IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY Volume 25, Number 10, October 1989 9 1989 Tissue Culture Association, Inc.

CHARACTERIZATION OF PROLIFERATION AND DIFFERENTIATION OF O P O S S U M K I D N E Y C E L L S IN A S E R U M - F R E E D E F I N E D M E D I U M LISA J. LEIDERMAN', J. ALLAN TUCKER, ANDVINCENT W. DENNIS Department of Medicine, Division of Nephrology (L. J. L., V. IV. D.), P. O. Box 3014, and Department of Pathology (J. A. T.), P. 0. Box 3712, Duke University Medical Center, Durham, North Carolina 27710

(Received 23 January 1989; accepted 15 May 1989)

SUMMARY

Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10/~g/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Ceils in serum-free medium seeded at 1 )< I(P per cm 2 grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium. K e y words: opossum kidney cell; serum-free; proliferation; differentiation; defined medium.

model system to study mechanisms of proximal tubular function, specifically in relation to phosphate transport. In this report, we describe the characterization of the proliferation and differentiation of OK cells in a

INTRODUCTION

Serum-free defined culture media have been developed for a variety of renal epithelial cells {Chuman et al., 1982: Chung et al. 1982; Jefferson et al., 1980; Taub et al., 1979; Tauh and Sato, 1980). These media are highly advantageous because they provide a controlled environment in which mechanisms of renal solute transport can be investigated. A number of transport systems have been demonstrated in cultured renal cells (Caversasio et al., 1986; Malstr6m and Murer, 1986; Rabito, 1983; Sepfilveda and Pearson, 1982). Sodium-dependent phosphate transport, which occurs across the apical surface of epithelial cells, has been demonstrated in the LLC-PKI (Malstr6m and Murer, 1986; Rahito, 1983), JTC-12.P~ (Malstr~m and Murer, 1986), and the opossum kidney cell lines (Caversasio et al., 1986; Leiderman et al., 1989; Malstr~m and Muter, 1986). The opossum kidney (OK) cell line (Koyama et al., 1978), which is of proximal tubular origin, is of particular interest because the transport of phosphate can be inhibited by parathyroid hormone (PTH) (Caversasio et al., 1986; Leiderman et al., 1989; Malstr~m and Murer, 1986). In the renal proximal tubule, P T H is the major endocrine regulator of phosphate transport (Dennis, 1988). The OK cell line therefore provides an appropriate

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20% I t n t 0 0.005 0.05 0.5 5 Insulin (/~g/ml)

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FIG. 1. Dose-response curves to each culture component. M e d i u m contained: A a nd B, insulin at varied concentrations

with BSA (1 mg/ml) and fetuin (1 mg/ml); C, BSA at varied concentrations with insulin (10 /~g/ml) and fetuin (1 mg/ml); D, fetuin at varied concentrations with insulin (10 #g/ml) and BSA (1 mg/ml). Each value is the mean --4- SEM of triplicate determinations.

t To whom correspondence should be addressed at Amgen Inc., Amgen Center, Thousand Oaks, CA 91320. 881

882

LEIDERMAN ET AL.

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FIG. 2. Addition of culture components. Each factor was added separately at its optimal concentration: fetuin (1 mg/ml), insulin (10 /~g/ml), and BSA il mg/ml). All factors combined produced maximal growth rate. Each value is the mean -tSEM of triplicate determinations.

serum-free medium. This medium will provide a means to study renal proximal tubular function under defined conditions. MATERIALS AND METHODS

Cell culture. OK cells were received from Dr. Paula Barrett, Department of Internal Medicine, Yale University Medical Center. Stock cultures (Passages 90 to 100) were maintained in flasks containing Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture (50:50%)

