IN VITRO Volume14, No. 6, 1978 All rightsreserved 9

N E O N A T A L R A T H E P A T O C Y T E S IN P R I M A R Y T I S S U E C U L T U R E FREE FROM DENSITY-DEPENDENT REGULATION OF GROWTH UBALDO ARMATO, PAOLA G. ANDREIS, ENRICA DRAGHI, ANDLORETA MENGATO

Tissue Culture Laboratory, Department of Human Anatomy, UniversityPadova, 1-35100Padova, Italy SUMMARY Studies employing [3H]thymidine and radioautography as well as colchicine and Feulgen staining of DNA showed that up to 19-fold increases in the degree of cell crowding in vitro, i.e. from 1.45 to 27.55 x 104 cells per specimen, did not change the rates of entry into D N A synthesis and mitosis of cultivated primary neonatal rat hepatocytes.

Key words: cultivated hepatocytes; neonatal rat; cell density; contact inhibition; growth regulation. INTRODUCTION It is commonly held that most normal fibroblast-like cells of the eukaryotes, when set into tissue culture, display a "density-dependent regulation" or "contact inhibition" of growth; i.e. the cells cease replicating once they have grown to a certain "saturation density" ~l,2j. A comparable phenomenon has been postulated to occur also in epithelia (1,3), but the in vitro evidence supporting such a claim is still rather scanty and/or disputable {4-7). As to the liver parenchymal epithelial cells, it has been suggested previously that such cells, when set into primary culture, may be free from contact inhibition of growth (8,9), although confirmatory quantitative data are not available in the literature. We became interested in this topic while studying the regulatory effects of purine cyclic nucleotides on the growth of neonatal rat hepatocytes in primary tissue culture (10-13). We were aware that at two points in the culture method employed--(a) when attaching ("squashing") isolated liver cells to polyethylene discs in the presence of clotting adult mouse plasma ~14j; and {bj once the plasma has clotted, when detaching such discs from the Perspex | plate il4)--there occasionally may occur, even in experienced hands, nonmeasurable cell wastage leading within the same culture set to separates, i.e. discs with cells, endowed with varying cell densities. Hence, we thought it worth establishing by quantitative and statistical methods whether differences in cell crowding among distinct liver culture specimens had any influence on the rates of entry of the cultivated hepatocytes into DNA synthesis and mitosis.

MATERIALSAND METHODS Materials used included: Hanks' basal salt solution (BSS) and Eagle's minimum essential medium IMEM)(Wellcome Ltd.); adult and fetal bovine serum (Flow Labs. t; trypsin 1:250 powder (Difco Ltd.); trypsin type II powder (from hog pancreas), collagenase type II (from Clostridium histolyticum), hyaluronidase type I ffrom ovine testes), thymidine (TdR), colchicine (Sigma Chemical Co.); cephaloridin (Eli Lilly Italia Spa.); streptomycin sulphate tFarmitalia Spa.); nystatin (Squibb Ltd. D; phosphate buffered saline A iPBS/A) (Oxoid Ltd.); polyethylene sheets (0.0375-mm thick) {Visqueen Ltd.); [methyl3H]thymidine t[Me-3H]TdR; specific activity: 29 Ci per mM) (The Radiochemical Centre). All compounds used were of the highest purity grade. Litters of 5-day-old Wistar albino rats (S. Morini Spa.) were used. Sterility procedures. Glassware and instruments were sterilized in a hot-air oven at 160 ~ C for 90 min. Liquids were either autoclaved at 15 pounds per square inch or filtered through 0.22sm mean pore diameter membranes (Millipore Spa.). All the procedures of the culture session were performed by strict aseptic technique. Trypsin stock solution. Trypsin powders (from Difco and Sigma) were mixed together at a 8:3 ratio (w/wj and next dissolved at 10% (w/v) in buffer (5.36 mM KC1, 0.44 mM KH2PO4, 0.42 mM Na2HPO4, and 5.55 mM D-glucose in twicedistilled water) by constant magnetic stirring (300 _+ 20 rev per min) for 15 min at room temperature I18 _+ 2 ~ C). The supernate was centrifuged at 10,000 x g at 4 ~ C for 1 hr and then sterilized by 479

