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A simple method for culturing chick embryo liver and kidney parenchymal cells for microscopic studies
TISSUE & CELL 1974 6 (4) 641-652 Published by Longman Group Ltd. Printed in Great Britain
IAN K. BUCKLEY and JUDIE R. WALTON
A SIMPLE METHOD FOR CULTURING CHICK EMBRYO LIVER AND KIDNEY PARENCHYMAL CELLS FOR MICROSCOPIC STUDIES ABSTRACT. Differentiated parenchymal cells were cultured from chick embryo livers or kidneys. Tissues were trypsinized, fragmented under a dissecting microscope, suspended in culture medium and separated into microscopic cell clumps with a syringe and wide bore needle. These cell clumps were grown in culture chambers and after light microscopic study were fixed and sectioned for electron microscopy. Both liver and kidney parenchymal cells appeared as clusters of epithelial cells containing round nuclei surrounded by numerous large mitochondria. Electron microscopy revealed well-differentiated cytoplasm which, in the liver cells, contained glycogen rosettes closely associated with smooth endoplasmic reticuhim.
Introduction
for use as microscopic models for studies in toxicology. We required some simple yet readily reproducible way of establishing these cells in chambers suitable for detailed light microscopy so that, having made light microscopic observations of the cells’ normal structure and behaviour, we could study their responses to particular perfused toxins. Then following fixation, embedding and section cutting, the same cells were to be examined electron microscopically to determine the corresponding toxin-induced changes in fine structure. The present method contributes a number of improvements, especially with respect to simplicity, rapidity and accurate microscopic assessment of the cultured cells.
FOR both cytophysiological
and cytopathological investigations there is need for simple methods for establishing cultures of various differentiated parenchymal cell types. Isolated from the nervous, hormonal and vascular influences of the parent organism, and living within a semi-synthetic extracellular fluid (culture medium) which can be readily manipulated and sampled, such cultured cells could provide extremely useful models for many cytological studies. For example, certain cell physiological and toxicological investigations would be greatly facilitated by using cultured liver parenchymal cells. In this regard, despite recent progress in methodology (e.g. Rose et al., 1968; Alexander and Grisham, 1970), the original difficulties experienced in culturing these cells (Bang and Warwick, 1965) have yet to be completely overcome. Our interest derived originally from the need to have cultured liver parenchymal cells
Methods and Materials
Culture technique As the method to be described applies to the culture of both liver and kidney parenchymal cells, only one description is made. Although designed to produce relatively small numbers of cells suitable for primary cultures in small culture chambers, the method could be adapted readily for the establishment of larger scale bottle cultures.
Department of Experimental Pathology, John Curtin School of Medical Research, The Australian National University, Canberra, A.C.T. 2600, Australia. Received 19 April 1974. Revised 16 September 1974. 641
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Cultures were made from the organs of lo-12 day old chick embryos. Working under a laminar flow hood (Clemco Ultraviolet Products, Artarmon, N.S.W.) and using aseptic techniques, the egg air space was entered with blunt forceps and the whole embryo transferred to a sterile Petri dish. With fresh instruments, the organ to be cultured was gently removed from the embryo and transferred to a second Petri dish containing approximately 7 ml 0.25 % trypsin made up in Hanks’ balanced salt solution (complete with calcium). Using a dissecting microscope and two fine pointed sterile probes, the gallbladder was dissected away from the liver (or the adrenal from the kidney). Then the capsule of the organ was carefully opened in several places to allow the trypsin solution free access to the underlying connective tissue septa. After allowing the organ to lie immersed in this solution at room temperature for 5-7 min, the fine pointed probes were employed to gently but progressively reduce the organ to tissue fragments of approximately 0.5 mm diameter. Using a 10 ml glass syringe fitted with a 13 cm 14 gauge needle, these fragments were serially drawn up into the syringe, along with the trypsin solution, then slowly expelled into a sterile graduated 10 ml centrifuge tube. Once the fragments had settled to the bottom of the tube, the supernatant trypsin solution was drawn off, discarded, and replaced by approximately 10 ml of Eagle’s basal culture medium (F9, Grand Island Biological Company, Grand Island, New York) containing 5% fetal calf serum (Commonwealth Serum Laboratories, Parkville, Victoria, Australia). With a fresh syringe and 14 gauge needle the fragments, suspended in this complete culture medium, were very gently aspirated back and forth through the needle. Doing this slowly, while keeping them continually suspended to avoid compression from crowding within the needle and crushing between the end of the plunger and barrel of the syringe, the tiny tissue fragments were progressively reduced to smaller and smaller clumps over a period of some 34 min. As soon as a good yield of cells and microscopic clumps had been obtained, aspiration was stopped and the remaining large fragments allowed to settle in the centrifuge tube. The supernatant medium, containing cells and tiny cell
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clumps, was then drawn into the same syringe and divided equally between two graduated centrifuge tubes each containing 1 ml fetal calf serum; this serum was designed to protect the parenchymal cells during subsequent centrifugation. These tubes were centrifuged at low speed (1200 rev/min) in an MSE ‘Minor’ bench top centrifuge for 1 min; this provided a centrifugal force of N 250 g, just sufficient to bring down the microscopic clumps and most of the intact cells. The supernatant medium which still contained some cells and much cell debris, was discarded and the cells in the pellet were gently resuspended in approximately 3 ml of fresh culture medium. The quality of the cell yield and quantity of cell debris were checked by direct microscopic observation, using a slide preparation and phase-contrast optics. The harvested cells, mostly in the form of microscopic cell clumps of various sizes, were used to seed previously incubated mediumfilled Rose culture chambers (Rose et al., 1958). This was done by injecting cells and cell clumps into the chamber cavities through 20 gauge needles. Cells not required for culture chambers were sometimes grown in T30 culture bottles. To provide for gas exchange between the medium contained in the culture chambers and the surrounding atmosphere, the Rose chambers were fitted with 38 mm2 lower coverslips cut from biaxially oriented gas-permeable polystyrene sheet (‘Polyflex’, Monsanto Chemicals (Australia) Limited, West Footscray, Victoria, Australia). Once chambers were seeded with cells, they were labelled and replaced, top coverglass down, in a thermal incubator adjusted to 38”C, for some 8-12 hr, until the cells had adhered to and spread thinly on the coverglass. To provide a gas phase suitable for the cultures, the incubator was gassed continuously with 5 % carbon dioxide in air. The time required to set up these cultures was approximately 30 min. Light microscopy
and photomicrography
Once the cells, either singly or in clumps, had spread thinly on the coverglass, the cultures were ready for microscopic examination and photomicrography. Cells most suitable for detailed light microscopic study were usually those at the margins ofcell clumps. These cells were not only the most thinly spread, making it possible to see considerable cytoplasmic
