ISOLATION AND PRIMARY CULTURE OF RAT RENAL CORTICAL EPITHELIAL CELLS

M a r y Ann S m i t h ' , J o h n Swann, and Daniel Acosta

University of Texas at Austin, College of Pharmacy, Division of Pharmacology and Toxicology, Austin, Texas 78712

SUMMARY: Methods are described for the isolation of rat renal cortical epithelial cells by a collagenase and hyaluronidase perfusion, followed by pressing fragments of renal cortex through an 80-mesh screen and further enzymatic dissociation in vitro. Primary monolayer cultures are derived from the resulting suspension of tubular fragments and cells. Fibroblast and endothelial cell overgrowth is suppressed by the use of medium lacking arginine and containing D-valine in place of L-valine. Further separation of fibroblasts from epithelial cells is achieved by a technique that takes advantage of the differential rate of attachment of the two cell types. The presence of glomerular cells in the cultures is diminished by a complete medium change 48 h after plating the ceils.

Key words: renal cortical epithelial cells; isolation; cell culture. I.

INTRODUCTION

II.

Many in vitro kidney preparations have been utilized for physiologic and toxicologic studies: these preparations include cell suspensions t9,19), tubule suspensions (5,22), slices {6,12}, isolated perfused kidney (2,23), cell lines {10,14), and primary cell cultures {4,20). Although each preparation offers advantages and suffers from particular drawbacks, cell culture alone offers the advantage of a longer duration of viability. The extended viability of cells in culture offers greater flexibility in the duration of experimental toxicant exposure. In addition, cell cultures retain the tissue attributes of cell-to-cell interactions and epithelial cell polarization that are characteristic of the intact organ {17). The use of cultured cell lines for toxicologic studies can pose several limitations when compared to primary cultures. The validity of toxicologic studies performed on essentially transformed cells may be questionable. Cell lines may undergo de-differentiation ~13) as a result of prolonged passage in vitro, and cell lines possess attributes that are not present in their presumed tissue of origin (24). This can result in an altered cellular response to toxicants. In addition, the use of renal cell lines can present problems in the extrapolation of data from cells maintained in culture for long periods of time to the intact animal. Primary cultures retain the utility of the cell culture method without the inherent drawbacks of cell lines.

A. Chemicals and culture medium 1. Dissociation medium NaC1, no. S-271, Fisher ' KC1, no. P-217 ' Na~HPO4, no. S-374' KH~PO4, no. P-285' Glucose, no. D-16' NaHC03, no. SX400, MCB ~ MgSO4" 7H~O, no. 2500 ~ MgCI~. 6H~O, no. M-331 Sodium succinate, no. S-2378, Sigma ~ L-Malic acid, no. M-10003 Sodium pyruvate, no. P2256 ~ Phenol red, sodium salt, no. P47583 Bovine albumin, fraction V, no. A-45033 Collagenase, class IV, no. 4188, Worthington 4 Hyaluronidase, no. H-21263 Deoxyribonuclease I, no. DN-25 ~ 2. Culture Medium Dry Powder, no. 86-5014, GIBCO 5 Sodium bicarbonate, no. 3506-05, Baker 6 Phenol red, sodium salt, no. P-47583 Hydrocortisone 21-hemisuccinate, no. H488P Insulin, no. 1-5500~ Transferrin, no. T-22523 Newborn bovine serum, sterile filtered, no. 1212377, Hazleton 7 Penicillin G potassium, no. 0510, Pfizer s Streptomycin sulfate, no. 1626a Amphotericin B, no. 43760, Squibb 9 Albumin, no. A-4503~

Present address: University of New Mexico, College of Pharmacy, Albuquerque, New Mexico 87131. Journal of Tissue Cuhure Methods

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MATERIALS

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~) 1988 Tissue Culture Association, Inc.

