IN VITRO Volume 13, No. 12, 1977 All rights reserved @

C U L T I V A T I O N O F M A M M A L I A N P I N E A L C E L L S : R E T E N T I O N O F O R G A N I Z A T I O N A N D F U N C T I O N

IN T I S S U E C U L T U R E

MARK A. NATHANSON,' SUE BINKLEY, AND S. ROBERT HILFER

Temple University, Department of Biology, Philadelphia, Pennsylvania 19122

SUMMARY

By means of a newly developed method of cultivating pineal tissue in vitro, the types of cells which comprise rat pineal glands have been identified. Previous in vitro studies have involved short-term culture more suitably called "organ culture" and provide no means of assessing the contribution of a putative "pineal" cell versus any other cell type found in the cultures. Short-term outgrowths of minced rat pineal glands provided a reproducible and easily dissociated source of pineal-derived cells. In monolayer culture these cells continued to have pineal enzyme activities which were sensitive to pineal-activating substances, and the cells aggregated to mimic the lobular organization of intact glands. Two types of aggre- gates were found, each composed of a single morphological cell type. In addition to the transient appearance of skeletal muscle straps, connective tissue and neural/glial tissue was consistently found. The cell types are discussed in relation to their in vivo counter- parts.

Key words: pineal; cell culture; N-acetyltransferase activity; hydroxyindole-0-methyl- transferase activity.

INTRODUCTION

It is well established that many cell types retain specialized functions in monolayer culture. How- ever, there are few bona fide examples of dissoci- ated cells in monolayer culture reorganizing to mimic the architecture of the intact organ. Par- ticipation of an organ's normal complement of cell types in a two-dimensional replica of organ structure is prerequisite to testing a number of questions concerning control of secretory activity and the origins of the various cell types.

I t has been demonstrated that mammalian pin- eal glands in organ culture can synthesize the in- dole, melatonin (1-31, and that human pineal tis- sue cultures form the peptide, arginine vasotocin (4). Several types of parenchymal cells, glia and connective tissue have been identified in cultures (5, 6) and sectioned material (7, 8) on the basis of morphology, staining characteristics, glycogen content and cell size. In this report we present a reproducible method for cultivating pineal tissue in vitro. Parenchymal cells, neural and/or glial elements and connective tissue segregate in these

1Present address: Department of Anatomy, Harvard University, 25 Shattuek St., Boston, Mass. 02115.

monolayer cultures to form an organization simi- lar to that of intact glands 17, 9-14) and contain the pineal enzymes responsible for melatonin syn- thesis.

MATERIAL AND METHODS

Culture method. Adult albino rats (Charles River) were killed by decapitation and the pineal glands were isolated into modified Ham's F-12 culture medium i l 5) lacking serum. (Avian pineal tissue has been cultured in an identical manner and with identical results.~ Adherent meninges and vascular tissue were removed from the glands and each gland was teased into 10 to 15 pieces with fine forceps or microknives. The pieces (ex- plants) from 2 to 4 glands were dispersed into 10- cm tissue culture plates (Falcon Plastics) contain- ing 7 ml of complete medium gassed with 5% CO2 in air. Complete medium consisted of modified Ham's F-12 medium supplemented with 10% pre- tested fetal bovine serum and penicillin-strepto- mycin 125 U per ml and 25/~g per ml, respec- tively), pH 7.0. Cultures were maintained in a 37~ humidified incubator with an atmosphere of 5% CO2 in air.

843

844 NATHANSON, BINKLEY, AND HILFER

In preliminary experiments, direct enzymatic digestion of pineal tissue provided low yields of viable cells. In contrast, explants of small pieces of pineal tissue gave rise to a large number of cen- trifugally migrating cells (zones of outgrowth). After 2 to 4 weeks of culture these migrating cells formed dense outgrowths which were easily dis- sociated into single cells.

Single-cell suspensions were prepared from zones of outgrowth by rinsing the plates with 37~ Hanks' saline, pH 7.0, followed by incuba- tion with 10 ml of 0.25% trypsin 1:250 (Difco Laboratories) in Hanks' for 10 min at 37~ Loos- ened strands and clumps were gently pipetted to facilitate dissociation, and the resultant suspen- sion was transferred to a 25-ml Erlenmeyer flask which was placed on a 37~ incubator shaker, oscillating at 80 rpm, for 10 min. The suspension was again pipetted, transferred to centrifuge tubes containing an equal amount of ice-cold complete medium and centrifuged at 183 • g for 3 min. The pellet was resuspended in ice-cold complete me- dium and centrifuged at 46 x g for 1 min to re- move aggregates. The supernate was again centri- fuged at 183 x g to pellet the single cells and, fi- nally, resuspended in ice-cold complete medium. The cells were counted in a hemacytometer and plated at a density of 2 x 104 to 105 cells per 10-cm tissue culture plate.