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detail, but once photographed, their marginal contours could be used for subsequent positive identification of particular cells required for electron microscopy (aide in&). Details of methods used for microscopy, photomicrography and cinephotomicrography were as described previously (Buckley and Porter, 1967). Cell perfusion and fixation
Prior to observations culminating in cell fixation, cultured cells were placed in a perfusion chamber. This was done by disassembling the original culture chamber, taking the coverglass with attached cells and reassembling it into a similar chamber, fitted with a gasket which provided for perfusion of liquid media (Buckley, 1971). Then the culture was promptly perfused with Eagle’s basal medium containing 5% fetal calf serum, as used for the original cultures. For fixation, the same perfusion apparatus was employed to perfuse the primary fixative solution. For the present observations, the primary fixative solution used was 3 % glutaraldehyde (Ladd Research Industries, Inc., Burlington, Vermont) made up in a N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES)-buffered solution containing the chlorides of calcium (0.1 M) and magnesium (0.02 M) adjusted to pH 7.2. After fixation in this solution for 7 min the chamber was disassembled and the cells, still attached to the coverglass, were post-fixed in cacodylate-buffered 1% osmium tetroxide for 7 min. Cell preparation for electron microscopy Fixed cells were dehydrated, embedded and sections of particular cells prepared for electron microscopy as described elsewhere (Buckley, 1971, 1973a). Sections were double stained with uranyl acetate and lead citrate. In this work, where attention was concentrated on the fine structure of particular thinly spread cultured cells from which comparatively few sections were obtainable, contamination with lead precipitate was often troublesome, despite the use of freshly prepared lead stain. It was found useful therefore, to filter the lead stain using a 13 mm Millipore syringe unit fitted with a VS (25 nm pore size) Millipore filter; staining sections in the fresh filtrate for 1-2 min
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was found to be a highly effective way of avoiding lead contamination. Results
The results obtained using the simple method outlined above will be illustrated by describing first a 1 day old culture from the liver of a 10 day old chick embryo and then a 2 day culture derived from the kidney of a 12 day old chick embryo. Liver parenchymal
cell culture
observations. Within 1224 hr of setting up the culture, liver parenchyma1 cells adhered to and spread out on the culture chamber coverglass. By low power phase-contrast microscopy it was seen that although occasional cells occurred singly, most occurred in cell clusters (Fig. la). These contained anything from two or three to approximately 100 cells per cluster, with an average of about 10 cells each. A few cells of connective tissue type were seen on the glass between these clusters (Fig. la). Cells within individual clusters were generally similar to one another, and were characteristically ‘epithelial’ in appearance. In contrast to connective tissue cells which separated from one another, these cells were neatly packed together and of fairly uniform dimensions, with similar sized round nuclei (Fig. la). Even within the larger clusters no organized arrangement of cells could be discerned. Most epithelial cell clusters comprised liver parenchymal cells (to be described in greater detail below) but in addition there were clusters of larger more thinly spread epithelial cells which may have been derived from bile duct epithelium. However, in this work attention was concentrated on the denser looking liver parenchymal cells. Considerable detail could be observed by examining thinly spread parenchymal cells using oil immersion phase-contrast microscopy (Fig. 1b). Individual cells contained one (or occasionally two) round nuclei each of which showed one or two dense nucleoli. Adjacent to some nuclei, an amorphous grey area, the Golgi complex, was seen. Surrounding this and the nucleus, the cytoplasm contained dense concentrations of organelles, most of which were mitochondria (Fig. 1b). As in cultured connective tissue cells (Buckley and Porter, 1967 ;
Light microscopic
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Buckley, 1974) these mitochondria were actively motile, undergoing a variety of deformational and migratory movements. In addition, the cytoplasm contained small dense round bodies which showed stop-start rapid darting movements characteristic of lysosomes (Buckley, 1973b) and occasional static lipid inclusions. In contrast to the situation in cultured connective tissue cells (Buckley, 1964) no endoplasmic reticulum could be observed light microscopically in these cells. The cell margins appeared as roundish projections on the glass surface; although these margins underwent gradual shifts in position, advancing or retracting, they were never seen to undergo the undulatory movements characteristic of many cultured cells. When the cells were fixed by glutaraldehyde perfusion, though all movements ceased abruptly, no other change could be detected in the appearance of their cytoplasm. Electron microscopic observations. Fig. I c represents a low power electron micrograph of a section of the cluster of living cells illustrated in Fig. 1b. Comparing the two figures, one can identify particular lipid inclusions and mitochondria and confirm the vesicular nature of the larger lysosomes (cf. Figs. lb, lc). Examining a portion of this cluster of cells at higher magnification, individual features
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are seen in greater detail (Fig. Id). The cells abut against one another as do epithelial cells. Large nuclei with prominent nucleoli are surrounded by numerous stubby mitochondria, lipid droplets and both dense and vesicular lysosomes. In addition, the cytoplasm contains numerous extremely dense granules. Fig. le, a more detailed view of adjacent portions of two cells illustrated in Fig. Id, shows part of two nuclei, an intercellular junction and nearby cytoplasm. Mitochondria show regularly disposed cristae and intracristal spaces. The surrounding cytoplasm contains, besides lysosomes and a lipid droplet, endoplasmic reticulum, some of which is in the smooth form and associated with the large dense granules. These granules are glycogen granules in rosette form associated with smooth endoplasmic reticulum in a way characteristic of differentiated liver parenchymal cells (Fig. le and le inset) (Bruni and Porter, 1965). In summary, it can be Seen that, by morphological criteria, these cultured cells are both well differentiated and healthy. Kidney parenchymal cell culture Light microscopic observations. As with the
liver parenchymal cells, cultured renal parenchymal cells spread on the supporting coverglass within 12-14 hr. Most occurred as epithelial cells in variable sized clusters with
Fig. I. Light and electron micrographs illustrating of cultured chick embryo liver parenchymal cells.
the structure
and tine structure
Fig. la. Very low power phase-contrast photomicrograph showing form and distribution of cultured chick embryo liver parenchymal cells, most of which are in clusters of various sizes. Note rounded nuclei and occasional isolated connective tissue cells (arrowheads). x 72. Fig. I b. Phase-contrast photomicrograph of embryo liver cells showing how cells, abutting coverglass surface. Each cell contains a single numerous mitochondria (m), occasional small lysosomes (v) and refractile lipid droplets (I). x
small cluster of living cultured chick on one another, stretch out on the round nucleus (n) about which are dense lysosomes (ly), some vesicular 700.
Fig. Ic. Very low power electron micrograph of a section through the right hand half of the cell cluster illustrated in Fig. lb. Overall shape, together with the disposition of the nuclei (n) and a large vesicular lysosome (v) identify the clumps. I, lipid droplet: m, mitochondrion; go, Golgi complex; ly, lysosome. x 2400.
