A New Method for Lateral Visualization of the Primary Cilia on the Surfaces of Cells Cultured on White Glass Rods

Abstract

Purpose: To investigate the motility of the primary cilia of corneal endothelial cells (CECs), which exist like a hair on the cell surface, using our new in vitro method.

Methods: A white glass rod was heated with a gas burner to produce a rod approximately 0.5 mm in diameter and 20 mm in length and then coated with collagen. A suspension of cultured human CECs (HCECs) was then added to the rod and cultured for 20 days. Cells on the rod's side were then observed using phase-contrast microscopy, and videos and images of the primary cilia were obtained. After fixing the cells cultured on the rod's surface, immunofluorescence staining was performed and fluorescence and phase contrast images were taken.

Results: Hair-like structures were observed on the surface of live HCECs on the rod's surface. Video images revealed that the structures sometimes swayed owing to slight convection of the medium, yet had no motile function, and immunostaining with acetylated α-tubulin antibody confirmed that the structures were primary cilia.

Conclusions: Our new method using white glass rods provided the ability to observe the movement of primary cilia in cultured living HCECs, and the findings clearly showed that the primary cilia of HCECs are passive rather than motile. This novel procedure can be applied widely to other cultured cells as a method to observe the movement of primary cilia from the lateral aspect of the cell.

Translational Relevance: This method may help to clarify the role of primary cilia in the anterior chamber.

Introduction

A primary cilium exists in most cells in the form of a hair-like structure on the surface of the cell; however, its specific function has yet to be elucidated fully. In particular, the function of the primary cilia of CECs has not been investigated in great detail. The findings in a previous report using an in vivo mouse model only revealed that when the CECs are scratched, primary cilia are expressed at the tips of the cells that migrate to the injured site.¹ 

We previously reported that the primary cilia of corneal endothelial cells (CECs) disappears when an isolated rabbit model cornea is stored at a low temperature in corneal preservation medium, simulating the temperature used for corneal preservation. 

It then reappears when the cornea is warmed up, just as it does when the temperature rises during transplantation.² Moreover, we recently reported the occurrence of primary cilia in the CECs of patients afflicted with bullous keratopathy.³ 

It has been reported that in motile tracheal cilia, microtubule pairs are arranged in a 9 + 2 arrangement, consisting of two central microtubules and nine pairs of peripheral microtubules, whereas in primary cilia, which are essentially immotile, microtubule pairs are arranged in a 9 + 0 arrangement and lack central microtubules.⁴ As an exception, it has also been reported that primary cilia in the node center cells that appear at the time of embryonic primary intestine entry have a motility function that produces water flow by rotational movement.^4–6 Primary cilia without automaticity are also known to act as a mechanosensor in the renal proximal tubules that senses the bending motion induced by water flow.⁷ Of note, primary cilia are also present in CECs, but their movement (automaticity or transitivity) has not been studied previously. 

We would like to clarify whether the primary cilia of the human CECs (HCECs) are motile or immotile, and if immotile, we would like to investigate whether any signals are received when there is a flow of body fluid. Thus, in this report, as a preliminary step, we investigated a new method to clarify motile or immotile in cultured HCECs. 

Methods

HCECs

The human cornea was handled in accordance with the tenets set forth in the Declaration of Helsinki. HCECs were obtained from human donor corneas provided by CorneaGen Eye Bank (Seattle, WA), and cultured as previously reported.⁸ All procedures followed tissue acquisition as previously described,⁸ including informed consent for eye donation for research. Moreover, all procedures were performed in accordance with the ARVO Statement for the Use of Human Materials in Ophthalmic and Vision Research, and the protocols of the research experiments performed in this study were approved by the Institutional Ethics Committee of Kyoto Prefectural University of Medicine, Kyoto, Japan. 

