Letter to the Editor
FACS as useful tool to study distinct hyalocyte populations

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Abstract

Hyalocytes, the cells of the vitreous body, are assumed to be involved in physiological as well as patho-physiological processes within the eye. However, current knowledge about the cells is still limited. As different morphological types of hyalocytes are described in the literature, it seems reasonable to try to isolate individual populations prior to characterization of single cell types. To achieve this, the present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte separation.

Subsequent to digestion of vitreous bodies using collagenase, the resulting cell suspension was analyzed and separated using FACS without any additional staining. Two-parameter dot plots of forward scatter (indicating size) against sideward scatter (indicating granularity) showed two distinct cell populations; staining with propidium iodide confirmed that both populations represent living cells. After sorting, cells of both populations were seeded on tissue culture plastic (tissue culture treated polystyrene). Only one population attached and proliferated, whereas the other population was non-adherent. Even when seeding the native cell mix, only one population of cells was observed after two passages, as indicated by FACS. Furthermore, ascorbic acid increased proliferation of these cells similarly to the proliferation of the separated cell population. These data point out that only one of the two populations adheres and proliferates on tissue culture plastic.

To conclude, the established isolation technique allows for separation of clearly defined hyalocyte populations. Moreover, clear hints were obtained that only one of the two populations adheres and proliferates under the commonly applied culture conditions.

Introduction

The vitreous body is the main compartment of the mammalian eye. This transparent, hydrogel-like substance consists of different types of collagens and glycosaminoglycans (Bishop, 2000) and is free of cells in its center; however, the cortex is lined by a single layer of cells, termed hyalocytes (Sebag, 1989). These cells exhibit two distinct morphologies, dependent on their localization on the vitreous surface. Hyalocytes on the inner limiting lamina appear elongated with a few stout processes, whereas cells on the epithelial surface of the ciliary body exhibit a stellate appearance with some short processes (Ogawa, 2002). The elongated hyalocytes of the posterior vitreous are furthermore substantially larger than the cells on the ciliary epithelium (Ogawa, 2002). Both cell types show a macrophage-like structure (Boltz-Nitulescu et al., 1979, Salu et al., 1985) and are, therefore, thought to belong to the reticuloendothelial system (Lazarus and Hageman, 1994). According to older literature, the cells are often referred to as resting cells (Salu et al., 1985, Gloor, 1973, Gloor, 1978); however, in more recent publications their physiological role becomes apparent. The cells of the posterior vitreous body are associated with the maintenance of the vitreous body as a transparent and avascular system (Zhu et al., 1997, Noda et al., 2004). Pathophysiologically, these cells are thought to be involved in diseases of the vitreoretinal interface, such as epiretinal membrane formation, diabetic macular edema, and macular holes (Badrinath et al., 1999, Heidenkummer and Kampik, 1996). Furthermore, hyalocytes have been shown to be able to produce the extracellular matrix components the vitreous is built of, especially collagens (Newsome et al., 1976, Kobuch et al., 2002) and glycosaminoglycans (Jacobson, 1984a, Jacobson, 1984b). Although these studies indicate that hyalocytes may have a housekeeping function within the vitreous, knowledge about hyalocytes is still limited.

To close this information gap, in vitro cell culture can be used to study cellular characteristics, cell functions, or the influence of specific factors on cells (Esteban and Maxwell, 2005, Dimova et al., 2005). In vitro cultivation of primary hyalocytes seems, thereby, to be a useful tool for investigations into these cells. As histological examinations indicated that these cells do not represent a homogenous population (Ogawa, 2002, Uehara et al., 1996), it seems reasonable to try to separate these different populations prior to characterization of each single cell type. However, no precise isolation method for different types of hyalocytes has been published. The commonly used isolation technique is based on mechanical dissociation of the posterior part of the vitreous and assumes that only a single type of hyalocytes is present in the excised tissue (Francois et al., 1973, Noda et al., 2004, Kobuch et al., 2002, Sommer et al., 2007).

The present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte characterization and separation. To this end, the primary cell suspension obtained after digestion of porcine vitreous bodies was studied using FACS. Subsequent to sorting the cells into distinct cell populations, adhesion and proliferation of the different populations were studied. To compare adherent and proliferating cells with each other, the influence of ascorbic acid on their proliferation behaviour was investigated.

Section snippets

Materials

Fetal calf serum (South America, Batch Nr. 40A0044K), Dulbecco's phosphate buffered saline (PBS), penicillin/streptomycin, and 0.25% trypsin–EDTA were obtained from Invitrogen (Karlsruhe, Germany). Dulbecco's modified Eagle medium (DMEM, low glucose, with glutamine and pyruvate) was obtained from Biochrom (Berlin, Germany). l-Ascorbic acid in cell culture quality, methylene blue, formaldehyde, boric acid, and propidium iodide were purchased from Sigma (Steinheim, Germany). Hoechst 33258 dye was

Results & discussion

In vitro cell culture proved to be a useful tool for the characterization of hyalocytes. As previous studies demonstrated that hyalocytes within the vitreous exhibit different morphologies (Ogawa, 2002, Uehara et al., 1996), a method to precisely isolate and separate these cells was deemed necessary. The present study demonstrated that FACS provides a means to separate two distinct populations of hyalocytes subsequent to digestion of vitreous bodies. Furthermore, the first evidence was found

Conclusions

To conclude, the established isolation technique using enzymatic digestion of the vitreous followed by FACS allows separation of clearly defined hyalocyte populations. The first evidence for different in vitro characteristics of the previously described hyalocyte populations was found. Moreover, clear hints were obtained that only one of the two populations, probably the one observed in the posterior part of the vitreous, adheres and proliferates on commonly used tissue culture plastic. The

Acknowledgements

This work was financially supported by grant 616/04 from the “Bayerische Forschungsstiftung”, Bavaria, Germany.

References (25)

  • B.P. Gloor

    Radioisotopes for research into vitreous and zonule

    Adv. Ophthalmol.

    (1978)
  • H.P. Heidenkummer et al.

    Morphologic analysis of epiretinal membranes in surgically treated idiopathic macular foramina. Results of light and electron microscopy

    Der Ophthalmologe

    (1996)
  • Cited by (0)

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