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occasional connective tissue cells between. As in liver cell clusters, there appeared to be two principal types of epithelial cells. One type of cluster comprised cells whose cytoplasm was packed with rod-shaped mitochondria. This type was often seen spreading out from recognizable segments of kidney tubule. The other epithelial cell type, less numerous and of unknown origin, contained relatively few organelles and thus appeared to have clear cytoplasm; this type was not used in the present study. The epithelial cells illustrated in Fig. 2a contain roundish nuclei, each with one or two dense nucleoli. The cell indicated by an arrowhead is shown in greater detail in Fig. 2b. Surrounding the nucleus of this cell are numerous elongate mitochondria. As seen in movie records of this cell, these mitochondria underwent minor deformational movements and small translational movements which were limited to the direction of the mitochondrial long axes. Small dense lysosomes showed characteristic high speed darting movements in various directions. Resulting from slow movements of its margins, the contours of this cell changed and at the same time it was overlapped by the moving cytoplasmic process extending from an adjacent connective tissue cell (cf. Figs. 2b and 2~). Again, at the time of fixation, direct microscopic observation showed that, apart from abrupt cessation of all cellular and subcellular movements, the structures of the fixed cell appeared unchanged (cf. Figs. 2b and 2~).
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Electron microscopic observations. Fig. 2d is a low power electron micrograph of the cell shown in Figs. 2b and 2c. Within the cytoplasm surrounding its single round nucleus, filamentous mitochondria and small dense lysosomes can be identified and related to the corresponding structures seen light-microscopically in the recently fixed cell (cf. Figs. 2c and 2d). As seen at higher magnification, the mitochondria are all of more or less uniform calibre with regularly disposed cristae (Fig. 2e). The surrounding cytoplasm shows numerous ribosomes and moderately large numbers of microtubules, the majority of which lie oriented more or less radially between the nucleus and the cell periphery. The cytoplasm immediately subjacent to the plasma membrane (seen here sectioned obliquely) contains a dense meshwork of fine dense filaments (Fig. 2e). Some details of this cytoplasm are illustrated more clearly in Fig. 2f. Here the fine cortical filaments are seen to be -70 A in diameter and to run shorter courses within the section than the wider (- 250 A diameter) microtubules. Ribosomes are largely in the form of polyribosomes. Mitochondria, showing prominent matrix granules, well-defined cristae and good demarcation between intracristal and matrix spaces, exhibit a normal healthy-appearing fine structure (Fig. 2f). Ultimate fate of culturedparenchymal
cells
The present method of culturing liver and kidney parenchymal cells provided large
Fig. Id. Higher magnification electron micrograph of the mid-section of the same cluster showing roundish nuclei (n), intercellular junctions (arrows), mitochondria (m), lipid droplets (I), lysosomes (ly) and collections of dense glycogen granules (arrowheads). x 3850. Fig. le. Higher magnification view of intercellular junction area (arrows) showing portions of two nuclei (n) and adjacent cytoplasm containing mitochondria (m), rough endoplasmic reticulum (er), lysosomes (ly) and a lipid droplet (I). Typical glycogen rosettes (g) are seen closely associated with profiles of the smooth endoplasmic reticulum (arrowheads). x 19,500. The inset shows this association in greater detail. x 21,000.
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numbers of well-differentiated healthy cells. From estimates of the number of living spread cells compared to the number of dead floating cells 12 hr after culture, it was calculated that approximately 80% of explanted cells survived the procedure. Initially nearly all of these appeared healthy. However, in all cultures, even at 24-48 hr when at their best, there were always a few cells (- 3-5 %) which showed early signs of damage. Then, just as occurs in cultures of chick embryo connective tissue cells (Buckley, 1964, 1973b), over succeeding weeks, the number of damaged parenchymal cells gradually increased. The earliest detectable signs indicating subtle
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parenchymal cell damage were similar to those found in cultured chick embryo connective tissue cells. These signs were an increase in the number, size and activity of lysosomes (especially those having a vesicular appearance) and an increase in the quantity of intracellular fat (Buckley, 1973b). In addition, there was a slow but progressive dedifferentiation of parenchymal cells. This dedifferentiation took the form of increasing separation between parenchymal cells, especially at the periphery of cell clusters, and of a greater degree of cell spreading, their peripheral cytoplasm becoming more thinned out and their margins less rounded in contour. However, notwithstanding thesevarious
Fig. 2. Light and electron micrographs cultured chick embryo kidney parenchymal
illustrating cells.
structure
and fine structure
of
Fig. 2a. Very low power phase-contrast photomicrograph showing marginal zone of large cluster of cultured kidney parenchymal cells. Note close association of cells and large round nuclei. An arrowhead indicates the cell shown in Figs. 2b and 2c. x 184. Fig. 2b. High power phase-contrast view of a living kidney parenchymal cell at the margin of this cluster. A nucleus (n) with dense nucleolus is surrounded by more or less radially oriented dense filamentous mitochondria (m), small dense lysosomes (ly) and homogeneous appearing cytoplasm. The contours of the cell margin, while largely rounded, show a number of small projections. x 940. Fig. 2c. Phase-contrast photomicrograph of the same cell taken 24 min later, 7 min after perfusion fixation with glutaraldehyde. During the 17 min prior to fixation, the cell was partially overlapped by a neighbouring cell (c) and it extended further on to the glass and changed its outline. At the same time its mitochondria (m) have moved further towards the cell periphery and, though most have remained more or less radially disposed. some have undergone pronounced conformational changes. ly, lysosomes. x 940. Fig. 2d. Very low power electron micrograph of a section through the same cell showing the overriding cell (c) and the roundish basal nucleus (n) surrounded by a few dense lysosomes (ly) and large numbers of mitochondria (m) the more peripheral ones of which are radially oriented. Although well separated from neighbouring cells over its basal aspects, it shows well marked intercellular junctions on either side. Arrowheads indicate the region shown in Fig. 2e. x 2450. Fig. 2e. Higher magnification electron micrograph of marginal zone of cytoplasm of the same cell. Healthy appearing elongate mitochondria (m) are flanked by ribosomerich cytoplasm containing moderate numbers of radially oriented microtubules (0. The obliquely sectioned cell cortex shows a well-developed filament meshwork (f). X 15,500. Fig. 2f. More detailed view of portion of this cytoplasm. Both microtubules (t) and mitochondria (m) run extended courses through the section. The mitochondria are uniform in calibre and show fairly regularly disposed cristae. Ribosomes are mostly in the form of polyribosomes and the cortical meshwork is made up of -70 A solid filaments(f); few solid filaments are seen deeper within the cytoplasm. x 27,500.
