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Laboratory science
Cultivation of lacrimal gland acinar cells in a microgravity environment
  1. S Schrader1,
  2. C Kremling1,
  3. M Klinger2,
  4. H Laqua1,
  5. G Geerling3
  1. 1
    Department of Ophthalmology, University of Lübeck, Lübeck, Germany
  2. 2
    Department of Anatomy, University of Lübeck, Lübeck, Germany
  3. 3
    Department of Ophthalmology, Julius-Maximilian-University, Würzburg, Germany
  1. Correspondence to Dr S Schrader, Universität zu Lübeck, Klinik für Augenheilkunde, Ratzeburger Allee 160, 23538 Lübeck, Germany; mail{at}stefanschrader.de

Abstract

Background: A rotary cell-culture system (RCCS) allows the creation of a microgravity environment of low shear force, high-mass transfer and three-dimensional cell culture of various cell types. The aim of the study was to evaluate the growth pattern and the secretory function of rabbit lacrimal gland acinar cells in a microgravity environment using an RCCS.

Methods: Lacrimal gland acinar cells from male New Zealand White rabbits were isolated and cultured in an RCCS up to 28 days. Cells were analysed by light and electron microscopy, and apoptosis was assessed by the TUNEL assay at days 7, 14, 21 and 28. Secretory function was tested by measuring the β-hexosaminidase activity.

Results: After 7 days of culture, spheroidal aggregates were found inside the RCCS. The spheroids consisted of acinus-like cell conglomerates. Apoptotic centres inside the spheroids were observed at all time points by means of the TUNEL assay. Evaluation of the secretory function revealed β-hexosaminidase release after carbachol stimulation which decreased over the culture period.

Conclusion: A simulated microgravity environment promotes the development of three-dimensional cell spheroids containing viable acinar cells up to 28 days. Due to the evolving central apoptosis, it is unlikely that such simple three-dimensional cell communities can serve as tissue equivalents for clinical transplantation, but they promise opportunities for further applications in basic and applied cell research on lacrimal gland cells.

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Aqueous tear deficiency due to lacrimal gland insufficiency is one of the major causes of dry eye. In severe cases such as Sjögren syndrome, Stevens–Johnson syndrome or ocular mucous membrane pemphigoid, the therapy with artificial tears can be insufficient to relieve severe discomfort. Transplantation of the lacrimal gland is hardly feasible due to the short ischaemia time of the tissue of less than 6 h and the small dimensions of the gland’s vascular supply. In the absence of a suitable lacrimal gland transplant, salivary glands have been used instead to treat patients with otherwise intractable severe dry eye.1 However, the substantial differences between tears and saliva in their electrolyte and protein composition result in persistent ocular surface disease and can induce a microcystic epithelial oedema. Engineering a lacrimal gland construct may offer a suitable alternative transplant with a tear-like secretion.

Several methods have been developed to maintain lacrimal gland cells in vitro,23456 but preservation of the acinar phenotype and secretory function has proven to be difficult. Rotary cell-culture systems (RCCS) were originally designed to predict the impact of the microgravity environment in space for the culture of cells.7 They have proven to support the development of three-dimensional tissue structures and the formation of cell–cell contacts89 and to provide an environment of low shear force and high mass transfer of nutrients and metabolic wastes.10 The aim of this study was to evaluate the effect of a microgravity environment on the growth pattern and the secretory function of lacrimal gland acinar cells.

Materials and methods

Animals

Six lacrimal glands from three male, adult, 2 kg New Zealand White rabbits (Charles River, Germany) were used for the study. The animals were treated in accordance with the ARVO statement for the use of animals in ophthalmic and vision research.

Cell preparation and culturing

For the preparation of lacrimal gland acinar cells, a method described by Guo et al was used.11 After removal, the glands were cut into small pieces, washed and enzymatically digested by collagenase (350 U/ml, Sigma C 0130; Sigma, St Louis, Missouri), DNAse (40 U/ml, Sigma D 5025) and hyaluronidase (300 U/ml, Sigma H 3884). The cell digest was then centrifuged and washed twice, before filtering through a 70 μm mesh. The retained cell suspension was centrifuged through a Ficoll gradient, and the obtained cell subpopulation was washed, centrifuged twice and resuspended in Hepato Stim culture medium complemented with EGF (5 ng/ml), l-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml). A total of 9−10×106 acinar cells were seeded per 10 ml vessel. Four vessels were mounted on the RCCS and rotated at approximately 18 rounds per minute (∼0.01 G).

