- Email: [email protected]
Isolation and cultivation of rat liver stellate cells
58
[6]
NORMAL CELLS
series of graded ethanol solutions and embedded in LX 112 (Ladd Research Industry Inc., Burlington, VT). Sections are contrasted with uranyl acetate and lead citrate and examined under an electron microscope. Presently, the only way to unequivocally assess the quality and purity of fat-storing cell preparations, under experimental conditions that alter the cellular composition and phenotype, is by applying morphological criteria using electron microscopy. Markers requiring intact membrane receptors or antigens 16,t7 can be applied only after an isolation method without pronase. Such markers include factor VIII-related antigen, Fc and mannose receptors, and the capacity to take up fluorescent acetylated low-density lipoproteins (LDL) for endothelial cells;~2,t6and phagocytosis of particulate material as well as Fc, C3, mannose, and galactose receptors for Kupffer cells. 12,~7,~s The presence of blebs in cell isolates can be tested biochemically9 and by electron microscopy. Concluding Remarks Isolated liver cells have been widely used to study the cellular distribution of retinoids, retinoid-binding proteins, and enzyme activities important in retinoid metabolism) ,6,t3,14 The results obtained are generally consistent with in vivo data. Cell isolation procedures are currently being used to define further the respective roles of the different liver cells types in retinoid metabolism. ~6A. Brouwer, E. Wisse, and D. L. Knook, in "The Liver. Biology and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Sehachter, and D. A. Shafritz, eds.), 2nd Eel., p. 665. Raven, New York, 1988. ~ E. A. Jones and J. A. Summerfield, in "The Liver. Biology and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Sehachter, and D. A. Shafritz, eds.), 2nd Ed., p. 683. Raven, New York, 1988. ~s H. Pertoft and B. Smedsrod, in "Cell Separation: Methods and Selected Applications," Vol. 4, p. 1. Academic Press, Orlando, 1987.
[6] I s o l a t i o n a n d C u l t i v a t i o n o f R a t L i v e r S t e l l a t e C e l l s B y RUNE BLOMHOFF a n d TROND BERG
Introduction The liver consists of parenchymal cells (hepatocytes) and three main types of nonparenchymal cells: endothelial cells, Kupffer cells, and stellate cells. The parenchymal cells are considerably larger than the nonparenchyMETHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Pr'-~, Inc. All rights of rt'production in any form reserved.
[6]
ISOLATION AND CULTIVATION OF STELLATE CELLS
59
TABLE I COMPOSITION OF RAT LIVERa
Cell type
Number of cells (96 of total)
Mass (% of total)
Parenchymal Endothelial Kupffer Steilate
65 18 10 7
94 2 3 1
a Data are taken from A. C. Munthe-Kaas, T. Berg, and R. Seljelid, Exp. Cell Res. 99, 146 (1976); R. Daoust, in "Liver Functions" (R. W. Brauer, ed.), Publ. 4, p. 3. American Institute of Biological Sciences, Washington, D.C., 1958; D. L. Knook, A. M. Seffelaar, and A. M. de Leeuw, Exp. CellRes. 139, 468 (1982); and D. L. Knook, N. Blansjaar, and E. C. Sleyster, Exp. CellRes. 109, 317 (1977).
mal cells and account for about 94% of the total liver mass (Table I). The nonparenchymal cells represent about 35% of the total number of cells. It has become recognized that the nonparenchymal cells in the liver play an important role in hepatic metabolism of various compounds. The nonparenchymal cells may also cooperate with the parenchymal cells in the handling of nutrients.
Endothelial Cells Endothelial cells form the continuous lining of the liver sinusoids and are a barrier between the blood and the parenchymal cells (Fig. 1). The endothelial cells in liver differ from most endothelial cells in the body with regard to both structure and function. The liver endothelial cells have fenestrations or pores which are about 0.1 a m in diameter. These fenestrations, which are grouped together in so-called sieve plates, influence the filtration of particles from the blood to the parenchymal cells and vice versa.l The pore sizes, and therefore control of filtration, can be influenced by certain hormones.2 Endothelial cells in liver have an important function t E. Wisse and D. L. Knook, in "Progress in Liver Diseases" (H. Popper and F. Schaffner, eds.), Vol. 6, p. 153. Grune & Stratton, New York, 1979. 2 j. H. Van Dierendonek, J. van der Meulen, R. B. De Zanger, and E. Wisse, Ultramicroscopy 4, 149 (1979).
60
NORMAL CELLS
[6]
in the clearance and degradation of various plasma components by receptor-mediated endocytosis.3-6
Kupffer Cells The Kupffer cells are the largest group of tissue macrophages in most mammals. A large part of the cell surface is exposed to the blood flow through the liver and to the space of Disse (Fig. 1). The main function of Kupffer cells is to phagocytose foreign particles, such as bacteria or colloids) They comprise about 25% of the nonparenchymal cells (Table I).
Perisinusoidal Stellate Cells Stellate cells (also called fat-storing cells, lipocytes, and Ito cells) are localized within the space of Disse parallel to the endothelial lining7 (Fig. 1). The stellate cells have a role in the metabolism of vitamin A,8 which is stored in the many fat droplets found in the cytoplasm of these cells. ~ Stellate cells also have a role in the synthesis of connective tissue components and may be involved in the pathological changes observed during the development of liver fibrosis. 7 Stellate cells represent about 7% of the cells in liver (Table I).
Role of Various Liver Cells in Retinoid Metabolism Most of the newly absorbed retinyl esters accompany chylomicron remnants when they are taken up by the liver parenchymal ceils via receptor-mediated endocytosis.9 The retinol taken up by parenchymal cells may subsequently be transferred to perisinusoidal steUate cells in liver for storage, lo Under normal circumstances about 50-80% of the total retinoids in the body are stored in the liver. Mass analysis of retinoids in isolated liver cells has revealed that 80-90% of the total retinol in liver is located in the perisinusoidal stellate cells as retinyl esters, with the rest in liver parenchy3 A. L. Hubbard, G. Wilson, G. Ashwell, and H. Stukenbrok, J. CellBiol. 83, 47 (1979). 4 R. Blomhoff, C.A. Drevon, W. Eskild, P. Helgerud, K. R. Norum, and T. Berg, J.Biol. Chem. 259, 8898 (1984), s j. F. Nagelkerke, K. P. Barto, and T. J. C. van Berkel, J. Biol. Chem. 258, 12221 (1983). 6 B. Smedmxt, H. Pertoft, S. Efiksson, J. R. E. Fraser, and T. C. Laurent, Biochem. £ 223, 617 (1984). 7 K. Wake, Int. Rev. Cytol. 66, 303 (1980). s R. Blomhoff, Nutr. Rev. 45, 257 (1987). 9 R. Blomhoff, P. Helgerud, M. Rasmussen, T. Berg, and K. R. Norum, Proc. NaIL Acad. Sci. U.S.A. 79, 7326 (1982). to R. Blomhoff, K. Holte, L. Naess, and T. Berg, Exp. CellRes. 150, 186 (1984).