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changes, large numbers of cells of recognizable parenchymal type persisted for at least 2-3 weeks, at which stage we usually discarded the cultures. Discussion As shown here, the present method represents a simple and rapid means of providing short-term cultures of fetal liver and kidney parenchymal cells. In the early stages of culture, the great majority of these cells are both highly differentiated and healthy. Accordingly, they provide excellent cell models for microscopic studies. Later, after several days in culture, some parenchymal cells begin to show subtle signs of damage and to dedifferentiate. Possible reasons for this will be discussed below, but first factors which appear to be responsible for the short-term effectiveness of the method will be outlined. These factors may be subdivided into those operating during cell isolation and those operating during culture, that is, the conditions of cell culture. In considering methods of cell isolation, we had in mind that highly specialized parenchymal cells, deprived of their blood sgpply and immersed calcium-free salt in enzyme-containing solutions would be extremely fragile and highly susceptible to mechanical damage. Accordingly, while following common tissue culture practice and using trypsin to disrupt connective tissue and intercellular bonds, we employed Hanks’ balanced salt solution as diluent. Although the calcium in this diluent reduces the rate of tryptic action, it maintains the integrity of the cell membranes and thus greatly reduces cell damage (Amos, 1965). Next, to minimize mechanically induced cell damage we used a method of cell separation which was extremely gentle. As a check on the gentleness of the method, we routinely examined the cell suspensions by phase-contrast microscopy to determine the relative proportions of intact cells and cell debris. In addition, phase-contrast microscopy was used to assess the state of health and differentiation of the isolated cultured cells. As it became clear that the incidence of cell damage was much lower when the cells remained in small clumps, mechanical treatment was stopped short of complete cell separation.
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Considering the cell culture conditions, there seemed to be no clear indication from the literature as to the optimum culture medium for liver parenchymal cells, so we used a comparatively simple one. Following a suggestion by Bissell and Tilles (1971) that high serum concentrations damaged liver parenchymal cells, we used a relatively low concentration. Overall confluence of parenchymal cells was avoided as this would have obscured the cytological detail needed for our studies. However, chambers were moderately heavily seeded with cells and the high cell densities believed to favour the maintenance of cell health (Knazek et al., 1972) were achieved locally, within the cell clusters. Although our culture medium lacked a free surface open to a gas phase, we compensated for this by providing a gaspermeable coverslip on one side of the chamber and, in accordance with the idea that high oxygen levels might inhibit liver parenchymal cells (Dickson, 1971; Iype, 1971), the culture chambers were incubated in an atmosphere of 5% carbon dioxide in air. Thus we provided cell culture conditions which, at least initially, maintained parenchymal cells in a state of differentiation and good health. And yet, as outlined above, over the successive days and weeks following the establishment of cultures, the parenchymal cells slowly deteriorated in quality. From this we concluded that the culture conditions we provided were non-optimal, leading ultimately to cellular injury, dedifferentiation and death. Mechanisms causing this damage and dedifferentiation affecting cultured chick embryo liver and kidney parenchymal cells are far from clear. As degeneration and death affects cultures of connective tissue cells from chick embryos (Ponten, 1970; Lima and Macieira-Coelho, 1972), but not of many other species (e.g. rat), it seems probable that much of the observed damage in chick embryo parenchymal cells is due to a species-determined response to conditions of culture (Ponten, 1970). Changes observed in cultured chick embryo cells include accumulation of fat and both an increase in the number of lysosomes and a change in their character, lysosomes enlarging greatly and becoming more vesicular (Buckley, 1973b). This change, together with the known transformation of the endoplasmic
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reticulum of cultured chick embryo cells to a more hydrated, less visible, form, suggests that these cells have lost full control of their water balance (Buckley, 1964, 1973b). However, the underlying causative mechanism for these changes in chick embryo
cells under conditions be determined.
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of culture has yet to
Acknowledgement
We wish to thank Mrs Gay Nicholls excellent technical assistance.
for
References ALEXANDER, R. W. and GRISHAM, J. W. 1970. Explant culture of rat liver. I. Method, morphology, and cytogenesis. Lab. Invest., 22, 50-62. AMOS, H. 1965. Personal communication. BANG, F. B. and WARWICK, A. C. 1965. Liver. In Cells and Tissues in Culture (ed. E. N. Willmer), Vol. II, pp. 607-630. Academic Press, New York. BISSELL, D. M. and TILLES, J. G. 1971. Morphology and function of cells of human embryonic liver in monolayer culture. J. Cell Biol., 50, 222-231. BRUNI, C. and PORTER, K. R. 1965. The fine structure of the parenchymal cell of the normal rat liver. Am. J. Path.,
46, 691-755.
BUCKLEY, I. K. 1964. Phase contrast Protoplasma,
observations
on the endoplasmic
reticulum
of living cells in culture.
59, 569-588.
BUCKLEY, 1. K. 1971. A simple technique for comparative light and electron microscopy of designated living cultured cells. Lab. Invest., 25, 295-301. BUCKLEY,I. K. 1973a. Studies in fixation for electron microscopy using cultured cells as microscopic models. Lab. Invest.,
29, 398-410.
BUCKLEY, I. K. 1973b. The lysosomes of cultured chick embryo cells: a correlated light and electron microscopic study. Lab. Invest., 29, 411-421. BUCKLEY, I. K. 1974. Subcellular motility: a correlated light and electron microscopic study using cultured cells. Tissue & Cell, 6, l-20. BUCKLEY, I. K. and PORTER, K. R. 1967. Cytoplasmic fibrils in living cultured cells. A light and electron microscope study. Protoplasma, 64, 349-380. DICKSON, J. A. 1971. Metabolism of adult rat liver cells in filter-well culture. E.upl Cell Res., 64, 17-28. IYPF.. P. T. 1971. Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver. J. Cell Physiol., 78, 281-288. KNAZEK, R. A., GULLINO, P. M., KOHLER, P. 0. and DEDRICK, R. L. 1972. Cell culture on artificial capillartes: an approach to tissue growth in vitro. Science, N. Y., 178, 65-66. LIMA, L. and MACIEIRA-COELHO,A. 1972. Parameters of aging in chicken embryo fibroblasts cultivated in vitro. Expl Cell Res., 70, 279-284. PONT~N, J. 1970. The growth capacity Int. J. Cancer, 6, 323-332.
of normal
and Rous-virus-transformed
chicken
fibroblasts
in virro.
ROSE, G. G., KUMEGAWA, M. and CATTONI, M. 1968. The circumfusion system for multipurpose culture chambers. II. The protracted maintenance of differentiation of foetal and newborn mouse liver in vitro. J. Cell Biol., 39, 430450.