Light and electron microscopy

Cultured cells were examined by light and electron microscopy at days 7, 14, 21 and 28. For light microscopy, cells were washed with PBS, fixed with 4% buffered paraformaldehyde and stained with haematoxylin–eosin and Methylene Blue. For electron microscopy, cells were immediately fixed by immersion in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde at pH 7.4 for 24 h. The specimens were postfixed in 1% OsO4 and stained “en bloc” with 2% uranylacetate. After dehydration in graded alcohols, the specimens were embedded in Araldite. Semithin sections (0.5 μm) were stained with Methylene Blue and Azure II to visualise the regions of interest. Ultrathin sections (70–90 nm) were cut and stained with lead citrate and examined under an electron microscope (EM 400; Phillips, Eindhoven, Netherlands). The findings were recorded both by conventional films (Agfa, Mortsel, Belgium) and a digital image system (analySIS, Soft Imaging System, Münster, Germany).

TUNEL assay

Apoptosis was assessed by staining the fragmented DNA with the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labelling (TUNEL)12 using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (S7100, Chemicon, Chandlers Ford, UK). In brief, speciemens were dewaxed and stripped from proteins by incubation with 20 μg/ml proteinase K for 15 min at room temperature, and the slides were then washed twice with dH2O for 2 min. Endogenous peroxidase was inactivated by covering the sections with 3% H2O2 for 5 min at room temperature. The sections were rinsed with PBS and immersed with equilibration buffer (75 μl/5 cm2) for 10 s. Soft plastic coverslips were always placed on the slides after application of the reagents to spread them evenly by capillary action over the area of the slide. Working strength TdT enzyme was then added to cover the sections (55 μl/5 cm2) and incubated at 37°C for 60 min. The reaction was terminated by transferring the slides into the stop/wash buffer for 10 min at room temperature. Then, the specimens were washed three times in PBS for 1 min, and then the AntiDigoxigenin conjugate was applied on the slides (65 μl/5 cm2) and incubated in a humidified chamber at room temperature for 30 min. The specimens were then washed four times in PBS for 2 min, and the peroxidase substrate was added to the surface of the specimens (75 μl/5 cm2) for 3–6 min. Then, the slides were washed three times in dH2O for 1 min each wash and incubated for 5 min. Finally, the specimens were counterstained with Methyl Green (0.5%).

Measurement of spheroid diameters

The mean diameters of the spheroids and apoptotic centres were determined as the mean of the largest and the shortest diameter using a light microscope (Zeiss Standard 20; Zeiss, Jena, Germany; magnification 10×) with a calibrated micrometre scale. Seventy-eight spheroids were measured at day 7, 77 at day 14, 80 at day 21 and 60 at day 28. The apoptosis ratio was determined by division of the apoptotic area diameter through the diameter of the spheroid and multiplying by 100. Differences between the mean diameters of the spheroids with and without apoptotic centres and the apoptosis ratio were evaluated by the analysis of variance (one-way ANOVA test) and the Student t test. Differences at the level of p⩽0.05 were considered statistically significant.

β-Hexosaminidase assay

The secretory response to carbachol was analysed at days 7, 14, 21 and 28, and at least three wells were set per condition. Before stimulation, the lacrimal gland cell spheroids were removed from the vessel and carefully rinsed with DMEM three times, then resuspended in DMEM and distributed drop by drop in wells of a 96-well plate. The suspension was then incubated at 37°C with 5% CO2 for 2 h. After removing a baseline sample, carbachol was added to a final concentration of 100 μM, and cells were incubated for 30 min. The samples were centrifuged for 5 min, and the supernatant medium was stored frozen at −20°C until use. For measurement of the β-hexosaminidase activity, 4-methylumbelliferyl N-acetyl-β-d-glucosaminide was used as a substrate, and fluorescence intensity was determined using a method described by Barrett and Heath.13 The experiments were repeated three times with lacrimal gland cells from three different rabbits.

Results

Morphology

After digestion and filtration of the lacrimal gland tissue, a suspension of single cells and small cell clusters was placed in the RCCS.

After 7 days of culture, small spheroidal aggregates with a mean diameter of 384.6 (111.8) μm (mean (SD)) were found inside the rotating vessels (fig 1A). The spheroids consisted of acinar cell conglomerates with fine granulation in their cytoplasm, typical for secreting cells (fig 1A, insert). The cell communities showed organisation into acinus-like structures with a central lumen as discernible by electron microscopy (fig 2A, arrowheads). Cells showed polarity with a basal nucleus and apical electron lucent and electron dense secretory granules. Microvilli were found on their apical surfaces, and the apical cell parts were connected by desmosoms (fig 2B, arrowhead). Not only in the acinus-like structures, but also on parts of the surface of the spheroids, the cells showed polarisation and short microvilli (fig 2C). However, in the centre of 24.4% of the spheroids, groups of apoptotic cells with a condensed small nucleus were observed. In spheroids, where no apoptotic centres were visible by light microscopy, the TUNEL assay revealed sporadic apoptotic cells inside the spheroids (fig 3A).