[6]
ISOLATION AND CULTIVATION OF STELLATE CELLS
61
\ "N
FIG. 1. Schematic diagram of the structure of the liver. PC, Parenchymal cell; EC, endothelial cell; KC, Kupffer cell; SC, stellate cell.
mal cells. 1~-12 The liver is a main organ for uptake of the retinol-retinolbinding protein (RBP) complex from plasma. 1a'~4Parenchymal as well as " R. Blomhoff, M. Rasmussen, A. N/Isson, K. R. Norum, T. Berg, W, S. Blaner, M. Kato, J. R. Mertz, D. S. Goodman, U. Eriksson, and P. A. Peterson, J. Biol. Chem. 260, 13560 (1985). i: W. S. Blaner, H. F. J, Hendriks, A. Brouwer, A. M. de Leeuw, D. L. Knook, and D. S. Goodman, J. LipidRes. 26, 1241 (1985). ~3R. Blomhoff, K.R. Norum, and T. Berg, J. Biol. Chem, 260, 13571 (1985). ~4T. Gjoen, T. Bjerkelund, H. K. Blomhoff, K. R. Norum, T. Berg, and R. Blomhoff, J. Biol. Chem. 262, 10926 (1987).
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NORMAL CELLS
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stellate cells take up approximately the same amount of retinol-RBP complex when expressed as uptake per gram of liver. We have recently used several approaches to elucidate the mechanism of transfer of retinol between liver parenchymal and stellate cells. Because parenchymal cells secrete retinol bound to RBP, and we had shown that stellate cells may take up retinol-RBP, we tested the possibility that RBP mediates the transfer of retinol from parenchymal to stellate ceils in the liver. In an in situ liver perfusion system we were able to show that antibodies against RBP completely blocked the transfer of retinol to stellate cells. ~5 These findings suggest that retinol secreted from parenchymal cells on RBP is taken up by stellate cells via RBP receptors. Although rat liver parenchymal cells may be easily recovered in high yield, until recently few simple methods had been developed for the preparation of the individual nonparenchymal cell types. The role of stellate cells in the hepatic metabolism of retinol has been determined owing to development of isolation and cultivation techniques for the nonparenchymal liver cells. Enzymatic Perfusion of Liver Most of the newly developed methods include either collagenase perfusion 16or a combined collagenase/pronase perfusion ~7of intact liver. Collagenase perfusion provides a suspension of all types of liver ceils. Nonparenchymal cells can then be prepared after removal of the larger parenchymal cells by low-speed differential centrifugation. Alternatively, protease or enterotoxin treatment of the total cell suspension may be used to selectively destroy the parenchymal cells2 s Based on our experience, coilagenase perfusion is the best method for enzymatic digestion of the liver. First, in contrast to the collagenase/protease perfusion method, it produces a suspension of all the liver cells, including the parenchymal cells, with high yield and viability. The cells can subsequently be separated into the individual cell types. This is especially important when studying the interplay of the various ceils on the hepatic metabolism of components. Second, although protease treatment destroys most cell surface proteins, very little proteolytic degradation of cell surface proteins takes place during the collagenase perfusion period. This is imp5R. Blomhoff, T. Berg, and K. R. Norum, Proc. Natl. Acad. Sci. U.S.A. 85, 3455 (1988). 16p. Seglen, Methods Cell Biol. 13, 29 (1976). 17D. L. Knook, A. M. Seffelaar, and A. M. de Leeuw, Exp. CellRes. 139, 468 (1982). 18R. Blomhoff, B. Smedsrod, W. Eskild, P. E. C_rranum,and T. Berg, Exp. CellRes. 150, 194 (1984).
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ISOLATION AND CULTIVATION OF STELLATE CELLS
63
portant when studying receptor-mediated endocytosis or when using antibodies to characterize cell surface proteins on the isolated cells.
Collagenase Perfusion of Liver Buffers Preperfusion buffer: 8.0 g NaC1, 0.4 g KC1, 60 mg Na2HPO4.2H20, 47 mg KH2PO4, 0.20 g MgSO4" 7I-I20, and 2.05 g NaHCO3 dissolved in water to 1000 ml final volume Perfusion buffer: 25-50 mg collagenase (130-150 units/mg solid) and 100/A of 1.0 M CaC12 made up to 50 ml with the preperfusion buffer Both the preperfusion and perfusion buffers are gassed with 95% 02/5% CO2 to pH 7.5 10 min before and during perfusion. Incubation buffer: 8.5 g NaC1, 0.4 g KC1, 60 mg Na2HPO 4. 2H20, 47 mg KH2PO4, 0.20 g MgSO4"7H20, 4.76 g HEPES, and 0.29 g CaC12" 2H20 dissolved in about 800 ml water and adjusted to pH 7.5 with NaOH; make up to 1000 ml with water (the osmolality of this solution is approximately 298 mOsm) Perfusion Apparatus. A simple perfusion apparatus is shown in Fig. 2. With this apparatus it is possible to perfuse the tissue with solutions preequilibrated at 37*. It also allows switching from the preperfusion buffer to the perfusion buffer without any air bubbles entering the system. Air bubbles cause air blocks and prevent efficient perfusion of the liver. The pump should be capable of a variable flow rate, from 0 to 50 ml/min. Perfusion. Expose the entire abdomen of a deeply anesthetized rat by a transverse cut across the lower abdomen and a longitudinal cut up to the sternum. Gently move the gut contents to the left side of the rat to expose the inferior vena cava and the vena porta. Pass a ligature around the vena porta about 1 cm from its junction with the liver capsule and tie loosely. With a very fine-pointed scissors, cut through the vena porta, about half its circumference, 0.5 cm on the caudal side of the ligature. Insert the cannula into the vena porta to a point 2 - 5 mm past the ligature. Do not enter the liver capsule as this will reduce the yield of cells considerably. During this operation the flow rate of the buffer should be reduced to about 10 ml/min. When the cannula is properly in place, tighten the ligature and tie securely. Immediately sever the inferior vena cava to permit a free flow of the preperfusion buffer and then increase the flow rate to 50 ml/min. This treatment disrupts the desmosomal cell junctions which are calcium-dependent. The rat should now be sacrificed by puncturing the diaphragm.