ROSE, G. G., POMERAT,C. M., SHINDLER, T. 0. and TRUNNELL, J. B. 1958. A cellophane-strip technique culturing tissue in multipurpose culture chambers. J. biophys. biochem. Cytol.. 4, 761-763.
for
IAN K. BUCKLEY and JUDIE R. WALTON
A SIMPLE METHOD FOR CULTURING CHICK EMBRYO LIVER AND KIDNEY PARENCHYMAL CELLS FOR MICROSCOPIC STUDIES ABSTRACT. Differentiated parenchymal cells were cultured from chick embryo livers or kidneys. Tissues were trypsinized, fragmented under a dissecting microscope, suspended in culture medium and separated into microscopic cell clumps with a syringe and wide bore needle. These cell clumps were grown in culture chambers and after light microscopic study were fixed and sectioned for electron microscopy. Both liver and kidney parenchymal cells appeared as clusters of epithelial cells containing round nuclei surrounded by numerous large mitochondria. Electron microscopy revealed well-differentiated cytoplasm which, in the liver cells, contained glycogen rosettes closely associated with smooth endoplasmic reticuhim.
Introduction
for use as microscopic models for studies in toxicology. We required some simple yet readily reproducible way of establishing these cells in chambers suitable for detailed light microscopy so that, having made light microscopic observations of the cells’ normal structure and behaviour, we could study their responses to particular perfused toxins. Then following fixation, embedding and section cutting, the same cells were to be examined electron microscopically to determine the corresponding toxin-induced changes in fine structure. The present method contributes a number of improvements, especially with respect to simplicity, rapidity and accurate microscopic assessment of the cultured cells.
FOR both cytophysiological
and cytopathological investigations there is need for simple methods for establishing cultures of various differentiated parenchymal cell types. Isolated from the nervous, hormonal and vascular influences of the parent organism, and living within a semi-synthetic extracellular fluid (culture medium) which can be readily manipulated and sampled, such cultured cells could provide extremely useful models for many cytological studies. For example, certain cell physiological and toxicological investigations would be greatly facilitated by using cultured liver parenchymal cells. In this regard, despite recent progress in methodology (e.g. Rose et al., 1968; Alexander and Grisham, 1970), the original difficulties experienced in culturing these cells (Bang and Warwick, 1965) have yet to be completely overcome. Our interest derived originally from the need to have cultured liver parenchymal cells
Methods and Materials
Culture technique As the method to be described applies to the culture of both liver and kidney parenchymal cells, only one description is made. Although designed to produce relatively small numbers of cells suitable for primary cultures in small culture chambers, the method could be adapted readily for the establishment of larger scale bottle cultures.
Department of Experimental Pathology, John Curtin School of Medical Research, The Australian National University, Canberra, A.C.T. 2600, Australia. Received 19 April 1974. Revised 16 September 1974. 641
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Cultures were made from the organs of lo-12 day old chick embryos. Working under a laminar flow hood (Clemco Ultraviolet Products, Artarmon, N.S.W.) and using aseptic techniques, the egg air space was entered with blunt forceps and the whole embryo transferred to a sterile Petri dish. With fresh instruments, the organ to be cultured was gently removed from the embryo and transferred to a second Petri dish containing approximately 7 ml 0.25 % trypsin made up in Hanks’ balanced salt solution (complete with calcium). Using a dissecting microscope and two fine pointed sterile probes, the gallbladder was dissected away from the liver (or the adrenal from the kidney). Then the capsule of the organ was carefully opened in several places to allow the trypsin solution free access to the underlying connective tissue septa. After allowing the organ to lie immersed in this solution at room temperature for 5-7 min, the fine pointed probes were employed to gently but progressively reduce the organ to tissue fragments of approximately 0.5 mm diameter. Using a 10 ml glass syringe fitted with a 13 cm 14 gauge needle, these fragments were serially drawn up into the syringe, along with the trypsin solution, then slowly expelled into a sterile graduated 10 ml centrifuge tube. Once the fragments had settled to the bottom of the tube, the supernatant trypsin solution was drawn off, discarded, and replaced by approximately 10 ml of Eagle’s basal culture medium (F9, Grand Island Biological Company, Grand Island, New York) containing 5% fetal calf serum (Commonwealth Serum Laboratories, Parkville, Victoria, Australia). With a fresh syringe and 14 gauge needle the fragments, suspended in this complete culture medium, were very gently aspirated back and forth through the needle. Doing this slowly, while keeping them continually suspended to avoid compression from crowding within the needle and crushing between the end of the plunger and barrel of the syringe, the tiny tissue fragments were progressively reduced to smaller and smaller clumps over a period of some 34 min. As soon as a good yield of cells and microscopic clumps had been obtained, aspiration was stopped and the remaining large fragments allowed to settle in the centrifuge tube. The supernatant medium, containing cells and tiny cell
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clumps, was then drawn into the same syringe and divided equally between two graduated centrifuge tubes each containing 1 ml fetal calf serum; this serum was designed to protect the parenchymal cells during subsequent centrifugation. These tubes were centrifuged at low speed (1200 rev/min) in an MSE ‘Minor’ bench top centrifuge for 1 min; this provided a centrifugal force of N 250 g, just sufficient to bring down the microscopic clumps and most of the intact cells. The supernatant medium which still contained some cells and much cell debris, was discarded and the cells in the pellet were gently resuspended in approximately 3 ml of fresh culture medium. The quality of the cell yield and quantity of cell debris were checked by direct microscopic observation, using a slide preparation and phase-contrast optics. The harvested cells, mostly in the form of microscopic cell clumps of various sizes, were used to seed previously incubated mediumfilled Rose culture chambers (Rose et al., 1958). This was done by injecting cells and cell clumps into the chamber cavities through 20 gauge needles. Cells not required for culture chambers were sometimes grown in T30 culture bottles. To provide for gas exchange between the medium contained in the culture chambers and the surrounding atmosphere, the Rose chambers were fitted with 38 mm2 lower coverslips cut from biaxially oriented gas-permeable polystyrene sheet (‘Polyflex’, Monsanto Chemicals (Australia) Limited, West Footscray, Victoria, Australia). Once chambers were seeded with cells, they were labelled and replaced, top coverglass down, in a thermal incubator adjusted to 38”C, for some 8-12 hr, until the cells had adhered to and spread thinly on the coverglass. To provide a gas phase suitable for the cultures, the incubator was gassed continuously with 5 % carbon dioxide in air. The time required to set up these cultures was approximately 30 min. Light microscopy
and photomicrography
Once the cells, either singly or in clumps, had spread thinly on the coverglass, the cultures were ready for microscopic examination and photomicrography. Cells most suitable for detailed light microscopic study were usually those at the margins ofcell clumps. These cells were not only the most thinly spread, making it possible to see considerable cytoplasmic