Figure 1

Light microscopy of haematoxylin–eosin and Methylene Blue stain. (A) Spheroidal aggregates containing viable lacrimal gland acinar cells after 7 days of culture. The acinar cells show fine granulation in their cytoplasm, typical for secreting cells (insert). (B) Organised cell communities with acinus-like structures inside spheroids after 14 days of culture. Some of the central lumina are filled with suggested secretory material (insert, arrowheads). (C) Spheroid with vital acinar cells in the periphery and a central apoptotic area after 21 days of culture (insert, arrowheads). (D) Section of a spheroid after 28 days of culture, presenting only a few vital cells in the periphery, but a massive central apoptotic area (arrowheads). Bars: 100 μm.

Figure 2

Transmission electron micrographs. (A) Acinus-like structures inside a spheroid after 7 days of culture, forming central lumina (arrowheads). The cells show polarity with a basal nucleus and apical electron lucent and electron dense secretory granules. (B) Apical cell parts connected by desmosomes (arrowhead), with microvilli on the apical surface of the cells. (C) Surface of a spheroid after 7 days of culture presenting polarised cells with a basal nucleus and short microvilli on the apical surface. (D) Acinus-like structure after 14 days of culture, showing a central lumen and suggested secretion into the central cavity. The number of secretory granules in the cytoplasm is reduced. Bars: 5 μm.

Figure 3

Staining of fragmented DNA with the terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labelling (TUNEL) method. (A) Image of a spheroid after 7 days of culture. Sporadic apoptotic cells inside the spheroids are shown by the TUNEL assay. (B) Spheroid without an apoptotic centre, but distributed apoptotic cells after 14 days of culture. (C) Central apoptosis, but also scattered smaller apoptotic areas in the periphery of a spheroid after 21 days of culture. (D) Massive apoptotic area inside a spheroid after 28 days of culture. Viable acinar cells are only rarely found between the apoptotic cells, mainly in the periphery. Bars: 100 μm.

On day 14, the mean diameter of the spheroids was 382.4 (92.2) μm. Acinus-like structures were frequently detected inside the spheroids. The central lumina were regulary filled with suggested secretory material (fig 1B arrowheads, fig 2D), but the number of cytoplasmatic secretory granules was reduced (fig 2D). Apoptotic centres were noted in 48.1% of the spheroids. Apoptotic cells also became more abundant in spheroids without apoptotic centres by means of the TUNEL assay (fig 3B).

On day 21, the mean diameter of the spheroids was 290.7 (62.8) μm. Viable cells with histotypic features of acinar cells were still found predominantly in the periphery of the spheroids. Also, on parts of the spheroid surface, still polarised cells with microvilli were noted, but large centres of apoptotic cells were observed in 20% (fig 1C, arrowheads). Massive central apoptosis was confirmed by the TUNEL assay, which also revealed scattered smaller apoptotic areas in the periphery of the spheroids (fig 3C).

On day 28, the mean diameter of the spheroids was 388.9 (179.2) μm, and 41.7% of the spheroids contained large apoptotic areas (fig 1D, arrowheads). Viable acinar cells were only rarely found between the apoptotic cells, mainly in the periphery of the spheroids (fig 3D).

Spheroid diameters and apoptotic centres

On days 14, 21 and 28, the mean diameter of spheroids containing apoptotic centres was significantly higher compared with spheroids without apoptotic centres (p⩽0.05). The mean ratio of apoptosis on the entire spheroid was 40% on day 7, 38.9% on day 14, 46.8% on day 21 and 89.7% on day 28. The analysis of variance showed a significant difference in apoptosis ratio between the four time points, and a significant increase in the apoptosis ratio was found between days 14 and 21, and between days 21 and day 28 (p⩽0.05) (table 1).

Table 1

Mean diameters of spheroids and apoptotic centres at the four time points (mean (SD))

Secretory response to carbachol

The lacrimal gland acinar cells showed a secretory response to carbachol stimulation which decreased over the culture period (fig 4). The analysis of variance (one-way ANOVA test) showed a significant difference in β-hexosaminidase secretion between the four time points (p⩽0.05). A significant reduction in the β-hexosaminidase secretion after carbachol stimulation was found between day 7 and 28 in all three experiments.