64
NORMAL CELLS
[6] SYRINGE TO REMOVE AIR BUBBLES
WATER
THE LIVER
WATER JACKET 02 / CO2
PERFUSION
BUFPER
U" LE
PERISTALTIC 'PUMP
WATER PUMP PREPERFUSION WATER BATH
FIo. 2. Recirculating liver perfusion apparatus.
Free the liver by carefully cutting it away from all attachments, taking care not to damage the liver capsule. Continue the preperfusion for l0 min with the liver in situ, the lobes lying in their natural position, thus ensuring a flow of buffer to all parts of the liver. Then switch to the perfusion buffer and allow 30 sec to elapse before transferring the liver to the perfusion dish so that recirculation of the perfusion buffer can take place. Continue the perfusion for l0 min, by which time it should be possible to see signs of the liver disintegrating
[6]
ISOLATION AND CULTIVATION OF STELLATE CELLS
65
within the capsule. Disconnect the cannula and place the liver into a petri dish containing ice-cold incubation buffer. Cut away all extraneous tissue from the liver, taking care not to damage the capsule. Transfer the liver to another dish of incubation medium and disrupt the capsule, using forceps, and free the cells into the medium by fairly vigorous agitation of the forceps in the medium. Transfer the capsule remnants to fresh medium and free any remaining cells in the same way. Discard the capsule remnants and combine the contents of the two dishes. The total volume of buffer into which the cells are isolated should be about 50 ml. Subsequent treatment of the cell suspension will depend on how the cells will be used. Differential Centrifugation of Total Liver Cell Suspension The parenchymal cells are easily separated from the nonparenchymal cells by differential centrifugation.~9 The average volume of the parenchymal cell is more than 10 times that of the average nonparenchymal cell, and centrifugation of a liver cell suspension at low speed (about 50 g) for 20-25 sec at 4 ° gives a fairly good separation of the parenchymal and nonparenchymal cells. The pellet, containing the bulk of the parenchymal cells, can be washed repeatedly to give a relatively pure suspension of parenchymal cells. (To get rid of all contaminating stellate cells, centrifugal elutriation is required. ~4) The supematant is enriched in nonparenchymal cells but still contaminated with some parenchymal cells. Dead parenchymal cells do not sediment during low-speed centrifugation and will therefore be selectively retained in the supernatant. Nevertheless, by repeated centrifugation at 600 g for 4 min at 4 °, a fairly pure preparation of nonparenchymal cells may be obtained from the initial supernatant after sedimentation of the parenchymal cells. Cell viability can be evaluated by the exclusion oftrypan blue (0.04% in incubation buffer): viable cells exclude trypan blue, whereas dead cells accumulate the blue dye. Viability of parenchymal as well as the nonparenchymal cells should always be more than 95%. The nonparenchymal cells collected using the above methods may be separated into their individual cell types by further steps which involve density-gradient centrifugation, centrifugal elutriation, or selective attachment to culture dishes. The method chosen depends on the purpose of the experiment. Very pure stellate cells may be isolated from the nonparenchymal cell suspension by centrifugal elutriation 17 or Percoll density-gradient centrifugation. ~° These methods will, however, lead to a low yield of stellate cells. About 1-3 × l0 s cells per gram liver may be isolated, 19 M. Nilsson and T. Berg, Biochim. Biophys. Acta 497, 171 (1977).
66
NORMAL CELLS
[6]
representing a recovery of about 8-25%. The yield of ceils may increase with the age of the rat. No method is available at present to isolate pure stellate cells in higher yield. As centrifugal elutriation needs a special and expensive centrifugation rotor, and Percoll density-gradient centrifugation is equally efficient, the latter is the method of choice for most purposes. Density-Gradient Centrifugation in Percoll Gradients For density-gradient centrifugation in Percoll gradients 1° (Fig. 3), nonparenchymal cells are first separated from parenchymal cells by differential centrifugation of a total liver cell suspension as described above. Percoll self-forming gradients are generated by mixing 7.5 ml Percoll solution [9 parts Percoll and 1 part 9% (w/v) NaC1] with 5 ml cell suspension and centrifuging of 40,000 g for 60 min at 4 °. In gradient fractions with densities of 1.025-1.035 g/ml almost all cells are stellate cells as judged by vitamin A autofluorescence. The cells contain considerable amounts of vitamin A, making identification by fluorescent microscopy easy. Alternatively, the cells can be identified by transmission electron microscopy (Fig. 4A).
~
collagenase perfusion
total liver cell suspension
Ip
~..
+
600.g for 4rain
50-g for 25 sec.
+
4
nonparenchyrnat cells
centrifugation
nonparen- parenchymal chyrnaI cells cells
Percoll mix with solution a
/st
centrifugation
40,000.g for 60 rnin I~
e[late cells ~ cell debris ~""1 Kupffer cells ~ endothelial cells
FIG. 3. Isolationprocedurefor perisinusoidalstellatecells fromliver.
[~]
ISOLATION AND CULTIVATION OF STELLATE CELLS
67
FIo. 4. (A) Transmission electron micrograph of a freshly isolated stellate cell; (B) light micrograph of stellate cells cultivated for 2 weeks; (C) fluorescence micrograph of a cultivated stellate cell.
68
NORMAL CELLS
FIG. 4.
