Pigineizt Cell Res 1997;. 10:85-87 Printed in the United States ofAmerica - all rights reserved.

Copyright 0 Munksgaard, 1997 PIGMENT CELL RF.SURCH

ISSN 0898-5785

Short Communication Melanocytes In Vitro:

How Do They Undergo Mitosis? STEFAN KIPPENBERGER,~~ AUGUST BERND,' JURGEN BEREITER-HAHN:

ANA RAMIREZ-BOSCA,l AND ROLAND KAUFMANN' 'Zentrum der Dermatologie und Venerologie, Klinikum der Johann Wolfgang Goethe Universitiit,

F'rankfurt/Main, Germany and 'AK Kinematische Zellforschung, Zoologisches Institut, Johann Wolfgang Goethe Universitiit, F'rankfuWMain, Germany

Human melanocytes of the adult skin are slow-cycling cells with a highly dendritic morphology. Nevertheless in vitro proliferation can be achieved using adequate stimula- tors. Time lapse studies revealed the morphologic changes during melanocyte mitosis: dendrites are drawn back into the cell body, the cell becomes spherical and detaches from the support. Cell division takes place while the cell is suspended. Consecutively the two cells reattach to the support and form new dendrites.

About 1% cells per culture are detached from the support and ca. 70% of these cells are viable and putative within mitosis. By every medium change mitotic cells become withdrawn supporting selection of Go-cells, Therefore we recommend centrifugation of exhausted medium in order to add mitotic cells back to the culture.

Key words: Melanocytes, Mitosis, In vitro

INTRODUCTION Melanocytes of adult skin are highly differentiated cells

with a low mitosis index in vivo (Jimbow et al., 1975). Their morphology is similar to nerve cells to whom they are on- tologically related (Weston, 1970). In human epidermis, mel- anocytes form pronounced dendrites that remain in contact with keratinocytes of the different strata. One melanocyte provides 36 keratinocytes with pigment, which is termed a melano-epidermal unit (Fitzpatrick and Breathnach, 1963). Although proliferation of melano- cytes in vivo is rare, it has been shown by histochemical, autoradiographic and electron microscope studies that dif- ferentiated melanocytes undergo mitosis under physiologi- cal conditions (Jimbow et al., 1975).

The exact mechanism of mitosis in melanocytes remains a matter of speculation. Proliferation of in vitro-cultured melanocytes is frequently induced by stimulators of the pro- tein kinase C and substances that lead to an increase in the intracellular CAMP level (Eisinger and Marko, 1982). Although these culture conditions cause a massive meta- bolic intervention, melanocytes maintain at least part of their dendritic appearance. Therefore it is possible to study how far the morphology of melanocytes alters during mitosis.

The present study intends to clarify the in vitro mecha- nism of melanocyte mitosis.

MATERIALS AND METHODS Cultivation of Melanocytes

Melanocytes were isolated from human fetal back skin and cultures were initiated as described previously (Ramirez-

Bosca et al., 1992). The medium composition was that of Bernd et al. (1994): F-10 basic medium (Gibco BRL, Eggenstein, Germany), 5% fetal calf serum (Serva, Heidel- berg, Germany), 1% penicillin/streptomycin (Biochrom KG, Berlin, Germany), 85 nM phorboldibutyrate (Sigma, St. Louis, MO), 0.1 mM isobutymethylxanthine (Sigma, St. Louis, MO), and 2.5 nM choleratoxin (Calbiochem-Behring, F'rankfurt, Germany). According to Valyi-Nagy et al. (19901, the popular phorbolester 4-0-methyl-12-0-tetradecanoyl- phorbol-13-acetate (TPA) was replaced by phorboldi- butyrate because of its better aqueous solubility. Cultures were incubated at 37°C in a 10% COz atmosphere. The mel- anocyte medium was renewed every 3 days. The exhausted medium was centrifuged (l,OOOg, 10 min, RT) and the re- sulting pellet of soluble cells underwent further examina- tions (see below).