Figure 4

β-Hexosaminidase release from acinar cells after 30 min of carbachol stimulation (100 μM). The cells showed a secretory response to carbachol stimulation which decreased over the culture period. The analysis of variance showed a significant difference in β-hexosaminidase secretion between the four time points (p⩽0.05). A significant reduction in the β-hexosaminidase secretion after carbachol stimulation was found between day 7 and 28 in all three experiments.

Discussion

In this study, we evaluated the growth pattern of lacrimal gland acinar cells in a microgravity environment. Microgravity bioreactors simulate a microgravity environment by adjusting the vessel rotation speed such that the cells are cultivated in a state of continuous free-fall. These conditions provide a relatively well-defined fluid dynamic environment, efficient mass transfer and low shear force.14

In our experiments, simulated microgravity promoted the development of spheroidal aggregates with a mean diameter of 384.6 (111.8) μm after 7 days. The spheroids consisted of organised lacrimal gland cells, and acini-like structures were observed. The mean diameter of the spheroids did not increase substantially during the culture period of up to 28 days. In the centre of the spheroids an area of apoptosis was noted at all time points. The development of apoptotic centres inside the spheroids correlated with their size, as on days 14, 21 and 28 the diameter of the spheroids containing apoptotic centres was significantly higher than in spheroids without apoptotic centres. Also, the duration of the culture period seemed to play a role, as the ratio of apoptosis inside the spheroids increased significantly between days 14 and 21 and also between days 21 and 28. Similar observations were made in a study by Khaoustov et al, where spheroids of human liver cells were formed in microgravity bioreactors, and central apoptosis was noted usually after 1 week in culture.15 The development of central apoptosis is also found in growing tumour populations without blood vessels in vivo due to poor diffusion to the centre of the cell aggregates.16 It is therefore likely that the areas of apoptosis observed in our experiments are also a result of poor oxygen supply or reduced diffusion of nutrients into the centre of the spheroids. This is supported by the short time lacrimal glands can tolerate ischaemia and highlights the need to establish a vascular supply for voluminous constructs of lacrimal gland cells. The morphological results agree with the secretory response to stimulation with carbachol, as the secretory response of the cells decreased during the culture period. This was also observed by us and others when lacrimal gland cells were expanded under two-dimensional conditions. After 3 days of culture on amniotic membrane, we found strong β-hexosaminidase secretion which was about three to four times that of the baseline. The secretory resonse decreased to approximately twice that of the baseline at day 7 and was substantially diminished beyond day 7.2 Similar observations were made by Andersson et al, who reported optimal secretory response of acinar cells after 2–3 days in culture,17 and Schechter et al, who noted a strong reduction in acinar cell secretion beyond day 7.5 The reason for the loss of secretory function during extended culture periods is uncertain. Schechter et al discussed the long-term absence of a basal secretomotor stimulation in culture as one of potential explanation.5

In our experiments, apoptosis observed inside the spheroids may contribute to the functional compromise. However, it is unknown whether the inner part of the spheroids significantly contributes at all to the β-hexosaminidase-activity measured in the culture medium or whether, due to a kind of diffusion barrier, this only reflects activity of the periphery of the spheroid. In addition, a lack of an extracellular matrix in our experimental set-up may also be involved, as it has been shown that cells will tend to retain their differentiated phenotype in vitro only under conditions that resemble their natural in vivo environment. In particular, the combination of high cell density and an appropriate substratum have been shown to be substantial to induce cell–cell and cell–matrix interactions1819 and are likely to support differentiation and secretory activity of lacrimal acinar cells.

In conclusion, an RCCS promotes the development of three-dimensional cell spheroids containing viable acinar cells up to 28 days. Due to the evolving apoptosis, it is unlikely that such simple three-dimensional cell communities of lacrimal gland acinar cells can serve as tissue equivalents for clinical transplantation, but it shows that a simulated microgravity environment is capable of forming three-dimensional, relatively large, lacrimal gland cell communities, which is essential for a possible lacrimal gland substitute, as high cell numbers will probably be needed for sufficient lubrication of the ocular surface. As a variety of cell functions, including proliferation, migration and differentiation, are regulated by cellular interactions with the extracellular matrix, the use of three-dimensional scaffolds as an extracellular matrix may help to retain a differentiated phenotype and cell function in vitro.20 It may also improve the mass transfer and oxygen supply in the centre of the tissue and thus avoid central apoptosis.

Acknowledgments

The authors thank AK Mircheff from the Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California, USA, for the opportunity to learn the techniques for purifying lacrimal gland acinar cells in his laboratory and for many helpful discussions.

REFERENCES

Footnotes

  • Funding This work was supported by a research grant of the University of Lübeck, Germany (A03-2007).

  • Competing interests None.