(continued)
[6]
[6]
ISOLATION AND CULTIVATION OF STELLATE CELLS
69
Cultivation of Stellate Cells When isolating cells for cultivation, all solutions should be sterile-illtered. The perfusion apparatus and tubings should be washed thoroughly in 70% ethanol and then in sterile water and buffer before use. All surgical equipment should be stored in 70% ethanol, and the abdomen of the rat shaved and washed with ethanol before surgery. Primary cultures of stellate cells may be obtained by several methods. The cells isolated by Percoll density-gradient centrifugation or centrifugal elutriation may be washed once or twice in cultivation medium and seeded. As noted earlier, these methods do have the drawback of yielding only a few stellate cells. We have recently used selective detachment during cultivation to obtain a much higher yield of cultivated stellate cells. This procedure is much shorter, and it is easier to obtain sterility. We are now regularly using this method for the cultivation of stellate cells. Nonparenchymal cells isolated by differential centrifugation of a total liver cell suspension should be washed twice in sterile incubation buffer. No extensive washing to remove contaminating parenchymal cells is needed. Cells are then suspended in Dulbecco's modified Eagle's medium (DMEM) containing L-glutamine (0.6 mg/ml), penicillin (100 U/ml), streptomycin (100 U/ml), and 20% fetal calf serum (FCS) and seeded at a density of about 104 cells/cm2. The cells are cultivated at 37 ° in a humidified atmosphere containing 5% CO2 in air. Most of the cells seeded will attach, and the 1-day-old cell culture contains mostly endothelial cells and some Kupffer and stellate cells. The medium is changed after overnight culture and then changed every second day. Under such conditions, endothelial and Kupffer cells do not proliferate, and they detach after 2 - 4 days in culture. The stellate cells, however, start to proliferate, and after 1 week the culture contains stellate cells exclusively, as judged by desmin immunostaining.2° After 1- 2 weeks the cultures are confluent. The cultivated stellate cells show a characteristic stellate shape with long cytoplasmic processes resembling those present in situ (Fig. 4B). Stellate cells can be subcultivated by trypsination through several passages. The cultures are washed in medium without FCS and trypsinated with DMEM containing trypsin (2.5 mg/ml) and EDTA (0.25 mg/ml) and no FCS. Detaching of the cells can be inspected by microscopy. After 5 - 10 min FCS is added to a final concentration of 20%, and the ceils are suspended carefully by pipetting up and down and washed for 10 min at 450 g. After dilution (1:4) in cultivation medium and replating, most cells reattach within 30 min, and the respreading process takes about 6 hr. 2oy. Yokoi, T. Namihisa, H. Kuroda, I. Komatsu, A. Miyazaki, S. Watanabe, and K. Usui, Hepatology (Baltimore) 4, 709 0984).
70
NORMAL CELLS
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Thereafter cells start to proliferate, and a confluent culture is obtained after about 1 week. The characteristic lipid droplets containing vitamin A are present in the primary culture but become smaller with increasing culture time, as judged by transmission electron microscopy. Vitamin A fluorescence (Fig. 4C) can be detected in cells cultured up to the second passage; however, the intensity of fluorescence decreases with time. The decrease in intensity is mainly due to the dilution of vitamin A concentration during cell division and the mobilization of retinol to the medium. When stellate cells are cultivated on uncoated plastic in vitro, they undergo a transition to proliferating myofibroblast-like cells, which is paralleled by a drop in retinyl ester content and a more than 10-fold increase in secretion of collagen (mainly type I). 21 Friedman et al. 22 demonstrated that cultivation of stellate cells on plastic coated with a basement membrane-like matrix inhibits such transformation. These results show that the extracellular matrix can modulate matrix production and dedifferentation of stellate cells in vitro. Identification and Characterization of Stellate Cells Freshly isolated stellate cells may be distinguished from endothelial and Kupffer cells by light microscopy. The lipid droplets of the cells can easily be detected, and the cells resemble a bunch of grapes. 23 Stellate cells may also be identified in a fluorescence microscope with a proper filter block. Retinol and its esters fluoresce after excitation at 330 nm. The lipid droplets of the stellate cells show intense, but rapidly fading vitamin A autofluorescence.7 When preparing cells for fluorescence microscopy, care should be taken to use dim light and not to expose the cells to sunlight as this will destroy the autofluorescence of the cells. The lipid droplets in stellate cells can also be detected by transmission electron microscopy.7 The number and size of the lipid droplets vary considerably. Typically, the nucleus is indented by the lipid droplets. In culture, the characteristic lipid droplets remain present for 1-2 weeks, although the larger ones seemed to be split into droplets of smaller size. The stellate cells produce several types of cytoskeletal and connective tissue proteins, such as vimentin, actin, tubulin, various collagens, fibronectin, and laminin. 23 Most of these are also present in other cell types z~ B. H. Davis, B. M. Pratt, and J. A. Madri, J. Biol. Chem. 262, 10280 (1987). S. L. Friedman, F. J. Roll, J. Boyles, and D. M. Bissell, Proc. Natl. Acad. Sci. U.S.A. 82, 8681 (1985). 23 A. M. de Leeuw, S. P. McCarthy, A. Geerts, and D. L. Knook, Hepatology (Baltimore) 4, 392 (1984).
[7]
SERTOLI CELLS
71
such as fibroblasts and may therefore not be used as a specific marker. However, staining with desmin antibodies represent a specific method for identification of the cells.2° Neither endothelial cells, Kupffer cells, nor fibroblasts are stained with desmin antibodies.
Acknowledgments Our research has been supported in part by grants from the Norwegian Cancer Society and The Norwegian Research Council for Science andthe Humanities.
[7] S e r t o l i C e l l s o f t h e T e s t i s : P r e p a r a t i o n Cultures and Effects of Retinoids
of Cell
B y ALICE F. K A R L a n d M I C H A E L D . GRISWOLD
Introduction Vitamin A in the form of retinol is essential for mammalian spermatogenesis) All cell types in the testis including Leydig cells, germinal cells, pefitubular myoid cells, and the Sertoli cells are potential candidates for the sites of vitamin A action. A major focus of scientific investigation has been on the Sertoli cells because they are the cells which provide physical and biochemical support to the differentiating germinal cells.2 The tight junctions between adjacent Sertoli cells separate the seminiferous tubules into a basal and an adluminal compartment so that the biochemical environment of the adluminal compartment in which the meiotic stages of the germinal cells are sequestered is to a large extent determined by the Sertoli cells. 2 A number of the secreted protein products of Sertoli cells have been isolated and characterized, and both antibodies and cDNA probes are available. One such protein, transferdn, has been utilized by a number of investigators as an index of Sertoli cell function.2 Actions of Retinoids on Cultured Sertoli Cells Sertoli cells contain relatively high levels of cellular retinol-binding protein (CRBP) but very little, if any, cellular retinoic acid-binding protein
t j. N. Thomson, J. McC. Howell, and G. A. Pitt,Proc. R. Soc. London B 159, 510 (1964). 2 M. D. Griswold, C. Morales, and S. R. Sylvester,OxfordRev. Reprod. Biol. I0, 124 (1988).