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detail, but once photographed, their marginal contours could be used for subsequent positive identification of particular cells required for electron microscopy (aide in&). Details of methods used for microscopy, photomicrography and cinephotomicrography were as described previously (Buckley and Porter, 1967). Cell perfusion and fixation
Prior to observations culminating in cell fixation, cultured cells were placed in a perfusion chamber. This was done by disassembling the original culture chamber, taking the coverglass with attached cells and reassembling it into a similar chamber, fitted with a gasket which provided for perfusion of liquid media (Buckley, 1971). Then the culture was promptly perfused with Eagle’s basal medium containing 5% fetal calf serum, as used for the original cultures. For fixation, the same perfusion apparatus was employed to perfuse the primary fixative solution. For the present observations, the primary fixative solution used was 3 % glutaraldehyde (Ladd Research Industries, Inc., Burlington, Vermont) made up in a N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES)-buffered solution containing the chlorides of calcium (0.1 M) and magnesium (0.02 M) adjusted to pH 7.2. After fixation in this solution for 7 min the chamber was disassembled and the cells, still attached to the coverglass, were post-fixed in cacodylate-buffered 1% osmium tetroxide for 7 min. Cell preparation for electron microscopy Fixed cells were dehydrated, embedded and sections of particular cells prepared for electron microscopy as described elsewhere (Buckley, 1971, 1973a). Sections were double stained with uranyl acetate and lead citrate. In this work, where attention was concentrated on the fine structure of particular thinly spread cultured cells from which comparatively few sections were obtainable, contamination with lead precipitate was often troublesome, despite the use of freshly prepared lead stain. It was found useful therefore, to filter the lead stain using a 13 mm Millipore syringe unit fitted with a VS (25 nm pore size) Millipore filter; staining sections in the fresh filtrate for 1-2 min
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was found to be a highly effective way of avoiding lead contamination. Results
The results obtained using the simple method outlined above will be illustrated by describing first a 1 day old culture from the liver of a 10 day old chick embryo and then a 2 day culture derived from the kidney of a 12 day old chick embryo. Liver parenchymal
cell culture
observations. Within 1224 hr of setting up the culture, liver parenchyma1 cells adhered to and spread out on the culture chamber coverglass. By low power phase-contrast microscopy it was seen that although occasional cells occurred singly, most occurred in cell clusters (Fig. la). These contained anything from two or three to approximately 100 cells per cluster, with an average of about 10 cells each. A few cells of connective tissue type were seen on the glass between these clusters (Fig. la). Cells within individual clusters were generally similar to one another, and were characteristically ‘epithelial’ in appearance. In contrast to connective tissue cells which separated from one another, these cells were neatly packed together and of fairly uniform dimensions, with similar sized round nuclei (Fig. la). Even within the larger clusters no organized arrangement of cells could be discerned. Most epithelial cell clusters comprised liver parenchymal cells (to be described in greater detail below) but in addition there were clusters of larger more thinly spread epithelial cells which may have been derived from bile duct epithelium. However, in this work attention was concentrated on the denser looking liver parenchymal cells. Considerable detail could be observed by examining thinly spread parenchymal cells using oil immersion phase-contrast microscopy (Fig. 1b). Individual cells contained one (or occasionally two) round nuclei each of which showed one or two dense nucleoli. Adjacent to some nuclei, an amorphous grey area, the Golgi complex, was seen. Surrounding this and the nucleus, the cytoplasm contained dense concentrations of organelles, most of which were mitochondria (Fig. 1b). As in cultured connective tissue cells (Buckley and Porter, 1967 ;
Light microscopic
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Buckley, 1974) these mitochondria were actively motile, undergoing a variety of deformational and migratory movements. In addition, the cytoplasm contained small dense round bodies which showed stop-start rapid darting movements characteristic of lysosomes (Buckley, 1973b) and occasional static lipid inclusions. In contrast to the situation in cultured connective tissue cells (Buckley, 1964) no endoplasmic reticulum could be observed light microscopically in these cells. The cell margins appeared as roundish projections on the glass surface; although these margins underwent gradual shifts in position, advancing or retracting, they were never seen to undergo the undulatory movements characteristic of many cultured cells. When the cells were fixed by glutaraldehyde perfusion, though all movements ceased abruptly, no other change could be detected in the appearance of their cytoplasm. Electron microscopic observations. Fig. I c represents a low power electron micrograph of a section of the cluster of living cells illustrated in Fig. 1b. Comparing the two figures, one can identify particular lipid inclusions and mitochondria and confirm the vesicular nature of the larger lysosomes (cf. Figs. lb, lc). Examining a portion of this cluster of cells at higher magnification, individual features
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are seen in greater detail (Fig. Id). The cells abut against one another as do epithelial cells. Large nuclei with prominent nucleoli are surrounded by numerous stubby mitochondria, lipid droplets and both dense and vesicular lysosomes. In addition, the cytoplasm contains numerous extremely dense granules. Fig. le, a more detailed view of adjacent portions of two cells illustrated in Fig. Id, shows part of two nuclei, an intercellular junction and nearby cytoplasm. Mitochondria show regularly disposed cristae and intracristal spaces. The surrounding cytoplasm contains, besides lysosomes and a lipid droplet, endoplasmic reticulum, some of which is in the smooth form and associated with the large dense granules. These granules are glycogen granules in rosette form associated with smooth endoplasmic reticulum in a way characteristic of differentiated liver parenchymal cells (Fig. le and le inset) (Bruni and Porter, 1965). In summary, it can be Seen that, by morphological criteria, these cultured cells are both well differentiated and healthy. Kidney parenchymal cell culture Light microscopic observations. As with the
liver parenchymal cells, cultured renal parenchymal cells spread on the supporting coverglass within 12-14 hr. Most occurred as epithelial cells in variable sized clusters with
Fig. I. Light and electron micrographs illustrating of cultured chick embryo liver parenchymal cells.
the structure
and tine structure
Fig. la. Very low power phase-contrast photomicrograph showing form and distribution of cultured chick embryo liver parenchymal cells, most of which are in clusters of various sizes. Note rounded nuclei and occasional isolated connective tissue cells (arrowheads). x 72. Fig. I b. Phase-contrast photomicrograph of embryo liver cells showing how cells, abutting coverglass surface. Each cell contains a single numerous mitochondria (m), occasional small lysosomes (v) and refractile lipid droplets (I). x
small cluster of living cultured chick on one another, stretch out on the round nucleus (n) about which are dense lysosomes (ly), some vesicular 700.
Fig. Ic. Very low power electron micrograph of a section through the right hand half of the cell cluster illustrated in Fig. lb. Overall shape, together with the disposition of the nuclei (n) and a large vesicular lysosome (v) identify the clumps. I, lipid droplet: m, mitochondrion; go, Golgi complex; ly, lysosome. x 2400.