Harvesting of cells. Water (controH, norepine- phrine (dissolved in 0.1 N HC1), 0.1 N HCI icon- troU or dibutyryl cyclic AMP were added to the culture medium in each plate for the last 6 hr of culture. At the end of 6 hr, cells were scraped from each plate with a rubber policeman and transferred lwith a minimal amount of culture medium~ to individual 1.5-ml plastic centrifuge tubes containing 10/~1 of 8 mM acetylcoenzyme A (16~ in 0.05 N sodium phosphate buffer, pH 6.8. Tubes were kept on ice until all samples had been collected. Samples then were centrifuged (4~ 154 x g for 10 minl; the supernate was removed and the pelleted cells were frozen in their plastic tubes on dry ice until assayed.

Enzyme assays. For the assays, 30 ~1 of ice-cold buffer was added to each sample. Samples then were sonicated and 10 ~1 of each sample was as- sayed for N-acetyltransferase activity, modified to increase the sensitivity of the assay by lengthening the incubation time to 20 min and by using only labeled serotonin ~a mixture of labeled and unla- beled serotonin was used in the original method) I17, 18). Hydroxyindole-0-methyltransferase (HIOMT) activity was measured by the method

of Klein, Berg and Weller (19) on 10/~1 of the same sample.

RESULTS

The single-cell suspensions gave rise to mono- layers containing the four major morphologically distinct cell types that have been described in the literature (5, 7). These are: type I, small, compact cells with round to oval nuclei, dense cytoplasm and a large ratio of nuclear to cytoplasmic area; type II , flattened cells with large variably shaped nuclei, and a lower ratio of nuclear to cytoplasmic area than that of type I cells; stellate cells, mor- phologically similar to neurons and glia; and con- nective tissue cells. After 7 to 14 days both type I and type II cells formed discrete aggregates throughout the monolayer. Each region consis- tently contained only one cell type. Thus the monolayer was composed of two major cell types in well defined locations surrounded by regions composed of a mixture of cell types. Throughout the culture period the type I and type II cells re- mained as monolayers while the surrounding cells increased in density.

After 25 to 30 days of culture, aggregates of type I and type II cells became subdivided into "lobules" which resembled closely those found in sections of whole glands (Fig. 1). The process of lobulation was observed over a period of a week or more to begin at the periphery of an aggregate and to proceed centripetally. The result was sub- division of the aggregates into smaller units sur- rounded by connective tissue cells. Where regions of type I cells abutted regions of type II cells, the lobules never contained cells of both types. Whereas type I and II aggregates were readily ob- served after short intervals of culture, connective tissue cells remained within the unaggregated mass and could be identified by their characteris- tic whorling morphology. Coincident with lobula- tion, connective tissue was found to occupy the spaces between adjacent lobules (Fig. lb, cD.

"Stellatc" ceils, morphologically identical to neural or glial cells, were consistently observed within the zones of outgrowth ~of the original ex- plant cultures) ~Fig. 2aL These stellate cells formed a basket-weave lattice of interconnected cell processes and were largely confined to dis- crete regions of the outgrowths. Similar, although not identical, cells were observed in the mono- layer cultures where they appeared as "stellate" cells in contact with and on top of the type II cells ~Fig. 2b). I t is unclear at the present time whether

PINEAL CELLS IN CULTURE 845

these latter cells are the originally noted stellate cells or growth artifacts of the type I I cells.

The occurrence and growth of the above four cell types was consistently reproducible; however, the appearance of a fifth, transitory, cell type should be noted. Contracti le skeletal muscle straps were found within the zones of outgrowth of most of our explant cultures. The muscle tissue was confined to the explants and was never found within the monolayer cultures. Several authors have previously noted the anomalous appearance of skeletal muscle in the pineal gland ~6, 7).

I t is unlikely that the procedures of dissociation and culture significantly altered the pineal cells. If explant cultures were allowed to continue grow- ing, they underwent lobulation and contained the same cell types and in the same relationships as in

FIG. 1. Parenchymal ceils found in monolayers of cul- tured pineal cells, a, Flattened cells (single arrow) and compact cells (double arrows}, corresponding to type II and type I cells, respectively, are found in cultures prior to organization of cells into lobules, x 146. b, Lobules of type I cells (in culture 31 days}, x 198. c, Lobules of type II cells (in culture 34 days}, x 198. b, c, Note the appear- ance of connective tissue (ct) between adjacent lobules. Bar: 1 ~.m.

846 NATHANSON, BINKLEY, AND H I L F E R

FIG. 2. Stellate cells, a, A region at the periphery of an explant showing the outgrowth of stellate cells along with the migrating pineal cells. Note the numerous cell processes which make contact with both pineal and adjacent stellate cells, x 222. b, Stellate cells (single arrow) found within a region of lobu- lated type II cells, x 164. Bar: 1/~m.

the m o n o l a y e r cul tures . Also, if s ingle-cel l suspen- In o rde r to d e m o n s t r a t e t h a t the cells were in- s ions were p r e p a r e d f r o m the lobu la t ed m o n o l a y e r deed func t iona l p inea l g l and cells, p inea l N- cu l tu res , they u n d e r w e n t lobu la t ion aga in a n d re- ace ty i t r ans fe ra se a n d H I O M T act ivi t ies were t a i ne d the cha rac te r i s t i c s desc r ibed above , s tud ied . T h e cells a lso were sub jec ted to t r e a t m e n t

