francis, 2023).

Ciliated samples stored in all other media showed robust maintenance of flow velocity across all time points examined (Fig. 4). However, beyond isolated spikes, some significant trends were observed. Samples stores in room temperature HBSS with FBS displayed significant increases in flow velocity after 30 mins (+45.8 ± 61.6%) which peaked after 2 h (93.1 ± 93.6%) before returning towards baseline values after 6–12 h (Fig. 4). A similar significant spike in flow velocity was seen for samples stored in room temperature RPMI w/o FBS at both 1 and 2 h (+39 ± 85.3%, +52 ± 55.9%) before returning to baseline values after 6–12 h (Fig. 4). The trend for room temperature storage causing spikes in flow velocity was probably the result of the elevations in CBF also seen in room temperature stored samples (Fig. 3). Significant differences were also seen for room temperature stored samples in M199 w/o FBS after 2 h (+61 ± 47.6%); DMEM with FBS after 6 h (+60.7 ± 52.7%); MEM w/o FBS after 6 h (+55.0 ± 46.9%); L15 media with FBS at the 1 h (+29.7 ± 34.4%) and 6 h time points (+31.7 ± 55.1%) (Fig. 4).

The importance of FBS supplementation of L15 was also seen in the velocity data. Samples stored in L15 w/o FBS at both 4 °C and room temperature all showed significant reductions in flow velocities starting after 30 mins storage (−32 ± 26.5% at 4 °C; −30.6 ± 36.9% at room temperature) and remained depressed up to 12 h storage (−51.7 ± 31% at 4 °C; −43.1 ± 15.3% at room temperature) (Fig. 4). Of note is that flow velocity was also significantly reduced for samples stored in 4 °C L15 with FBS after 2 h (−48.0 ± 17.2%), which remained significantly depressed at all subsequent times points up to 12 h (−38.3 ± 27.7%).

Storage media and the maintenance of cilia generated flow (directionality)

Directionality is a ratio indicating the degree of flow linearity and provides an easy assessment for the presence or absence of cilia generated flow. Except for samples stored in saline, samples stored in all other media showed little change in directionality indicating the presence of robust linear flow across the surface of all the ciliated epithelia examined (Fig. 5).

Samples in saline showed a significant decrease in directionality after 1 h room temperature storage (−32.4 ± 34.3%) and after 2 h 4 °C storage (−57.3 ± 36.3%), and further decreased up to 12 h storage (−82.9 ± 13.1% at 4 °C; −80.5 ± 10.5% at room temperature) (Fig. 5). The disappearance of linear flow across the surface of saline stored samples would logically arise due to the disappearance of cells with motile cilia in these samples (Fig. 2).

Ciliated samples stored in all other media showed mostly constant directionality. However, beyond isolated small significant outliers, some small but significant trends were observed (Fig. 5). Samples stored in 4 °C HBSS w/o FBS showed small but significant reductions in directionality starting after 30 min storage (−2.1 ± 2%) which is maintained up to 12 h (−4.1 ± 4.1%). Samples in room temperature RPMI with FBS showed small but significant reductions in directionality at the 6 and 12 h time points (−8.1 ± 5.4%, −4.4 ± 4.3%). Conversely, samples in room temperature DMEM with FBS showed small but significant increases in directionality starting after 30 min storage (+3.2 ± 2.0%), which was also significant at the 1 and 6 h time points (+2.9 ± 2.1%, +3.1 ± 2.2%). Samples in room temperature M199 with FBS also showed small but significant increases in directionality at the 30 min and 2 h time points (+3.1 ± 1.9%, +3.5 ± 1.5%). Finally, Samples in 4 °C L15 w/o FBS showed small but significant reductions in directionality starting after 2 h storage (−3.5 ± 4.1%) which is maintained up to 12 h (−3.6 ± 4.5%).

Saline vs DPBS

Unsurprisingly, saline was not able to maintain cilia function, with large and significant decreases in all cilia functional parameters seen following storage of samples in saline (Figs. 2–5). As saline contains no nutrients/growth factors, saline storage was expected to negatively impact cell viability and thus cilia function (Chen et al., 2017). However, the impact of this effect was fast and dramatic with decreases in all motile parameters after just 30 min in saline (becoming significant after just 1 h). It was also interesting to note that 4 °C storage did nothing to help prevent this degradation of cilia function in saline, suggesting saline should not be used for assessment of cilia function except when immediately imaging fresh tissue. Conversely DPBS, while only slightly more complex in composition than saline (but also lacking nutrients/growth factors), appeared very capable in maintaining cilia motility during storage. As highlighted by ciliated samples, even when stored for up to 12 h in room temperature DPBS, still displaying cilia motility on par with that found in samples stored in more complex media. The difference between saline and DPBS for maintaining cilia motility indicates that a relatively simple factor is essential for maintaining cell viability/cilia function in these samples, such as the presence of calcium, magnesium, or phosphate buffering of pH changes, future studies are needed to pinpoint what mechanism is critical. The finding that DPBS can maintain ciliated tissues and cilia function for up to 12 h will however prove very useful as it provides a relatively ’blank canvas’ for future studies exploring factors regulating cilia function by removing a lot of extraneous variables from the experimental equation (i.e., the large number of different factors present in more complex cell culture media formulations not to mention those found in FBS).