METHODS IN ENZYMOIJ3GY,VOL. 190
Copytght© !990 by AcademicPre~ Inc. All rightsof reproductionin any formreserved~
[6]
NORMAL CELLS
series of graded ethanol solutions and embedded in LX 112 (Ladd Research Industry Inc., Burlington, VT). Sections are contrasted with uranyl acetate and lead citrate and examined under an electron microscope. Presently, the only way to unequivocally assess the quality and purity of fat-storing cell preparations, under experimental conditions that alter the cellular composition and phenotype, is by applying morphological criteria using electron microscopy. Markers requiring intact membrane receptors or antigens 16,t7 can be applied only after an isolation method without pronase. Such markers include factor VIII-related antigen, Fc and mannose receptors, and the capacity to take up fluorescent acetylated low-density lipoproteins (LDL) for endothelial cells;~2,t6and phagocytosis of particulate material as well as Fc, C3, mannose, and galactose receptors for Kupffer cells. 12,~7,~s The presence of blebs in cell isolates can be tested biochemically9 and by electron microscopy. Concluding Remarks Isolated liver cells have been widely used to study the cellular distribution of retinoids, retinoid-binding proteins, and enzyme activities important in retinoid metabolism) ,6,t3,14 The results obtained are generally consistent with in vivo data. Cell isolation procedures are currently being used to define further the respective roles of the different liver cells types in retinoid metabolism. ~6A. Brouwer, E. Wisse, and D. L. Knook, in "The Liver. Biology and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Sehachter, and D. A. Shafritz, eds.), 2nd Eel., p. 665. Raven, New York, 1988. ~ E. A. Jones and J. A. Summerfield, in "The Liver. Biology and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Sehachter, and D. A. Shafritz, eds.), 2nd Ed., p. 683. Raven, New York, 1988. ~s H. Pertoft and B. Smedsrod, in "Cell Separation: Methods and Selected Applications," Vol. 4, p. 1. Academic Press, Orlando, 1987.
[6] I s o l a t i o n a n d C u l t i v a t i o n o f R a t L i v e r S t e l l a t e C e l l s B y RUNE BLOMHOFF a n d TROND BERG
Introduction The liver consists of parenchymal cells (hepatocytes) and three main types of nonparenchymal cells: endothelial cells, Kupffer cells, and stellate cells. The parenchymal cells are considerably larger than the nonparenchyMETHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Pr'-~, Inc. All rights of rt'production in any form reserved.
[6]
ISOLATION AND CULTIVATION OF STELLATE CELLS
59
TABLE I COMPOSITION OF RAT LIVERa
Cell type
Number of cells (96 of total)
Mass (% of total)
Parenchymal Endothelial Kupffer Steilate
65 18 10 7
94 2 3 1
a Data are taken from A. C. Munthe-Kaas, T. Berg, and R. Seljelid, Exp. Cell Res. 99, 146 (1976); R. Daoust, in "Liver Functions" (R. W. Brauer, ed.), Publ. 4, p. 3. American Institute of Biological Sciences, Washington, D.C., 1958; D. L. Knook, A. M. Seffelaar, and A. M. de Leeuw, Exp. CellRes. 139, 468 (1982); and D. L. Knook, N. Blansjaar, and E. C. Sleyster, Exp. CellRes. 109, 317 (1977).
mal cells and account for about 94% of the total liver mass (Table I). The nonparenchymal cells represent about 35% of the total number of cells. It has become recognized that the nonparenchymal cells in the liver play an important role in hepatic metabolism of various compounds. The nonparenchymal cells may also cooperate with the parenchymal cells in the handling of nutrients.
Endothelial Cells Endothelial cells form the continuous lining of the liver sinusoids and are a barrier between the blood and the parenchymal cells (Fig. 1). The endothelial cells in liver differ from most endothelial cells in the body with regard to both structure and function. The liver endothelial cells have fenestrations or pores which are about 0.1 a m in diameter. These fenestrations, which are grouped together in so-called sieve plates, influence the filtration of particles from the blood to the parenchymal cells and vice versa.l The pore sizes, and therefore control of filtration, can be influenced by certain hormones.2 Endothelial cells in liver have an important function t E. Wisse and D. L. Knook, in "Progress in Liver Diseases" (H. Popper and F. Schaffner, eds.), Vol. 6, p. 153. Grune & Stratton, New York, 1979. 2 j. H. Van Dierendonek, J. van der Meulen, R. B. De Zanger, and E. Wisse, Ultramicroscopy 4, 149 (1979).
60
NORMAL CELLS
[6]
in the clearance and degradation of various plasma components by receptor-mediated endocytosis.3-6
Kupffer Cells The Kupffer cells are the largest group of tissue macrophages in most mammals. A large part of the cell surface is exposed to the blood flow through the liver and to the space of Disse (Fig. 1). The main function of Kupffer cells is to phagocytose foreign particles, such as bacteria or colloids) They comprise about 25% of the nonparenchymal cells (Table I).
Perisinusoidal Stellate Cells Stellate cells (also called fat-storing cells, lipocytes, and Ito cells) are localized within the space of Disse parallel to the endothelial lining7 (Fig. 1). The stellate cells have a role in the metabolism of vitamin A,8 which is stored in the many fat droplets found in the cytoplasm of these cells. ~ Stellate cells also have a role in the synthesis of connective tissue components and may be involved in the pathological changes observed during the development of liver fibrosis. 7 Stellate cells represent about 7% of the cells in liver (Table I).
Role of Various Liver Cells in Retinoid Metabolism Most of the newly absorbed retinyl esters accompany chylomicron remnants when they are taken up by the liver parenchymal ceils via receptor-mediated endocytosis.9 The retinol taken up by parenchymal cells may subsequently be transferred to perisinusoidal steUate cells in liver for storage, lo Under normal circumstances about 50-80% of the total retinoids in the body are stored in the liver. Mass analysis of retinoids in isolated liver cells has revealed that 80-90% of the total retinol in liver is located in the perisinusoidal stellate cells as retinyl esters, with the rest in liver parenchy3 A. L. Hubbard, G. Wilson, G. Ashwell, and H. Stukenbrok, J. CellBiol. 83, 47 (1979). 4 R. Blomhoff, C.A. Drevon, W. Eskild, P. Helgerud, K. R. Norum, and T. Berg, J.Biol. Chem. 259, 8898 (1984), s j. F. Nagelkerke, K. P. Barto, and T. J. C. van Berkel, J. Biol. Chem. 258, 12221 (1983). 6 B. Smedmxt, H. Pertoft, S. Efiksson, J. R. E. Fraser, and T. C. Laurent, Biochem. £ 223, 617 (1984). 7 K. Wake, Int. Rev. Cytol. 66, 303 (1980). s R. Blomhoff, Nutr. Rev. 45, 257 (1987). 9 R. Blomhoff, P. Helgerud, M. Rasmussen, T. Berg, and K. R. Norum, Proc. NaIL Acad. Sci. U.S.A. 79, 7326 (1982). to R. Blomhoff, K. Holte, L. Naess, and T. Berg, Exp. CellRes. 150, 186 (1984).