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FIG. 3. Growth curves of OK cells. Cells were grown in serum-free medium (--) (insulin, 10 /~g/ml; BSA, 1 mg/ml; fetuin, 1 mg/ml) and serum-containing medium t--) (10% FBS) for a period of 8 d. Each value is the mean -+- SEM of triplicate determinations. (DMEM-F12, GIBCO, Grand Island, NY), 2 m M glutamine, and 10% fetal bovine serum (FBS) (GIBCO). Serum-free culture medium consisted of DMEM-F12 supplemented with insulin (10 gg/ml), bovine serum albumin (BSA) fraction V (1 mg/ml), bovine Pedersen fetuin (1 mg/ml), and 2 m M glutamine. All supplements other than glutamine (GIBCO) were purchased from Sigma Chemicals, St. I_~uis, MO. No antibiotics were used. Random cultures tested negatively from mycoplasma (Bionique Laboratories, Saranac Lake, NY). Cells taken from stock cultures were seeded on plastic

Fit}. 4. Phase contrast photomicrographs of OK cells. Cultures were grown in (.4) serum-free medium (insulin, 10 ~g/ml; BSA, 1 mg/ml; fetuin, 1 mg/ml) and (B) serum-containing medium. Domes were prevalent in both cultures X 200.

883

GROWTH AND DIFFERENTIATION OF OPOSSUM KIDNEY CELLS

TABLE 1 PROLIFERATION OF OPOSSUM KIDNEY CELLS~

Passage

Days in Culture

Serum-Free Medium, Cell Number )< 104/cm z

Serum-Containing Medium. Cell Number X 104/cm 2

1 2 3

7-8 14-16 22-24

36.68 -4- 2.4 36.79 -4- 3.7 33.04 -4- 1.5

35.36 -4- 1.9 38.46 -4- 3.1 34.10 -4- h l

~ medium consisted of DMEM-F12 with insulin (10 tag/ml), BSA ql mg/mlL and fetuin 11 mg/mlL Serum-containing medium consisted of DMEM-F12 with 10% FBS. Values represent the means -4- SEM of two experiments each set up with triplicate cultures.

cultureware (Costar Corporation, Cambridge, MA) at a density of 1.0 to 1.5 )< 10~/cm~ in either maintenance medium or serum-free medium. Cultures were incubated at 37 ~ C in humidified atmosphere containing 5% CO~: 95% air. Assays were performed on confluent monolayers which were obtained after an incubation period of 6 to 8 d. Cells were detached from the cultureware with 0.05% trypsin-0.53 M E D T A (ethylenediamine tetraacetic acid) (GIBCO). Trypsin treatment was terminated with soybean trypsin inhibitor (Sigma). Cells were counted with a hemacytometer. Viability as determined by trypan blue was greater than 95%. Phosphate transport. Confluent monolayers in 12-well plastic plates (Costar) were preincubated for 1 h at 37~ C in DMEM-F12 medium containing bovine parathyroid hormone fragment (bPTH1-34) (Sigma) at 10-6 M or the vehicle (dH20). Cultures were subsequently washed with Earle's buffered salt solution (pH 7.4) containing 15 m M HEPES, 143 mmol/liter sodium, 5.36 mmol/liter potassium, 0.8 mmol/liter magnesium, 1.8 mmol/liter calcium, 125 mmol/liter chloride and 0.2 m M phosphate. Transport was initiated by adding to the monolayers 1 ml of the above solution containing NaH232PO4 (0.5 to 1.0 /aCi/ml) tNew England Nuclear, Boston, MA). After an incubation of 3 min at 37 ~ C, the reaction was terminated by washing with ice-cold Earle's solution. Monolayers were solubilized in 0.1 N NaOH

and aliquots of 0.5 ml were counted in a liquid scintillation counter. Protein determinations were assessed with a kit (Sigma ~employing the method of Peterson ~1977). Cyclic AMP assay. Confluent monolayers grown in 24-well plastic plates (Costar) were preineubated for 1 h at 37~ C in DMEM-F12 medium containing 1 m M isobutylmethylxanthine IIBMX) with either bovine parathyroid hormone fragment (b PTH1-34) lSigma) at 10-' M or the vehicle ldH20). Cultures were subsequently washed with 50 m M tris [Tris(hydroxymethyl)aminomethane hydrochloride] buffer ~pH 7.5~ containing 4 m M EDTA and resuspended in 1.25 ml of 6% trichloracetic acid. Monolayers were then scraped from the wells and fragmented via repeated pipetting. After centrifugation, the supernatant was passed through a 0.6 )< 5.0-cm column of Dowex 50w -- x8 I H + L 200 to 400 mesh, and assayed for cyclic AMP lcAMP) using a [12sIodine] radioimmunoassay kit ~Dupont, Wilmington, DE). Electron microscopy. Confluent monolayers were fixed in 4% glutaraldehyde and processed as previously described (Leiderman et al., 1989). Perpendicularly cut sections were examined with a Philips E M 410 electron microscope.