TABLE 1

ENZYME ACTIVITY IN INTACT GLANDS AND IN CELL AND ORGAN CULTURES OF PINEAL CELLS

N-Acetyltransferase HIOMT Treatment Activity Activity

pmol/plate-hr a pmol /plate-hr a Cell culture b Water 809_+59 33_+1

HC1 ~5x10 -~ N~ c 815-+57 34_+3 Norepinephrine i 10 -4 M~ c 1053_+175 Dibutyryl cyclic AMP i 10 -3 M~ c 1187+--70

Pineal glands pmol / pineal-hr a pmol / pineal-hr a A. In vivo d Light 175_+42

Dark 2778+_226 47+_2 B. Organ cultures e HC1 ~5x10 -5 N) c 133+_31

Norepinephrine {10 -4 M~ c 2581+_477

a Enzyme activity values are the average for four plates or glands + 1 S.C.; all enzyme activity values are signifi- cantly different from the blanks ~lacking tissue j; blanks were corrected to equal zero enzyme activity.

b Additions were made in 50/~1 to cultures made from dissociated cells prepared as described in the text. The concentrations refer to final concentrations in the culture medium.

d The in vivo measurements were made on pineal glands obtained from rats by killing them in either light or dark of a light-dark cycle ~ LD12:12, killing times 1000 and 1600, lights-on 2400}.

e Organ cultured glands were obtained from rats and cultured for 18 hr after which norepinephrine or its diluent ( HCI( was added to the cultures ~in 5 ~l~.

PINEAL CELLS IN CULTURE 847

with compounds which stimulate pineal N-acetyl- transferase activity. Evidence from in vivo and or- gan culture experiments has been used to support the idea that pineal N-acetyltransferase activity is modulated by/3-adrenergic innervation from the superior cervical ganglion; norepinephrine is be- lieved to be the neurotransmitter which stimulates N-acetyltransferase activity through a cyclic AMP mechanism (19, 20).

N-acetyltransferase and H I O M T activities were detected in homogenates of mass cultures (Table 1). Norepinephrine and dibutyryl cyclic AMP slightly stimulated enzyme activity in the cell cultures. The assays reported here were per- formed on cell cultures maintained in vitro for a total of 51 days. During this extended period the pineal cells retained the enzyme systems necessary to convert serotonin into melatonin. We consider this to be a clear demonstration that the cell cul- tures contained functional pineal cells. The re- duced enzyme activities and response to chemicals relative to pineal glands in 2-day organ culture may be due to" Ca) the disproportional occurrence of one cell type with respect to another; (b) loss of enzyme activity during the cell harvesting proced- ure; and (c) loss of the cellular architecture nor- mally found in the intact gland.

DISCUSSION

The cultures described in this report were de- rived from explants of minced rat pineal glands. Since this procedure allowed cells from the inter- ior, as well as the periphery, to migrate from the explants, the monolayer cultures represent the progeny of a random mixture of pineal cells. Our results suggest that at most two different types of parenchymal cells are found in pineal glands. I t is noteworthy that during the course of this in- vestigation the distinction between types I and II cells was not always clear, particularly before most of the cells within an aggregate had lobu- lated. Such an observation is entirely coincident with a growth process (i.e. lobulation) and ac- counts for the variable appearance of the cells.

The occurrence of connective tissue, stellate and two types of parenchymal cells reflects the di- versity of cell types found in pineal glands. One of us (M.N.) has been cloning fibroblasts derived from rat skeletal muscle for other purposes. In clonal culture these fibroblasts frequently formed a lobular organization similar to the type I I pineal cells. Whereas type II cells may represent an as- pect of connective tissue growth in vitro, the simi-

larity of type II cells to certain fibroblasts in clonal culture points out the necessity for identify- ing cell types by biochemical as well as morpho- logical criteria. The occurrence of stellate cells in contact with type II cells also may suggest a glial origin of the type II cells. Further studies utilizing cloned pineal cells are necessary to distinguish be- tween these alternatives and to correlate structure with function.

The appearance of N-acetyltransferase and H I O M T activity was not found to be correlated with the appearance of lobules. Enzyme activity and inducibility also were found in cultures of prelobulate cells.

We conclude from our experiments that: Ca) Pineal cells derived from single-cell suspensions may be cultured in vitro for extended periods if the suspensions are prepared from previously cul- tured explants. (b) Within the monolayers grown from these suspensions, four morphologically dis- tinct cell types can be identified: two parenchymal cell types, stellate cells and connective tissue cells. These cells are similar to those found in intact glands. (c) Cells of the monolayer form lobules similar to the lobular morphology seen in intact glands (although, of course, two- rather than three-dimensional). (d) The monolayer cultures and zones of outgrowth contain the same morpho- logical cell types and identical organization. (e) The monolayer cultures have enzyme activity characteristic of pineal glands.

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848 NATHANSON, BINKLEY, AND HILFER

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We thank J. Nathanson for critically reading the manuscript, K. Hal l for assistance with the enzyme assays and figures, and S. Goddes for assistance with typing. Supported by N S F Grant GB-43215 to S.B. and N S F Grant GB- 20919 to S .R .H.