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occasional connective tissue cells between. As in liver cell clusters, there appeared to be two principal types of epithelial cells. One type of cluster comprised cells whose cytoplasm was packed with rod-shaped mitochondria. This type was often seen spreading out from recognizable segments of kidney tubule. The other epithelial cell type, less numerous and of unknown origin, contained relatively few organelles and thus appeared to have clear cytoplasm; this type was not used in the present study. The epithelial cells illustrated in Fig. 2a contain roundish nuclei, each with one or two dense nucleoli. The cell indicated by an arrowhead is shown in greater detail in Fig. 2b. Surrounding the nucleus of this cell are numerous elongate mitochondria. As seen in movie records of this cell, these mitochondria underwent minor deformational movements and small translational movements which were limited to the direction of the mitochondrial long axes. Small dense lysosomes showed characteristic high speed darting movements in various directions. Resulting from slow movements of its margins, the contours of this cell changed and at the same time it was overlapped by the moving cytoplasmic process extending from an adjacent connective tissue cell (cf. Figs. 2b and 2~). Again, at the time of fixation, direct microscopic observation showed that, apart from abrupt cessation of all cellular and subcellular movements, the structures of the fixed cell appeared unchanged (cf. Figs. 2b and 2~).
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Electron microscopic observations. Fig. 2d is a low power electron micrograph of the cell shown in Figs. 2b and 2c. Within the cytoplasm surrounding its single round nucleus, filamentous mitochondria and small dense lysosomes can be identified and related to the corresponding structures seen light-microscopically in the recently fixed cell (cf. Figs. 2c and 2d). As seen at higher magnification, the mitochondria are all of more or less uniform calibre with regularly disposed cristae (Fig. 2e). The surrounding cytoplasm shows numerous ribosomes and moderately large numbers of microtubules, the majority of which lie oriented more or less radially between the nucleus and the cell periphery. The cytoplasm immediately subjacent to the plasma membrane (seen here sectioned obliquely) contains a dense meshwork of fine dense filaments (Fig. 2e). Some details of this cytoplasm are illustrated more clearly in Fig. 2f. Here the fine cortical filaments are seen to be -70 A in diameter and to run shorter courses within the section than the wider (- 250 A diameter) microtubules. Ribosomes are largely in the form of polyribosomes. Mitochondria, showing prominent matrix granules, well-defined cristae and good demarcation between intracristal and matrix spaces, exhibit a normal healthy-appearing fine structure (Fig. 2f). Ultimate fate of culturedparenchymal
cells
The present method of culturing liver and kidney parenchymal cells provided large
Fig. Id. Higher magnification electron micrograph of the mid-section of the same cluster showing roundish nuclei (n), intercellular junctions (arrows), mitochondria (m), lipid droplets (I), lysosomes (ly) and collections of dense glycogen granules (arrowheads). x 3850. Fig. le. Higher magnification view of intercellular junction area (arrows) showing portions of two nuclei (n) and adjacent cytoplasm containing mitochondria (m), rough endoplasmic reticulum (er), lysosomes (ly) and a lipid droplet (I). Typical glycogen rosettes (g) are seen closely associated with profiles of the smooth endoplasmic reticulum (arrowheads). x 19,500. The inset shows this association in greater detail. x 21,000.
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numbers of well-differentiated healthy cells. From estimates of the number of living spread cells compared to the number of dead floating cells 12 hr after culture, it was calculated that approximately 80% of explanted cells survived the procedure. Initially nearly all of these appeared healthy. However, in all cultures, even at 24-48 hr when at their best, there were always a few cells (- 3-5 %) which showed early signs of damage. Then, just as occurs in cultures of chick embryo connective tissue cells (Buckley, 1964, 1973b), over succeeding weeks, the number of damaged parenchymal cells gradually increased. The earliest detectable signs indicating subtle
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parenchymal cell damage were similar to those found in cultured chick embryo connective tissue cells. These signs were an increase in the number, size and activity of lysosomes (especially those having a vesicular appearance) and an increase in the quantity of intracellular fat (Buckley, 1973b). In addition, there was a slow but progressive dedifferentiation of parenchymal cells. This dedifferentiation took the form of increasing separation between parenchymal cells, especially at the periphery of cell clusters, and of a greater degree of cell spreading, their peripheral cytoplasm becoming more thinned out and their margins less rounded in contour. However, notwithstanding thesevarious
Fig. 2. Light and electron micrographs cultured chick embryo kidney parenchymal
illustrating cells.
structure
and fine structure
of
Fig. 2a. Very low power phase-contrast photomicrograph showing marginal zone of large cluster of cultured kidney parenchymal cells. Note close association of cells and large round nuclei. An arrowhead indicates the cell shown in Figs. 2b and 2c. x 184. Fig. 2b. High power phase-contrast view of a living kidney parenchymal cell at the margin of this cluster. A nucleus (n) with dense nucleolus is surrounded by more or less radially oriented dense filamentous mitochondria (m), small dense lysosomes (ly) and homogeneous appearing cytoplasm. The contours of the cell margin, while largely rounded, show a number of small projections. x 940. Fig. 2c. Phase-contrast photomicrograph of the same cell taken 24 min later, 7 min after perfusion fixation with glutaraldehyde. During the 17 min prior to fixation, the cell was partially overlapped by a neighbouring cell (c) and it extended further on to the glass and changed its outline. At the same time its mitochondria (m) have moved further towards the cell periphery and, though most have remained more or less radially disposed. some have undergone pronounced conformational changes. ly, lysosomes. x 940. Fig. 2d. Very low power electron micrograph of a section through the same cell showing the overriding cell (c) and the roundish basal nucleus (n) surrounded by a few dense lysosomes (ly) and large numbers of mitochondria (m) the more peripheral ones of which are radially oriented. Although well separated from neighbouring cells over its basal aspects, it shows well marked intercellular junctions on either side. Arrowheads indicate the region shown in Fig. 2e. x 2450. Fig. 2e. Higher magnification electron micrograph of marginal zone of cytoplasm of the same cell. Healthy appearing elongate mitochondria (m) are flanked by ribosomerich cytoplasm containing moderate numbers of radially oriented microtubules (0. The obliquely sectioned cell cortex shows a well-developed filament meshwork (f). X 15,500. Fig. 2f. More detailed view of portion of this cytoplasm. Both microtubules (t) and mitochondria (m) run extended courses through the section. The mitochondria are uniform in calibre and show fairly regularly disposed cristae. Ribosomes are mostly in the form of polyribosomes and the cortical meshwork is made up of -70 A solid filaments(f); few solid filaments are seen deeper within the cytoplasm. x 27,500.