R ESULTS The growth of OK cells as a function of the concentration of culture components is shown in Fig. 1. No change occurred in cell number with respect to insulin concentration IA). There was, however, a marked increase in total cellular protein when insulin concentrations were 0.5 tag/ml or greater (BL The maximal protein level was equivalent to that in cells grown in serumcontaining medium. Figure 2 shows each component added separately at its optimal concentration. Maximal growth rate was achieved with the total combination of factors. The OK cells grown in serum-free medium for 8 d had a doubling time of 17 h whereas cells grown in medium containing serum had a doubling time of 14.5 h ~Fig. 3). Domes or hemicysts, which appeared at about 4 to 5 d,

TABLE 2 PHOSPHATE UPTAKE IN OPOSSUM KIDNEY CELLSa Serum-Free Medium Culture Periled. days

7-8 7-8 7-8 22-24 22-24 22-24

Serum-Containing Medium

Control

PTH, t0-6 M

Control

PTH, 10-6 M

4.06 + 0.03 4.87 + 0.05 7.16 -4- 0.20 3.32 -4- 0.02 3.80 -4- 0.19 3.84 _ 0.08

3.14 _ 0.12 3.23 + 0.08 4.72 -4- 0.43 2.67 -4- 0.09 3.42 -4- 0.07 2.89 -4- 0.08

4.38 -4- 0.20 5.05 -4- 0.07 8.47 + 0.08 3.56 -4- 0.09 3.41 -4- 0.11 3.42 _ 0.10

3.55 -4- 0.06 3.81 + 0.13 6.09 -4- 0.33 2.90 -4- 0.05 3.19 -4- 0.23 3.11 ___0.08

~ medium consisted of DMEM-F12 with insulin i10/ag/ml), BSA ~1 mg/ml), and fetuin ~1 mg/mlL Serumcontaining medium consisted of DMEM-F12 with 10% FBS. Values denote the means + SEM of triplicate cultures. Units are nmol/mg protein. Inhibitionby PTH significant at P < 0.05, paired-sample t test.

884

LEIDERMAN ET AL.

TABLE 3 cAMP PRODUCTION IN OPOSSUM KIDNEY CELLS~ Serum-FreeMedium Culture Period, days

7-8 7-8 7-8 22-24 22-24 22-24

Serum-ContainingMedium

Control

PTH. 10-6 M

14.16 _ 1.3 11.93 4- 0.6 19.10 4- 1.1 9.1 4- 1.4 6.7 4- 2.4 11.8 4- 1.6

316.9 4- 79 320.7 4- 32 269.8 4- 36 141.2 4- 12 107.4 4- 12 29.2 4- 2.5

Control

16.3 4- 1.9 16.7 _4- 1.2 25.4 + 4.5 13.3 -4- 2.2 12.0 + 1.6 9.8 4- 1.9

PTH, 10-s M

338.6 4- 31 214.4 4- 9.1 257.0 4- 52 97.8 4- 9.8 108.0 4- 28 78.8 4- 5.3

aSerum-free medium consisted of DMEM-F12 with insulin ~10/~g/ml), BSA il mg/ml), and fetuin ~1 mg/ml). Serumcontaining medium consisted of DMEM-F12 with 10% FBS. Values denote the means +_ SEM of triplicate cultures. Units are pmol/mg protein.