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\ "N
FIG. 1. Schematic diagram of the structure of the liver. PC, Parenchymal cell; EC, endothelial cell; KC, Kupffer cell; SC, stellate cell.
mal cells. 1~-12 The liver is a main organ for uptake of the retinol-retinolbinding protein (RBP) complex from plasma. 1a'~4Parenchymal as well as " R. Blomhoff, M. Rasmussen, A. N/Isson, K. R. Norum, T. Berg, W, S. Blaner, M. Kato, J. R. Mertz, D. S. Goodman, U. Eriksson, and P. A. Peterson, J. Biol. Chem. 260, 13560 (1985). i: W. S. Blaner, H. F. J, Hendriks, A. Brouwer, A. M. de Leeuw, D. L. Knook, and D. S. Goodman, J. LipidRes. 26, 1241 (1985). ~3R. Blomhoff, K.R. Norum, and T. Berg, J. Biol. Chem, 260, 13571 (1985). ~4T. Gjoen, T. Bjerkelund, H. K. Blomhoff, K. R. Norum, T. Berg, and R. Blomhoff, J. Biol. Chem. 262, 10926 (1987).
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[6]
stellate cells take up approximately the same amount of retinol-RBP complex when expressed as uptake per gram of liver. We have recently used several approaches to elucidate the mechanism of transfer of retinol between liver parenchymal and stellate cells. Because parenchymal cells secrete retinol bound to RBP, and we had shown that stellate cells may take up retinol-RBP, we tested the possibility that RBP mediates the transfer of retinol from parenchymal to stellate ceils in the liver. In an in situ liver perfusion system we were able to show that antibodies against RBP completely blocked the transfer of retinol to stellate cells. ~5 These findings suggest that retinol secreted from parenchymal cells on RBP is taken up by stellate cells via RBP receptors. Although rat liver parenchymal cells may be easily recovered in high yield, until recently few simple methods had been developed for the preparation of the individual nonparenchymal cell types. The role of stellate cells in the hepatic metabolism of retinol has been determined owing to development of isolation and cultivation techniques for the nonparenchymal liver cells. Enzymatic Perfusion of Liver Most of the newly developed methods include either collagenase perfusion 16or a combined collagenase/pronase perfusion ~7of intact liver. Collagenase perfusion provides a suspension of all types of liver ceils. Nonparenchymal cells can then be prepared after removal of the larger parenchymal cells by low-speed differential centrifugation. Alternatively, protease or enterotoxin treatment of the total cell suspension may be used to selectively destroy the parenchymal cells2 s Based on our experience, coilagenase perfusion is the best method for enzymatic digestion of the liver. First, in contrast to the collagenase/protease perfusion method, it produces a suspension of all the liver cells, including the parenchymal cells, with high yield and viability. The cells can subsequently be separated into the individual cell types. This is especially important when studying the interplay of the various ceils on the hepatic metabolism of components. Second, although protease treatment destroys most cell surface proteins, very little proteolytic degradation of cell surface proteins takes place during the collagenase perfusion period. This is imp5R. Blomhoff, T. Berg, and K. R. Norum, Proc. Natl. Acad. Sci. U.S.A. 85, 3455 (1988). 16p. Seglen, Methods Cell Biol. 13, 29 (1976). 17D. L. Knook, A. M. Seffelaar, and A. M. de Leeuw, Exp. CellRes. 139, 468 (1982). 18R. Blomhoff, B. Smedsrod, W. Eskild, P. E. C_rranum,and T. Berg, Exp. CellRes. 150, 194 (1984).
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ISOLATION AND CULTIVATION OF STELLATE CELLS
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portant when studying receptor-mediated endocytosis or when using antibodies to characterize cell surface proteins on the isolated cells.
Collagenase Perfusion of Liver Buffers Preperfusion buffer: 8.0 g NaC1, 0.4 g KC1, 60 mg Na2HPO4.2H20, 47 mg KH2PO4, 0.20 g MgSO4" 7I-I20, and 2.05 g NaHCO3 dissolved in water to 1000 ml final volume Perfusion buffer: 25-50 mg collagenase (130-150 units/mg solid) and 100/A of 1.0 M CaC12 made up to 50 ml with the preperfusion buffer Both the preperfusion and perfusion buffers are gassed with 95% 02/5% CO2 to pH 7.5 10 min before and during perfusion. Incubation buffer: 8.5 g NaC1, 0.4 g KC1, 60 mg Na2HPO 4. 2H20, 47 mg KH2PO4, 0.20 g MgSO4"7H20, 4.76 g HEPES, and 0.29 g CaC12" 2H20 dissolved in about 800 ml water and adjusted to pH 7.5 with NaOH; make up to 1000 ml with water (the osmolality of this solution is approximately 298 mOsm) Perfusion Apparatus. A simple perfusion apparatus is shown in Fig. 2. With this apparatus it is possible to perfuse the tissue with solutions preequilibrated at 37*. It also allows switching from the preperfusion buffer to the perfusion buffer without any air bubbles entering the system. Air bubbles cause air blocks and prevent efficient perfusion of the liver. The pump should be capable of a variable flow rate, from 0 to 50 ml/min. Perfusion. Expose the entire abdomen of a deeply anesthetized rat by a transverse cut across the lower abdomen and a longitudinal cut up to the sternum. Gently move the gut contents to the left side of the rat to expose the inferior vena cava and the vena porta. Pass a ligature around the vena porta about 1 cm from its junction with the liver capsule and tie loosely. With a very fine-pointed scissors, cut through the vena porta, about half its circumference, 0.5 cm on the caudal side of the ligature. Insert the cannula into the vena porta to a point 2 - 5 mm past the ligature. Do not enter the liver capsule as this will reduce the yield of cells considerably. During this operation the flow rate of the buffer should be reduced to about 10 ml/min. When the cannula is properly in place, tighten the ligature and tie securely. Immediately sever the inferior vena cava to permit a free flow of the preperfusion buffer and then increase the flow rate to 50 ml/min. This treatment disrupts the desmosomal cell junctions which are calcium-dependent. The rat should now be sacrificed by puncturing the diaphragm.