HCECs Culture on the Surface of White Glass Rods

White glass rods (Art Loco, Tokyo, Japan) commercially available for glass crafts were heated by a gas burner to produce rods of approximately 0.5 mm in diameter and 20 mm in length, with the center of the rod weakly heated and bent into a U or V shape (Fig. 1A). The rods were then transferred to culture plates, coated with collagen (Nitta Gelatin Inc., Osaka, Japan), and transferred again to uncoated culture dishes. Next, a suspension of HCECs (i.e., 1 × 10⁴ cells/mL; 5 cell lines) was added and cultured at 37°C for 20 days (Fig. 1B). An inverted phase contrast microscope (Leica DMIRB; Leica Microsystems GmbH, Wetzlar, Germany) was then used to focus on the cells on the side of the glass rod for observation, and a video and still images of the primary cilia were obtained at room temperature. Next, cells cultured on the surface of the glass rod were fixed in 4% paraformaldehyde fixative for 15 minutes. Immunofluorescence staining was then performed using an acetylated α-tubulin antibody (66200-1-Ig mouse monoclonal antibody; Proteintech Japan Co., Ltd., Tokyo, Japan) that specifically recognizes primary cilia,⁹ and fluorescence and phase contrast images were captured via the use of the Leica DMIRB. In the 30 cells in which primary cilia was found, Image J software was used to measure the length of the cilia in the captured image. 

Figure 1.

 

White glass rods used in this study. (A) A thick white glass rod heated to create a rod with a diameter of approximately 0.5 mm and a length of approximately 20 mm. The center of the rod was heated gently and bent into a U or V shape to prevent it from rolling (scale in millimeters). (B) A white glass rod with cultured HCECs attached in the culture medium was placed in a 35-mm culture dish.

Fixed Samples

Corneal endothelium specimens were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, washed three times in phosphate buffer, and postfixed for 1 hour in 2% aqueous osmium tetroxide. After dehydration in ethanol at increasing concentrations, the specimens were transferred to t-butyl alcohol, dried using a freeze-drying device (VFD-21S; Vacuum Device Co., Ltd., Mito, Japan), and sputter coated with gold in a magnetron sputter (MSP-mini; Vacuum Device Co.). The specimens were then observed using a tabletop scanning electron microscope (TM4000 Plus; Hitachi High-Tech Corporation, Tokyo, Japan). 

Results

Phase contrast microscopy images revealed a hair-like structure on the surface of the living cells on the glass rods in the culture medium. However, there was no regular automaticity like flagella or airway cilia (Fig. 2, Supplementary Fig. S1). Video imaging of the phase contrast microscopy images revealed that the primary cilia sometimes seemed to sway owing to a slight convection of the culture medium, yet there was no motor function, and when the movement of the tip of the primary cilia in the video in which the most movement was measured using Image J software, the average movement speed was found to be 0.30 µm/second (Supplementary Fig. S1). Moreover, measurement by Image J revealed that the combined total mean length of the primary cilia in the obtained images of each of the five cell lines was 6.0 ± 4.5 µm (n = 30 cilia) (Supplementary Table S1). However, because the lateral curve of the cells was the area that was observed, it is possible that short cilia could not be measured. At the time of observation, the culture dishes were removed from the cell incubator and observed under the microscope at room temperature for approximately 2 minutes, so we posit that there was some stress on the cells. 

Figure 2.

 

Phase contrast microscopy images of living CECs on the side of the white glass rod. Hairy protrusions that appeared to be primary cilia were observed on the surface of living HCECs on the surface of the glass rods in the culture medium. Yellow arrows point to primary cilia.

For confirmation, phase contrast microscopy observation of the sample after fixation and immunostaining revealed a hair-like structure protruding from the cell surface, similar to what was observed in the unfixed sample. After switching from phase contrast to fluorescence observation on the same screen of the Leica DMIRB, green fluorescence was observed, thus indicating that the protruding structure was positive for acetylated α-tubulin, which identifies primary cilia. Fluorescence horizontal images of the lower surface of the glass rod showed that cells were distributed uniformly on the surface of the glass rod as on the plate. The primary cilia structure was observed as strong fluorescence, although a weak positive reaction to tubulin was widely observed within the cells (Fig. 3). Scanning electron microscopy also revealed protrusions on the sides of the glass rod (Fig. 4). 

Figure 3.

 

Phase contrast microscopy images and immunostaining images of fixed CECs on the side of the white glass rod. (A, D) After fixation and immunostaining, phase contrast microscopy revealed a hair-like structure protruding from the cell surface in the samples. (B, E) Switching to fluorescence observation on the same angle, the hair-like structure was found to be positive for acetylated α-tubulin, thus confirming that it was the primary cilia. (C, F) That finding was also observed in the merged images. (G) The bottom surface of the white glass rod showing fluorescence.