Trypan Blue Exclusion Test The cell pellet was washed once with PBS (w/o calcium

and magnesium) in order to remove traces of serum. Con- secutively the pellet becomes suspended in 320 p1 PBS and 180 pl trypan stock solution (0.5%) (Boehringer, Mannheim, Germany). After incubation (5 min, RT), the assay was

Address reprint requests to Dr. S. Kippenberger, Abteilung fur Dermatologie 1, Zentrum der Dermatologie und Venerologie, Klinikum der J.U! Goethe Univcrsitat, Theodor-Stern-Kai 7,60590 FrankfiirU Main, Germany

Received December 24,1996; accepted January 27,1997.

86 S. Kippenberger et al.

mixed and a drop of cell suspension was pipetted into a Neubauer chamber. Cells were counted and viable cells (col- orless cells) were related to dead cells (blue cells).

RESULTS Morphology

Alterations in melanocyte morphology during mitosis were detected by a video camera system connected to an inverse light microscope. Melanocyte culture flasks were fued to the object tray and filmed. The temperature was

Fig. 1. Mitosis of a melanocyte in vitro. The time interval between each picture is 40 sec. A The ini- tial situation. The cell undergoing mitosis is marked by an arrow. B and C: The dendrites become inte- grated into the cell body. The cell turns out to be spherical. By soft tapping at the mounting table the cell can be moved which indicates a complete detachment from the support. D: The detached cell di- vides. E and F: The cells reattach and begin to develop dendrites.

maintained at 37°C. Figure 1 shows the chronology of events during melanocyte mitosis.

Viability of Solubilized Melanocytes In order to test whether soluble melanocytes are viable

or not they were incubated with trypan blue. Non-viable cells are distinguished from viable cells by color take up (see Fig. 2). About 1% melanocytes per culture are solubi- lized in the medium. About 70% of these solubilized cells are viable and putative mitotic cells (see Fig. 3).

Fig. 2. Trypan blue exclusion test of suspended melanocytes. The uptake of blue color indicates cell death. In contrast a vital colorless cell is shown.

Melanocytes In Vitro 87

Sotubfe cells (complete)

Soluble cells (Trypan negative)

T I

Fig. 3. Cell count of solubilized cells of four strains of primary melanocytes from different donors. About 1% of cells are detached from the support. About 70% of these cells are putative within mitosis.

DISCUSSION The present time lapse studies provide evidence that

melanocytes retract their dendrites into the cell body when they undergo mitosis in vitro (see Fig. 1). Spheri- cal cells detach from the support and divide. The newly divided cells reattach to the support and start to form new dendrites. Bertaux et al. (1988) observed in their experiments on a skin equivalent round dopa-positive cells in the epidermis. Before that they stimulated mel- anocyte proliferation by UVB or UVA in combination with 8-MOP At that time they had no explanation for this phenomenon. Maybe these round dopa-positive cells represented melanocytes during mitosis.

Our findings have a useful practical aspect for cell cul- tures: when renewing the medium, the exhausted medium is usually discarded, although it consists of solubilized mi- totic cells. Therefore a selection of non-proliferative adher- ent cells in the culture dish is likely. In order to maintain proliferative melanocyte cultures we centrifuge the ex- hausted medium and solubilize the pellet in fresh medium

before adding it again to the culture. The pelleted cells could also be seeded separately into a new culture dish. Prelimi- nary results failed to show a synchronization effects of newly plated cells by BrdU staining but nevertheless vital proliferative cultures can be obtained from detached mi- totic melanocytes.

CONCLUSIONS Human melanocytes in vitro undergo prominent morpho-

logical changes during mitosis. Their dendrites were drawn back into the cell body and the complete cell loses contact to the substrate. Regularly these solubilized cells become discarded during medium exchange. A selection of non-mi- totic cells is likely. In order to reach a maximum expansion in cell count, we recommend centrifugation of exhausted medium, and consecutively add the mitotic cell back to the culture system.

ACKNOWLEDGMENTS We are grateful to Dr. Adrian Sewell (Universitats-

Kinderklinik, Ikmkfwt) for critically reading the manuscript.

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