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[6] SYRINGE TO REMOVE AIR BUBBLES
WATER
THE LIVER
WATER JACKET 02 / CO2
PERFUSION
BUFPER
U" LE
PERISTALTIC 'PUMP
WATER PUMP PREPERFUSION WATER BATH
FIo. 2. Recirculating liver perfusion apparatus.
Free the liver by carefully cutting it away from all attachments, taking care not to damage the liver capsule. Continue the preperfusion for l0 min with the liver in situ, the lobes lying in their natural position, thus ensuring a flow of buffer to all parts of the liver. Then switch to the perfusion buffer and allow 30 sec to elapse before transferring the liver to the perfusion dish so that recirculation of the perfusion buffer can take place. Continue the perfusion for l0 min, by which time it should be possible to see signs of the liver disintegrating
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ISOLATION AND CULTIVATION OF STELLATE CELLS
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within the capsule. Disconnect the cannula and place the liver into a petri dish containing ice-cold incubation buffer. Cut away all extraneous tissue from the liver, taking care not to damage the capsule. Transfer the liver to another dish of incubation medium and disrupt the capsule, using forceps, and free the cells into the medium by fairly vigorous agitation of the forceps in the medium. Transfer the capsule remnants to fresh medium and free any remaining cells in the same way. Discard the capsule remnants and combine the contents of the two dishes. The total volume of buffer into which the cells are isolated should be about 50 ml. Subsequent treatment of the cell suspension will depend on how the cells will be used. Differential Centrifugation of Total Liver Cell Suspension The parenchymal cells are easily separated from the nonparenchymal cells by differential centrifugation.~9 The average volume of the parenchymal cell is more than 10 times that of the average nonparenchymal cell, and centrifugation of a liver cell suspension at low speed (about 50 g) for 20-25 sec at 4 ° gives a fairly good separation of the parenchymal and nonparenchymal cells. The pellet, containing the bulk of the parenchymal cells, can be washed repeatedly to give a relatively pure suspension of parenchymal cells. (To get rid of all contaminating stellate cells, centrifugal elutriation is required. ~4) The supematant is enriched in nonparenchymal cells but still contaminated with some parenchymal cells. Dead parenchymal cells do not sediment during low-speed centrifugation and will therefore be selectively retained in the supernatant. Nevertheless, by repeated centrifugation at 600 g for 4 min at 4 °, a fairly pure preparation of nonparenchymal cells may be obtained from the initial supernatant after sedimentation of the parenchymal cells. Cell viability can be evaluated by the exclusion oftrypan blue (0.04% in incubation buffer): viable cells exclude trypan blue, whereas dead cells accumulate the blue dye. Viability of parenchymal as well as the nonparenchymal cells should always be more than 95%. The nonparenchymal cells collected using the above methods may be separated into their individual cell types by further steps which involve density-gradient centrifugation, centrifugal elutriation, or selective attachment to culture dishes. The method chosen depends on the purpose of the experiment. Very pure stellate cells may be isolated from the nonparenchymal cell suspension by centrifugal elutriation 17 or Percoll density-gradient centrifugation. ~° These methods will, however, lead to a low yield of stellate cells. About 1-3 × l0 s cells per gram liver may be isolated, 19 M. Nilsson and T. Berg, Biochim. Biophys. Acta 497, 171 (1977).
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[6]
representing a recovery of about 8-25%. The yield of ceils may increase with the age of the rat. No method is available at present to isolate pure stellate cells in higher yield. As centrifugal elutriation needs a special and expensive centrifugation rotor, and Percoll density-gradient centrifugation is equally efficient, the latter is the method of choice for most purposes. Density-Gradient Centrifugation in Percoll Gradients For density-gradient centrifugation in Percoll gradients 1° (Fig. 3), nonparenchymal cells are first separated from parenchymal cells by differential centrifugation of a total liver cell suspension as described above. Percoll self-forming gradients are generated by mixing 7.5 ml Percoll solution [9 parts Percoll and 1 part 9% (w/v) NaC1] with 5 ml cell suspension and centrifuging of 40,000 g for 60 min at 4 °. In gradient fractions with densities of 1.025-1.035 g/ml almost all cells are stellate cells as judged by vitamin A autofluorescence. The cells contain considerable amounts of vitamin A, making identification by fluorescent microscopy easy. Alternatively, the cells can be identified by transmission electron microscopy (Fig. 4A).
~
collagenase perfusion
total liver cell suspension
Ip
~..
+
600.g for 4rain
50-g for 25 sec.
+
4
nonparenchyrnat cells
centrifugation
nonparen- parenchymal chyrnaI cells cells
Percoll mix with solution a
/st
centrifugation
40,000.g for 60 rnin I~
e[late cells ~ cell debris ~""1 Kupffer cells ~ endothelial cells
FIG. 3. Isolationprocedurefor perisinusoidalstellatecells fromliver.
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ISOLATION AND CULTIVATION OF STELLATE CELLS
67
FIo. 4. (A) Transmission electron micrograph of a freshly isolated stellate cell; (B) light micrograph of stellate cells cultivated for 2 weeks; (C) fluorescence micrograph of a cultivated stellate cell.
68
NORMAL CELLS
FIG. 4.