Room temperature storage causes increased cilia motility

While robust cilia motility and cilia generated flow was maintained for up to 12 h in all media more complex than saline, some variations in cilia function were observed between sample groups. Most notable was the observation that for many samples room temperature storage results in significant elevations of cilia motility (CBF and cilia generated flow) compared to samples stored at 4 °C. These findings match previous observations from freshly harvested human respiratory epithelia, where increased CBF was seen after: storage in 22 °C RPMI media (Sommer et al., 2010), room temperature storage in M199 media (Bricmont et al., 2023) and room temperature storage in DMEM (when compared to 4 °C storage) (Reula et al., 2021).

Temperature is well known to influence CBF in a range of cilia models, with increased temperature causing a corresponding linear increase in CBF (Chen, Lemieux & Wong, 2016; Christopher et al., 2014; Schipor et al., 2006). However, a role for storage temperature later impacting cilia motility when imaged at 37 °C has not been previously acknowledged. It may be hypothesized that whereas cells stored at 4 °C remains relatively static, room temperature storage causes cells to develop abnormal cellular physiology, which may be related to abnormal enzymatic function(s) inherent with suboptimal temperatures (Knapp & Huang, 2022). More work is needed to determine if this elevation in CBF is permanent or may disappear if cells are given more time at 37 °C to recover from storage. However, even with this elevation in CBF following room temperature storage, ciliated samples in these groups still displayed robust cilia motility and cilia generated flow. Thus, room temperature storage may still be a viable transport option when looking to standardize handling protocols for ciliated sample shipping to facilities for functional diagnosis of cilia function. Such elevations in CBF may not be expected to influence diagnostic testing of diseases characterized by dramatic defects in cilia motility, such as PCD where just about all cilia motility is lost (Despotes et al., 2024; Shapiro et al., 2018).

FBS supplementation not essential except when using L15

Another important observation was that except for L15, FBS supplementation appears relatively unimportant for the maintenance of cilia motility when stored in the different media. The finding that FBS supplementation was essential for maintaining cilia function in L15 was unexpected, was found for samples stored at both 4 °C or room temperature, and was characterized by significant reductions in ciliated cell abundance, CBF, and cilia flow velocity.

L15 is a commonly used media for mammalian cell experiments as it doesn’t require CO₂ for pH control, so the finding that L15 without FBS was significantly worse than other media in maintaining cilia function was surprising and has not been previously reported. One noticeable difference between L15 and other media is its energy source (carbohydrate), L15 is the only media lacking glucose and instead contains galactose (Table S1). Glucose and galactose are epimers which differ in the position of one hydroxyl group, and enter glycolysis through different routes (Chandel, 2021). L15 contains galactose because it’s metabolized more slowly and doesn’t produce as many acidic by-products than glucose (Conte et al., 2021), which helps L15 maintain a stable pH in the absence of CO₂ buffering. No significant adverse effects of L15 using galactose instead of glucose as an energy source has been previously reported for mammalian cell studies. However, it has been shown that yeast cells display altered metabolism and metabolic gene expression if grown in galactose rich environments (Escalante-Chong et al., 2015). Thus, it’s possible that similar changes in metabolism and gene expression could also occurs in mammalian cells when the dominant energy source is switched from glucose to galactose. This could severely impact ciliated cells which have high energetic requirements (Villar, Vergara & Bacigalupo, 2021) resulting in the observed reduction in cilia motility when the tissue was stored in L15 alone. Supplementation with FBS, which contains glucose (108–166 mg/dL) (Lee et al., 2023) could prevent this altered metabolism and thus maintains cilia motility. Such a hypothesis requires more work but may indicate a major issue with L15, especially if used without FBS with cell cultures that have high energetic requirements, and this may impact other cellular processes beyond simple cilia motility.

Other possible effects impacting cilia motility

Other factors that differ for the media studied which are known to influence cilia motility include osmotic stresses, pH changes, and possible chemosensory stimulation. While the osmolarity of the different media studied are necessarily similar by design, due to differences in formulations they may not be identical, and evidence exists for osmolarity influencing CBF. Elevated osmolarity reduced CBF in freshly isolated human and murine samples (Horvath & Sorscher, 2008) while hypertonic nebulized saline reduced CBF in isolated sheep trachea (Kelly et al., 2023). However, these osmolarity studies utilized much greater changes in osmolarity than one can expect to find between the different media used in this study, so more work would be required to determine if it plays a role here. pH is also well known to impact CBF with elevations in pH resulting in significant elevations in CBF (Sutto, Conner & Salathe, 2004), the different media in this study utilize a range of methods to buffer pH changes, meaning it’s possible that pH changes are also responsible for some of the changes observed. Finally, cilia are recognized to be chemosensory (Shah et al., 2009), thus it cannot be ruled out that the different media formulations may result in different cilia chemosensory effects causing subsequent changes in cilia motility.