were numerous both in confluent monolayers grown in serum-free as well as serum-containingmedium (Fig. 4). The OK cells were grown both in serum-free medium and serum-containing medium for a period of 22 to 24 d iTable 1). Cells were seeded on Day 0 at a density of 1 X l(Y'/crhL After a 7- to 8-day period, confluent cultures were trypsinized and cells were counted. These cells were then seeded again at the same density and enumerated following a similar culture period. This procedure was repeated once again resulting in a total growth period of 24 d. At all time points the number of cells that were present in serum-free medium was equivalent to that in serum-containing medium. The differentiated characteristics of OK cells include sodium-dependent phosphate transport and the inhibition of phosphate transport by P T H (Caversasio et al., 1986; Leiderman et al., 1989; MalstriJm and Murer, 1986). In addition, OK cells produce cAMP in response to P T H (Caversasio et al., 1986L Tables 2 and 3 demonstrate PTH-inhibitable, phosphate uptake, and cAMP production in monolayers grown in serum-free medium for 8 d as well as after a 24-d period. The values at both time points are comparable to those obtained from monolayers grown in serum-containingmedium. Electron micrographs of confluent monolayers grown in serum-free medium as well as serum-containing medium over a period of 24 d are shown in Figs. 5 and 6. Cells grown both in serum-free ~5 A and 6 A~ as well as serum-containing medium 15 B and 6 B~ possessed tight junctions as well as scattered microvilli along their apical surfaces.

cAMP in response to P T H were preserved in serum-free medium for the same length of time. In addition, electron microscopy revealed normal epithelial cell morphology as assessed by polarized monolayers, with the presence of microvilli and tight junctions.

D ISCUSSION We have developed a serum-free defined medium which supports the proliferation and differentiation of OK cells. The basic medium, DMEM-F12 combined with the mitogenic hormone insulin, BSA, a binding or transport protein, and fetuin, an attachment and spreading factor, was shown to support the growth of OK cells over a 24-d period. The differentiated characteristics of OK cells, including P T H - i n h i b i t a b l e , sodiumdependent phosphate transport, and the generation of

FI6. 5. Electron photomicrographs of OK cells. Confluent monolayers were grown in IA) serum-free medium tinsulin, 10 /~g/ml; BSA, 1 mg/ml; fetuin, 1 mg/ml} and (B) serum-containing medium t10% FBSL Microvilli were scattered along the apical surface of the cells. X4286

GROWTH AND DIFFERENTIATION OF OPOSSUM KIDNEY CELLS

885

the role of fetuin as a spreading and/or growth factor for OK cells in serum-free medium. Insulin has been shown to be mitogenic for a number of kidney cells grown in serum-free medium (Chuman et al., 1982; Chung et al. 1982; Taub et al., 1979). Although the presence of insulin in our serum-frce medium resulted in increased protein levels in OK cells, no apparent effect on cell number was demonstrated. It is possible that fetuin or a contaminant thereof may have played a role in mitogenesis, thus making the proliferative effects of insulin. The serum-free defined medium developed for the OK cell line should prove to be a valuable tool for studies relating to mechanisms of renal proximal tubular function. The investigation of renal cellular proliferation and differentiation with respect to the effects of hormones, growth factors, and nephrotoxic agents will be able to be carried out under more defined, controlled conditions which will eliminate the multitude of complexities associated with the use of serum.

FiG. 6. Electron photomicrographs of OK cells. Tight junctions were present both in ceils grown in IA) serum-free medium as well as (B) serum-containing medium. X26 429