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filtration. Aliquots of 2.5 ml were stored at - 3 0 ~ C. Tissue dissociating solution. To make up the liver cell dissociating solution, Hank's BSS was added to: (al 2.5% (v/v) trypsin stock solution; (b) 0.025% (w/v) of both collagenase and hyaluronidase; and (c) 1 to 2 ml of a NaHCO3 solution (8% w/v) to bring the final pH to 7.6. Culture growth medium. Before use Eagle's M E M was fortified with 10% (v/v) fetal bovine serum inactivated at 56 ~ C for 30 min and with cephaloridin (50/~g per ml), streptomycin (50/~g per ml), and nystatin (25 IU per ml), and brought to pH 7.2 to 7.3 by 1 to 2 ml NaHCO3 (8% w/vL Preparation of dissociated liver cells. Rats were killed by cervical dislocation and put on melting ice. All the livers were drawn within 20 min after killing and pooled in a glass Petri dish (10 cm in size) containing 10 ml P B S / A cooled at 4 ~ C. The organs were chopped into pieces of 2 to 4 mm ~ by means of scissor-like movements of two scalpel blades. The outpouring blood was completely washed off by several changes of cold PBS/A. The liver pieces next were transferred into a 100ml Erlenmeyer flask containing 15 ml tissue dissociating solution. The tissue pieces underwent gentle magnetic stirring (100 _+20 rev per min) for 20 min at 18 _+ 2 ~ C. The supernate then was collected, mixed with 20% (v/v) inactivated {at 56 ~ C for 30 mint adult bovine serum and stored at 4 ~ C. Three to six further stirrings, each lasting 20 min, were carried out with additional aliquots (15 ml) of the tissue dissociating solution. The supernates thus obtained also were mixed with adult bovine serum and next pooled with the first supernate. The cell suspension was centrifuged at 50 x g for 5 min at 4 ~ C. The packed cells were resuspended in 5 ml growth medium and centrifuged again as above. The final cell suspension was made up ir~ 0.8 to 1.2 ml growth medium. Primary liver cultures. Cultures were set up according to Fulton's "squash method" (15). Sixteen small (40.02 ml) drops of the liver cell suspension were placed on a Perspex | plate ( l l - c m each side, 0.6-cm thick) sterilized by 1 hr of prior exposure to ultraviolet rays. Each drop then was covered with a polyethylene disc (1.3-cm diameter} previously sterilized for 1 hr in peroxidefree diethyl ether and next moistened on one side with clotting, sterile adult mouse plasma (15,16). The Perspex | plate was inverted and gently pressed against several sheets of sterile blotting paper to absorb the excess plasma. After the plasma had clotted, the discs were detached from

the plate by means of a drop of P B S / A (16). Each disc then was floated, ceils downwards, in a glass well (1.4-cm diameter) attached to the bottom of a modified Petri dish (20-cm size, 16 wells) and containing 0.7 ml growth medium. The "squash" procedure was repeated two to four more times, so that the separates (discs) eventually set up were from 48 to 80 in total. They were incubated at 35 ~ C in a gas phase of 5% (v/v) CO2 in air. The viability of cultured liver cells was checked daily by phase-contrast microscopy under a Leitz Orthoplan | microscope (16). Treatment of cultures. To fulfill the purposes of the present work, the above detailed general procedure was modified as follows. Before the "squash" procedure was carried out, the final suspension of isolated liver cells was divided into at least four equal parts, each of which was next diluted with P B S / A according to one of the following scheduled ratios: 0;1/6;1/5;1/4;1/3;1/2;1/1; 2/3. The isolated cells underwent "squashing" only after dilution. By this way the degree of variability in cell crowding within and among the different sets of liver cultures was significantly increased. At the end of the 4th day in vitro the cultures were fed with normal fresh growth medium. The specimens were divided into two equal stocks 20 to 24 hr later: (a) The first was labeled for 1 hr at 35 ~ C with 1.0 ~Ci per ml [Me-3H]TdR, next washed with several changes of "cold" T d R (10 mM in PBS/A) and fixed in acetic ethanol (3"1 v/v) to undergo radioautographic processing (10-14). (b) The second stock of separates was incubated for 4 hr at 35 ~ C with colchicine (0.12 mM) in the presence of 1.0 x 10-4% (v/v) ethanol and then fixed and stained according to the Feulgen technique for DNA.