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3. 36% HCI, no. A-144 ~ B. Glassware and plastics Beakers, 20 ml, no. B2660-20, Baxter '° Beakers, 400 ml, no. B2660-4001° Beakers, 600 ml, no. B2660-6001° Beakers, 1000 ml, no. 2660-1L 1° Culture tubes, 16 )< 125 mm, no. T1310-751° Graduated cylinder, 25 ml, no. C9054-251° Graduated cylinder, 100 ml, no. C9054-100 ~° Morton Culture Tube Closures, no. T1390-161° Pipettes, serological, 10 ml, no. P4315-10 ~° Pipettes, pasteur, 5.75 in., no. P5215-1 ~° Star head magnetic stirring bar, 17 )< 13 mm, no. $8309-21° Tissue culture dishes, 35 mm, no. 3001, Falcon '~ Trypsinizing flask, 35 ml, no. 355212, Wheaton 12 Volumetric flask, 1000 ml, no. F4615-1L 1° C. Equipment Incubator, CO~, model 351920, Hotpack ~3 Microscope, inverted phase contrast, Leitz TM Dissection hood, with UV germicidal lamp, no. 11000, Labconco 's Laminar flow hood, model TM-48, Pure Aire" Centrifuge, model H N - S I I , I E C " Water bath, no. B 6648, Precision Scientific's Pipet-aid, Becton Dickinson '9 Hotplate-stirrer, no. 3832, Cole Parmer 2° Mega-Pure Still, Coming ~ D. Instruments Surgical scissors: 6.5, 4.5 in., no. D2640 ~° Stevens tonotomy scissors, straight, 4.25 in., no. 360140, Ford Dixon 22 Halstead mosquito forceps, mouse tooth, curved, 5 1/8 in., no. 105-112 ~ Spatula, 6 in., no. S1510-4 ~° Forceps, blunt pointed, straight, 5 in., no. 08-8901 E. Animals Sprague-Dawley rats, 7-to-14-d-old, Animal Resources Center 2s F. Miscellaneous Dispensing vessel, stainless steel, no. XX6700003, Millipore~4 Filter holder, syringe type, 25 mm, no. 4320, Gelman 2s Syringes, sterile disposable, 10 cc, no. 56049 Hypodermic needle, 27 gauge, 0.5 in., no. 51099 Membrane filter, 0.2/Jm, 90 mm, no. 648582s OptiVISOR, no. DA-5 ~ Filter holder, 90 mm, no. YY30090002" Pump, no. XX550000024 Thermometer, 0 to 100 ° C '° Laboratory sieve, U. S. Sieve designation 80, no. S122780~o III. PROCEDURE A. Preparation of media 1. Dissociation medium: a modification of Hanks' calcium-free balanced salt solution (BSS ~. a. For 1 liter of medium, combine the following in deionized, double distilled water: Chemical Gram NaCI 8.8 208

KCI Na2HPO4 KH2PO, glucose NaHCO3 MgSO4.7H20 MgC12 - 6H20 sodium succinate L-malic acid sodium pyruvate phenol red