Several studies have suggested that the biological activity of ietuin with regard to its role in cellular development in vitro is related to the presence of contaminants (Rizzino and Sato, 1978; Salomon et al., 1982; Salomon et al., 1984}. Fetuin, as prepared by the method of Pedersen {1947), has been shown to contain a high molecular weight species which has similar properties to those of alpha2-macroglobulin (Salomon et al., 1982; Salomon et al., 1984). This high molecular weight component, embryonin, as well as alphazmacroglobulin have been demonstrated to promote the growth of mouse embryocarcinomal cells in a serum-free medium in addition to enhancing collagen production in cultures of normal rat kidney and rat mammary epithelial cells (Salomon et al., 1982; Salomon et al., 1984). In contrast, Gaillard et al. {1985) have shown that alpha2macroglobulin could not substitute for the growthmodulating effect of fetuin with regard to proliferation and differentiation of Oh17 preadipose cells in serumfree medium. In addition, Florini and Roberts (1979) demonstrated that highly purified fetuin, relatively devoid of active contaminants, promoted the proliferation of muscle cells in serum-free medium. In light of these reports, it will be of interest to pursue the investigation of

REFERENCES Caversasio, J.; Rizzoli, R.; Bonjour, J.-P. Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line. J. Biol. Chem. 261:3233-3237; 1986. Chuman, L,; Fine, L. G.; Cohen, A. H., et al. Continuous growth of proximal tubular kidney epithelial cells in hormonesupplemented serum-free medium. J. Cell Biol. 94:506-510; 1982. Chung, S. D.; Alavi, N.; Livingston, D., et al. Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium. J. Cell Biol. 95:118-126; 1982. Dennis, V. W. Phosphate homeostasis. In: Windhager, E. E., ed. Handbook of physiology,Rockville, MD: American Physiology Society: in press. Florini, J. R.; Roberts, S. B. A serum-free medium for the growth of muscle cells in culture. In Vitro 15:983-992; 1979. Gaillard, D.; Ailhaud, G.; N~grel, R. Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormonesupplemented medium. Biochim. Biophys. Acta 846:185-191; 1985. Jefferson, D. n . ; Cobb, n . H.; Gennaro, J. F., et al. Transporting renal epithelium: culture in hormonally defined serum-free medium. Science210:912-914; 1980. Koyama, H.; Goodpasture, C.; Miller, M. M., et al. Establishment and characterization of a cell line from the American opossum {Didelphys virginiana). In Vitro 14:239-246; 1978. Leiderman, L. J.; Tucker, J. A.; Dennis, V. W. Growth and differentiation of opossum kidney cells on microscopically transparent microporous membranes. Tissue & Cell 21:355-360; 1989. nalstri~m, K.; nurer, H. Parathyroid hormone inhibits phosphate transport in OK cells but not LLC-PK, and JTC-12.P3 cells. Am. J. Physiol. 251:C23-C31; 1986. Pedersen, K. O. Ultracentrifugal and electrophoretic studies on fetuin. J. Physiol. Colloid Chem. 51:164-171; 1947. Petersen, G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346-356; 1977. Rabito, C. A. Phosphate uptake by a kidney cell line (LLC-PK~). Am. J. Physiol. 245{14):F22-F31; 1983. Rizzino, A.; Sato, G. Growth of embryonal carcinoma cells in serumfree medium. Proc. Natl. Acad. Sci. USA 75:1844-1848; 1978. Salomon, D. S.; Bauo, n . ; Smith, K. B., et al. Isolation and characterization of a growth factor (embryonin) from bovine

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fetuin which resembles alpha2-macroglobulin. J. Biol. Chem. 257:14093-14101; 1982. Salomon, D. S.; Smith, K. B.; Losonczy, I., et al. Alpha2macroglobulin, a contaminant of commercially prepared Pedersen fetuin: isolation, characterization and biological activity. Cell Cult. Methods Mol. Cell. Biol. 3:125-153; 1984. Sepfilveda, F. V.; Pearson, J. D. Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney. J. Cell Physiol. 112:182-188; 1982.

Taub, M.; Chuman, L.; Safer, M. H., et al. Growth of Madin-Darby canine kidney epithelial cell (MDCK} line in hormonesupplemented, serum-free medium. Proc. Natl. Acad. Sci. USA 76:3338-3342; 1979. Taub, n . ; Sato, G. Growth of functional primary cultures of kidney epithelial cells in defined medium. J. Cell. Physiol. 105:369-378; 1980.

The authors thank Ms. LaRue D. Poe for her technical assistance and Ms. Lynne Grossman for typing the manuscript.