Quantitative histology and statistical analysis. The cellular density of each disc was evaluated by counting at x 500 all the nuclei present in 12 randomly chosen fields, each of 29,400/~m ~. The percentage of these nuclei pertaining to hepatocytes also was scored. The [Me-3H]TdR-labeling index per 1 hr (L.I. per hr) and the colchicine metaphase index % (C.M.I.%) (12) were estimated by counting 600 hepatocytes in each disc. The L.I. per hr or C . M . I . % of each specimen was correlated with its degree of cell crowding. The sampled values thus obtained were plotted after grouping into 10 classes of increasing cellular density, the interval of each class being one-tenth the total interval occurring between the minimum and the maximum value of cell density observed

CELL CROWDING AND HEPATOCYTE GROWTH

481

FIG. 1. Monolayered primary rat hepatocytes (H) on the 5th day in vitro after fixation in glutaraldehyde 2.5% (w/v) and staining with uranyl acetate. F:Fibroblast-like cells. The cytoplasm of parenchymal ceils, although resembling honeycomb because of liposome extraction during the histological processing, is rather dark due to its richness in organdies, a, x400; b, x1300. (17). To these grouped values the one-way analy- ference microscopy revealed that hepatocytic sis of variance (17), Bartlett's X2 test (17), and the colonies were much thicker in height than the weighted linear regression analysis (18) were ap- mesenchymal cell monolayers. (b) Under phasecontrast microscopy the hepatocytic cytoplasm plied for statistical evaluation. appeared very dark and granular, because of its The total mean cell number per disc of aH the [Me-3H]TdR - and of all the colchicine-incubated rich endowment with organelles, and contained numerous small droplets of neutral fat (13). (c) preparations also was estimated. These two mean values were compared by the Student's t test for Immunofluorescent studies showed that the unpaired samples. hepatocytic cytoplasm contained detectable amounts of albumin, fibrinogen and other adult rat serum proteins even after 15 days in vitro (19); R ESULTSAND D ISCUSSION immunofluorescence also demonstrated that Primary cultures set up from dissociated neo- hepatocytes are the cell type richest in tubulin natal rat liver cells contained hepatocytes and also (unpublished data). (d) Electron microscopy several types of mesenchymal cells, thus some- showed that hepatocytes possessed, among the what mirroring the complex histology of the in various other organelles, a very well developed vivo liver (10,11,14). rough endoplasmic reticulum (19) containing releHepatocytes were usually grouped in monolay- vant amounts of proteins to be secreted. (e) Autoered, homogeneous colonies made up by 10 to 500 radiography showed that, unlike the coexisting elements, surrounded by more or less closely fibroblast-like cells and the so-called "clear cells," packed connective tissue cells (Fig. 1 ) (11,13,14). hepatocytes took up intensely and specifically Several criteria can help to recognize the paren[3H]bilirubin from the growth medium (14). (f) chymal cells: (a) Observation by Nomarski interPreliminary toxicological studies demonstrated

482

ARMATO

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F;G. 2. The relationship between the heptocytic L.I. per hr and the degree of cellular density per separate. The ceils were treated and radioautographed as detailed in the text. The means _+ 1; of grouped sampled values are shown. The numbers in parentheses indicate the number of separates, each from a distinct set of liver cultures, belonging to each class of values. The degree of variability in the intraclass determinations of L.I. per hr as compared to the interclass ones was found to be coincident both by the one-way analysis of variance [F = 0.9537 with 9/101 degrees of freedom (dr)] and by Bartlett's X2 test (X2 = 12.2763 with 9 dr). Continuous line, weighted mean of all data; dotted line, fitting the results by the equation: Y = 5.1696 - 0.0042x according to linear weighted regression analysis.

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FIG. 3. The correlation between the hepatocytic C.M.I. % and the degree of cell crowding per separate. The cells were treated as described in the text. The means _+ 14 of grouped data are shown. The numbers in brackets indicate the number of separates, each from a different set of liver cultures, pertaining to each group of values. The degree of variability among the inter- and intraelass determinations of the C.M.I.% was found not to be significant both by the one-way analysis of variance (F = 0.7933 with 9/114 df) and by Bartlett's X2 test (X2 = 5.8339 with 9 df). Continuous line, weighted mean of all values; dotted line, fitting the findings by the equation: Y ~ 2.2471 + 0.0192x according to the linear weighted regression analysis.

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CELL CROWDING AND HEPATOCYTE GROWTH that phenobarbitone-exposed hepatic ceils significantly increased their capacity to survive cytotoxic concentrations of viublastine sulphate (20L In this study the whole of [Me-3H]TdR-labeled liver cultures possessed a mean of 137,000 +_6560 cells per disc of which 24 to 30% were hepatocytes, whereas the whole of colchicine-treated cultures had 84,800 _+ 5600 cells per separate, 39 to 44% being hepatocytes. It appears, therefore, that the 4-hr incubation with 0.12 mM colchicine caused the detachment of about 38~ (P