O.4 0.05 O.O6 1.0 0.35 0.1 0.1 0.1 0.005 0.2 0.01

b. Adjust the pH of the solution to approximately 7.2 with 1 N HCI or 1 N NaHCO3, and adjust the volume of the solution to 1 liter. Sterilize the BSS by filtration under positive pressure through a 0.2-/~m membrane filter. c. Store in 100-ml aliquots at 4 ° C. 2. Culture medium: a modification of DulbeccoVogt's modification of Eagle's minimum essential medium ~DMEM) lacking arginine and containing D-valine. a. Prepare GIBCO dry powder in deionized double distilled water adding 2 g sodium bicarbonate, 10 mg insulin, 50 mg hydrocortisone hemisuccinate, 5 mg transferrin, and 10 mg of phenol red. Adjust the pH and the volume of the solution as indicated for BSS. b. Sterilize the D M E M as indicated for BSS. c. Store in 90-ml alquots at 4 ° C. 3. Serum-Antibiotics a. To each thawed 100-ml bottle of newborn bovine serum, add 200,000 U of penicillin G, 200 mg of streptomycin, and 4 mg of amphotericin B in the laminar flow hood. b. Mix well and store in 10-ml aliquots at - - 10° C. 4. Sterilize the glassware, instruments, 80-mesh screen, dissection hood, and 0.1 and 1.0 N HCI and NaHCO3 solutions by established procedures. B. Isolation and culture of kidney cells 1. Warm one 100-ml alquot of BSS, one 90-ml aliquot of D M E M and one 10-ml aliquot of serumantibiotics to 37 ° C in the water bath. Place a thermometer in a l-liter beaker, fill it with about 200 ml of water, and place it on the hotplate-stirrer. 2. In the laminar flow hood, add 100 mg albumin, 10 000 U of collagenase, 6000 U of hyaluronidase, and 1000 U of deoxyribonuclease to the BSS. When these have dissolved, adjust the pH of the dissociation medium to 7.4 with 1 N HCI or 1 N NaHCO3, as required, using a sterile, plugged Pasteur pipette and rubber bulb (1 to 4 drops is usually sufficient). 3. Add 100 mg albumin and the 10-ml aliquot of serum-antibiotics to the D M E M in the laminar flow hood. When these have dissolved, adjust the pH of the culture medium to 7.4 as indicated for BSS. 4. Pour about 20 ml of dissociation medium into a 20ml beaker in the dissection hood and place it on ice Journal oJ/Tissue Culture Methods

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5.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

in a 400-ml beaker. Fill the 10-cc syringe with dissociation medium and attach a 27-gauge needle. One at a time, decapitate 14 Sprague-Dawley rats (7 to 14 d old) and expose the lower aorta and the renal arteries. Remove the heart to prevent backflow of blood after perfusion. With the syringe, perfuse each rat with 0.75 to 1.5 ml of dissociation medium through the lower aorta at a point below the joining of the right and left renal artery to the aorta. With the forceps and tonotomy scissors, remove each kidney and place it in the ice-cold dissociation medium. Place the petri dishes on ice in 400-ml beakers. Transfer the collected kidneys to one petri dish and fill the other dish with dissociation medium. With the mouse-tooth forceps, hold each kidney by the renal pelvis and remove slices of cortex, using the tonotomy scissors. Transfer the slices to the petri dish containing dissociation medium. Discard the medullary region that remains after dissection. Place the 80-mesh screen above a 600-ml beaker. Pour the collected slices of cortex onto the screen and press them through the screen using the large spatula. Rinse the screen with dissociation medium using the syringe and needle. Collect any tissue that adheres to the bottom of the sieve. Transfer the cortical fragments to the trypsinizing flask. Add about 20 ml of dissociation medium to the flask. Close the trypsinizing flask with sterile foil to minimize pH rise. Maintaining a temperature of 37 ° C, stir the fragment suspension at low speed on the hot platestirrer for 10 min. During dissociation, adjust the pH of the suspension to 7.4 with 0.1 N HCI or 0.1 N NaHCO3, as required, using a sterile, plugged Pasteur pipette and rubber bulb i1 to 4 drops is usually sufficient). At the end of the first dissociation, allow the trypsinizing flask to stand undisturbed for 2 min to allow the fragments to settle. Carefully remove the supernatant {which contains mainly debris and red blood cells) with a sterile Pasteur pipette, and discard it. Add 20 ml of dissociation medium to the flask and incubate for 20 min with gentle stirring at 37° C. At the end of the second dissociation, allow the trypsinizing flask to stand undisturbed for 2 min. Carefully remove the supernatant with a Pasteur pipette and divide the liquid between two culture tubes. Cap the tubes with morton closures. Centrifuge the suspension at 50 X g for 5 min; discard the supernatant. Add 2 ml of culture medium to each tube and gently resuspend the pellet using a 10-ml pipette. Cap the tubes and centrifuge the suspension again at 200 X g for 5 min to wash the cells free of the enzymes. Discard the supernatant from each tube. Add 2 ml of culture medium to each tube and gently resuspend the pellet using a 10-ml pipette.