Figure 4.

 

Scanning electron microscopy images of fixed CECs on the white glass rod. Primary cilia are observed on the CECs located at the (A) top surface and (B) side of the white glass rod.

Discussion

It has previously been reported that in anesthetized Gt(ROSA)26Sor^{tm1(Sstr3/GFP)Bky} mice, primary cilia in the corneal stroma can be visualized by confocal microscopy.¹⁰ In GFP non-transferred tissues or cells, confocal laser microscopy and scanning electron microscopy allows for three-dimensional observation of the primary cilia in fixed cells,^1,2 yet not in living cells. In this study, we were able to observe the passive movement of the primary cilia of cultured living HCECs via our newly devised method using a white glass rod, and our findings revealed that the primary cilia of HCECs are not motile. 

In a report on observing the movement (automaticity) of primary cilia, a tissue called a node is formed in the abdomen of the embryo at the stage of primordial colonization at 7.5 days postconception mice embryos. It is known that each cell in the center of the node has one motile hair that rotates counterclockwise to produce water flow.⁵ In the case of motile cilia, the movement of intraventricular cilia in mouse brain slices has been observed using beads.¹¹ Moreover, the movement (probably passive) of cilia with forced expression of green fluorescent protein in the renal proximal tubules of mice has reportedly been observed.⁷ 

The cells used in this present study were primary cells in culture, yet it was difficult to introduce green fluorescent protein into all cells and observe them as in the above-described mouse proximal tubules. Moreover, and to the best of our knowledge, this study is the first to report the observation of the primary cilia of cultured cells from a lateral aspect. Observation of the cells from the side revealed that the primary cilia of cultured HCECs do not move automatically, as has been suggested previously. However, in some cases, the long cilia do move passively with the slightest flow or Brownian motion of the culture medium (Supplementary Fig. S1). 

Initially, we made transparent glass rods from Pasteur pipettes commonly used in the culture room. However, because we posited that the glass rods might roll and damage the cells and cilia on the surface when the cells are cultured, we subsequently devised a method to gently heat and bend the glass rod into a U or V shape to prevent it from rolling. In addition, transparent glass rods caused light to be refracted inside, which was an obstacle during phase contrast imaging and fluorescence observation. Therefore, we decided to purchase commercially available white glass rods commonly used for glass crafts and heat them with a gas burner to produce thin white glass rods (Fig. 1). 

Confocal laser microscopy and scanning electron microscopy allow for three-dimensional observation of the cilia of cells cultured and fixed on a plate, but they cannot be used to observe live cells. Our newly developed method enables us to capture still and moving images of the cilia of living cells from the lateral aspect. 

Our search of the published literature on previous similar studies revealed reports of culture cells on the inner surface of dialysis tubes for antibody secretion,¹² as well as reports of culture cells on the inner surface of biological use tubes.^13,14 However, we were unable to find any reports of cultured cells on the surface of rods and observation of those cells from a lateral aspect. Hence, the novel method that we present in this study may be applicable to other culture cells and be useful as an in vitro observation model of motile cilia, such as the tracheal epithelium. Furthermore, there is a possibility that this new method can be applied widely as a way to observe some changes on the cell surface from a lateral aspect. 

In the future, we intend to use this method to observe the movement of cilia of CECs in a constant flow of water considering the flow of aqueous humor in the anterior chamber and examine whether the cilia of CECs function as mechanosensors that sense water flow. 

Acknowledgments

The authors thank John Bush for his critical review of the article. 

Supported by JSPS KAKENHI Grant Number JP20K09832. 

Disclosure: H. Tanioka, None; H. Deguchi, None; S. Kinoshita, None; C. Sotozono, None 

Supplementary Material

Supplementary Figure S1. Video imaging of phase contrast microscopy images of living CECS located on the side of the white glass rod. Video imaging revealed that the primary cilia sometimes appeared to sway due to a slight convection of the culture medium, yet there was no motor function. When the movement of the tip of the primary cilia in this video was measured using Image J software, the average movement speed was 0.30 µm/second.