(continued)
[6]
[6]
ISOLATION AND CULTIVATION OF STELLATE CELLS
69
Cultivation of Stellate Cells When isolating cells for cultivation, all solutions should be sterile-illtered. The perfusion apparatus and tubings should be washed thoroughly in 70% ethanol and then in sterile water and buffer before use. All surgical equipment should be stored in 70% ethanol, and the abdomen of the rat shaved and washed with ethanol before surgery. Primary cultures of stellate cells may be obtained by several methods. The cells isolated by Percoll density-gradient centrifugation or centrifugal elutriation may be washed once or twice in cultivation medium and seeded. As noted earlier, these methods do have the drawback of yielding only a few stellate cells. We have recently used selective detachment during cultivation to obtain a much higher yield of cultivated stellate cells. This procedure is much shorter, and it is easier to obtain sterility. We are now regularly using this method for the cultivation of stellate cells. Nonparenchymal cells isolated by differential centrifugation of a total liver cell suspension should be washed twice in sterile incubation buffer. No extensive washing to remove contaminating parenchymal cells is needed. Cells are then suspended in Dulbecco's modified Eagle's medium (DMEM) containing L-glutamine (0.6 mg/ml), penicillin (100 U/ml), streptomycin (100 U/ml), and 20% fetal calf serum (FCS) and seeded at a density of about 104 cells/cm2. The cells are cultivated at 37 ° in a humidified atmosphere containing 5% CO2 in air. Most of the cells seeded will attach, and the 1-day-old cell culture contains mostly endothelial cells and some Kupffer and stellate cells. The medium is changed after overnight culture and then changed every second day. Under such conditions, endothelial and Kupffer cells do not proliferate, and they detach after 2 - 4 days in culture. The stellate cells, however, start to proliferate, and after 1 week the culture contains stellate cells exclusively, as judged by desmin immunostaining.2° After 1- 2 weeks the cultures are confluent. The cultivated stellate cells show a characteristic stellate shape with long cytoplasmic processes resembling those present in situ (Fig. 4B). Stellate cells can be subcultivated by trypsination through several passages. The cultures are washed in medium without FCS and trypsinated with DMEM containing trypsin (2.5 mg/ml) and EDTA (0.25 mg/ml) and no FCS. Detaching of the cells can be inspected by microscopy. After 5 - 10 min FCS is added to a final concentration of 20%, and the ceils are suspended carefully by pipetting up and down and washed for 10 min at 450 g. After dilution (1:4) in cultivation medium and replating, most cells reattach within 30 min, and the respreading process takes about 6 hr. 2oy. Yokoi, T. Namihisa, H. Kuroda, I. Komatsu, A. Miyazaki, S. Watanabe, and K. Usui, Hepatology (Baltimore) 4, 709 0984).
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Thereafter cells start to proliferate, and a confluent culture is obtained after about 1 week. The characteristic lipid droplets containing vitamin A are present in the primary culture but become smaller with increasing culture time, as judged by transmission electron microscopy. Vitamin A fluorescence (Fig. 4C) can be detected in cells cultured up to the second passage; however, the intensity of fluorescence decreases with time. The decrease in intensity is mainly due to the dilution of vitamin A concentration during cell division and the mobilization of retinol to the medium. When stellate cells are cultivated on uncoated plastic in vitro, they undergo a transition to proliferating myofibroblast-like cells, which is paralleled by a drop in retinyl ester content and a more than 10-fold increase in secretion of collagen (mainly type I). 21 Friedman et al. 22 demonstrated that cultivation of stellate cells on plastic coated with a basement membrane-like matrix inhibits such transformation. These results show that the extracellular matrix can modulate matrix production and dedifferentation of stellate cells in vitro. Identification and Characterization of Stellate Cells Freshly isolated stellate cells may be distinguished from endothelial and Kupffer cells by light microscopy. The lipid droplets of the cells can easily be detected, and the cells resemble a bunch of grapes. 23 Stellate cells may also be identified in a fluorescence microscope with a proper filter block. Retinol and its esters fluoresce after excitation at 330 nm. The lipid droplets of the stellate cells show intense, but rapidly fading vitamin A autofluorescence.7 When preparing cells for fluorescence microscopy, care should be taken to use dim light and not to expose the cells to sunlight as this will destroy the autofluorescence of the cells. The lipid droplets in stellate cells can also be detected by transmission electron microscopy.7 The number and size of the lipid droplets vary considerably. Typically, the nucleus is indented by the lipid droplets. In culture, the characteristic lipid droplets remain present for 1-2 weeks, although the larger ones seemed to be split into droplets of smaller size. The stellate cells produce several types of cytoskeletal and connective tissue proteins, such as vimentin, actin, tubulin, various collagens, fibronectin, and laminin. 23 Most of these are also present in other cell types z~ B. H. Davis, B. M. Pratt, and J. A. Madri, J. Biol. Chem. 262, 10280 (1987). S. L. Friedman, F. J. Roll, J. Boyles, and D. M. Bissell, Proc. Natl. Acad. Sci. U.S.A. 82, 8681 (1985). 23 A. M. de Leeuw, S. P. McCarthy, A. Geerts, and D. L. Knook, Hepatology (Baltimore) 4, 392 (1984).
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such as fibroblasts and may therefore not be used as a specific marker. However, staining with desmin antibodies represent a specific method for identification of the cells.2° Neither endothelial cells, Kupffer cells, nor fibroblasts are stained with desmin antibodies.
Acknowledgments Our research has been supported in part by grants from the Norwegian Cancer Society and The Norwegian Research Council for Science andthe Humanities.
[7] S e r t o l i C e l l s o f t h e T e s t i s : P r e p a r a t i o n Cultures and Effects of Retinoids
of Cell
B y ALICE F. K A R L a n d M I C H A E L D . GRISWOLD
Introduction Vitamin A in the form of retinol is essential for mammalian spermatogenesis) All cell types in the testis including Leydig cells, germinal cells, pefitubular myoid cells, and the Sertoli cells are potential candidates for the sites of vitamin A action. A major focus of scientific investigation has been on the Sertoli cells because they are the cells which provide physical and biochemical support to the differentiating germinal cells.2 The tight junctions between adjacent Sertoli cells separate the seminiferous tubules into a basal and an adluminal compartment so that the biochemical environment of the adluminal compartment in which the meiotic stages of the germinal cells are sequestered is to a large extent determined by the Sertoli cells. 2 A number of the secreted protein products of Sertoli cells have been isolated and characterized, and both antibodies and cDNA probes are available. One such protein, transferdn, has been utilized by a number of investigators as an index of Sertoli cell function.2 Actions of Retinoids on Cultured Sertoli Cells Sertoli cells contain relatively high levels of cellular retinol-binding protein (CRBP) but very little, if any, cellular retinoic acid-binding protein
t j. N. Thomson, J. McC. Howell, and G. A. Pitt,Proc. R. Soc. London B 159, 510 (1964). 2 M. D. Griswold, C. Morales, and S. R. Sylvester,OxfordRev. Reprod. Biol. I0, 124 (1988).
METHODS IN ENZYMOIJ3GY,VOL. 190
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