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changes, large numbers of cells of recognizable parenchymal type persisted for at least 2-3 weeks, at which stage we usually discarded the cultures. Discussion As shown here, the present method represents a simple and rapid means of providing short-term cultures of fetal liver and kidney parenchymal cells. In the early stages of culture, the great majority of these cells are both highly differentiated and healthy. Accordingly, they provide excellent cell models for microscopic studies. Later, after several days in culture, some parenchymal cells begin to show subtle signs of damage and to dedifferentiate. Possible reasons for this will be discussed below, but first factors which appear to be responsible for the short-term effectiveness of the method will be outlined. These factors may be subdivided into those operating during cell isolation and those operating during culture, that is, the conditions of cell culture. In considering methods of cell isolation, we had in mind that highly specialized parenchymal cells, deprived of their blood sgpply and immersed calcium-free salt in enzyme-containing solutions would be extremely fragile and highly susceptible to mechanical damage. Accordingly, while following common tissue culture practice and using trypsin to disrupt connective tissue and intercellular bonds, we employed Hanks’ balanced salt solution as diluent. Although the calcium in this diluent reduces the rate of tryptic action, it maintains the integrity of the cell membranes and thus greatly reduces cell damage (Amos, 1965). Next, to minimize mechanically induced cell damage we used a method of cell separation which was extremely gentle. As a check on the gentleness of the method, we routinely examined the cell suspensions by phase-contrast microscopy to determine the relative proportions of intact cells and cell debris. In addition, phase-contrast microscopy was used to assess the state of health and differentiation of the isolated cultured cells. As it became clear that the incidence of cell damage was much lower when the cells remained in small clumps, mechanical treatment was stopped short of complete cell separation.
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Considering the cell culture conditions, there seemed to be no clear indication from the literature as to the optimum culture medium for liver parenchymal cells, so we used a comparatively simple one. Following a suggestion by Bissell and Tilles (1971) that high serum concentrations damaged liver parenchymal cells, we used a relatively low concentration. Overall confluence of parenchymal cells was avoided as this would have obscured the cytological detail needed for our studies. However, chambers were moderately heavily seeded with cells and the high cell densities believed to favour the maintenance of cell health (Knazek et al., 1972) were achieved locally, within the cell clusters. Although our culture medium lacked a free surface open to a gas phase, we compensated for this by providing a gaspermeable coverslip on one side of the chamber and, in accordance with the idea that high oxygen levels might inhibit liver parenchymal cells (Dickson, 1971; Iype, 1971), the culture chambers were incubated in an atmosphere of 5% carbon dioxide in air. Thus we provided cell culture conditions which, at least initially, maintained parenchymal cells in a state of differentiation and good health. And yet, as outlined above, over the successive days and weeks following the establishment of cultures, the parenchymal cells slowly deteriorated in quality. From this we concluded that the culture conditions we provided were non-optimal, leading ultimately to cellular injury, dedifferentiation and death. Mechanisms causing this damage and dedifferentiation affecting cultured chick embryo liver and kidney parenchymal cells are far from clear. As degeneration and death affects cultures of connective tissue cells from chick embryos (Ponten, 1970; Lima and Macieira-Coelho, 1972), but not of many other species (e.g. rat), it seems probable that much of the observed damage in chick embryo parenchymal cells is due to a species-determined response to conditions of culture (Ponten, 1970). Changes observed in cultured chick embryo cells include accumulation of fat and both an increase in the number of lysosomes and a change in their character, lysosomes enlarging greatly and becoming more vesicular (Buckley, 1973b). This change, together with the known transformation of the endoplasmic
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reticulum of cultured chick embryo cells to a more hydrated, less visible, form, suggests that these cells have lost full control of their water balance (Buckley, 1964, 1973b). However, the underlying causative mechanism for these changes in chick embryo
cells under conditions be determined.
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of culture has yet to
Acknowledgement
We wish to thank Mrs Gay Nicholls excellent technical assistance.
for
References ALEXANDER, R. W. and GRISHAM, J. W. 1970. Explant culture of rat liver. I. Method, morphology, and cytogenesis. Lab. Invest., 22, 50-62. AMOS, H. 1965. Personal communication. BANG, F. B. and WARWICK, A. C. 1965. Liver. In Cells and Tissues in Culture (ed. E. N. Willmer), Vol. II, pp. 607-630. Academic Press, New York. BISSELL, D. M. and TILLES, J. G. 1971. Morphology and function of cells of human embryonic liver in monolayer culture. J. Cell Biol., 50, 222-231. BRUNI, C. and PORTER, K. R. 1965. The fine structure of the parenchymal cell of the normal rat liver. Am. J. Path.,
46, 691-755.
BUCKLEY, I. K. 1964. Phase contrast Protoplasma,
observations
on the endoplasmic
reticulum
of living cells in culture.
59, 569-588.
BUCKLEY, 1. K. 1971. A simple technique for comparative light and electron microscopy of designated living cultured cells. Lab. Invest., 25, 295-301. BUCKLEY,I. K. 1973a. Studies in fixation for electron microscopy using cultured cells as microscopic models. Lab. Invest.,
29, 398-410.
BUCKLEY, I. K. 1973b. The lysosomes of cultured chick embryo cells: a correlated light and electron microscopic study. Lab. Invest., 29, 411-421. BUCKLEY, I. K. 1974. Subcellular motility: a correlated light and electron microscopic study using cultured cells. Tissue & Cell, 6, l-20. BUCKLEY, I. K. and PORTER, K. R. 1967. Cytoplasmic fibrils in living cultured cells. A light and electron microscope study. Protoplasma, 64, 349-380. DICKSON, J. A. 1971. Metabolism of adult rat liver cells in filter-well culture. E.upl Cell Res., 64, 17-28. IYPF.. P. T. 1971. Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver. J. Cell Physiol., 78, 281-288. KNAZEK, R. A., GULLINO, P. M., KOHLER, P. 0. and DEDRICK, R. L. 1972. Cell culture on artificial capillartes: an approach to tissue growth in vitro. Science, N. Y., 178, 65-66. LIMA, L. and MACIEIRA-COELHO,A. 1972. Parameters of aging in chicken embryo fibroblasts cultivated in vitro. Expl Cell Res., 70, 279-284. PONT~N, J. 1970. The growth capacity Int. J. Cancer, 6, 323-332.
of normal
and Rous-virus-transformed
chicken
fibroblasts
in virro.
ROSE, G. G., KUMEGAWA, M. and CATTONI, M. 1968. The circumfusion system for multipurpose culture chambers. II. The protracted maintenance of differentiation of foetal and newborn mouse liver in vitro. J. Cell Biol., 39, 430450.
ROSE, G. G., POMERAT,C. M., SHINDLER, T. 0. and TRUNNELL, J. B. 1958. A cellophane-strip technique culturing tissue in multipurpose culture chambers. J. biophys. biochem. Cytol.. 4, 761-763.
for