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17. Strain the suspension through a stainless steel screen housed in a 25-mm Gelman filter holder connected to a syringe barrel. Collect the suspension in a 600-ml beaker. Place the beaker, loosely covered with foil, in the incubator until the addition of the suspensions from the third and fourth dissociations. 18. Repeat steps 15 through 17 for the third and fourth dissociations t20 min eachl. Pool the strained suspensions in the 600-ml beaker. 19. Measure the volume of the pooled suspension with a 25-ml graduated cylinder and return the suspension to the 600-ml beaker. Add sufficient culture medium to bring the volume of the suspension to 85 ml. C. Cultivation of ceils 1. Plating a. Swirl the beaker gently to evenly disperse the cells and tubules. Plate 2 ml of the suspension in each 35-mm culture dish. b. Incubate the dishes in a humidified environment of 5% CO~:95% air at 37 ° C. 2. Three-hour pour-off a. Three hours after plating, gently swirl each dish and pour the medium containing unattached epithelial cells and tubule fragments into another culture dish. Discard the original culture dishes. Incubate the dishes for 45 h. 3. Forty-eight hour medium change a. Forty-eight hours after plating, aspirate the medium in each dish and replace it with fresh medium. IV.

DISCUSSION This culture technique produces a mixture of cells and tubular fragments, which can be readily visualized at the time of plating with phase contrast microscopy. With time, the fragments attach to the dishes and outgrowth of attached, flattened cells with epithelial morphology, ensues. By the 48-h medium change, the fragments have lost their tubular morphology and are visible only as phase-bright objects surrounded by a confluent group of cells with typical epithelial morphology. Depending on plating density, a confluent cell monolayer is achieved by 72 h after plating. Our technique utilizes a modified Dulbecco's modified Eagle's medium to enrich the content of epithelial cells in culture and to prevent fibroblast growth I l l L The substitution of I)-valine for L-valine suppresses fibroblast growth. Mammalian proximal epithelial ceils express D-amino acid oxidase, which, with a transaminase, converts the D-enantiomer into the essential amino acid, J~valine. Fibroblasts do not express D-amino acid oxidase and will not grow in medium lacking L-valine ~7). In addition, a pour-off technique separates non-epithelial cells from epithelial cells t21 }. The first medium change, 48 h after plating, minimizes attachment of glomerular cells 18). The utility of primary culture lends itself to toxicologic investigation; several researchers have used 21)9

SMITH ET AL.

primary cultures for toxicologic studies. Cherian (3) used primary cultures to study the toxicity of metals; Smith et al. (18) have used primary cultures to investigate the cytotoxicity o! mercury. In our laboratory, this culture technique has been used in studies of the cytotoxicity of mercury, cadmium, and cephaloridine (1,15,16). It is hoped that this technique will permit standardization of the kidney cell culture technique so that the potential nephrotoxicity of xenohiotics can be evaluated. V.

REFERENCES

1. Acosta, D.; Sorensen, E. M. B.; Amfforo, D. C., et al. An in vitro approach to the study of target organ toxicity of drugs and chemicals. In Vitro. 21:495-504; 1985. 2. Besarab, A.; Jarrell, B. E.; Hirsch, S., et al. Use of the isolated perfused kidney model to assess the acute pharmacologic effects of eyclosporine and its vehicle, cremophor EL. Transplantation 44:195-201; 1987. 3. Cherian, M. G. Rat kidney epithelial cell culture for metal toxicity studies. In Vitro 21:505-508; 1985. 4. 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. 5. Cojocel, C.; Malta, K.; Pasino. D. A., et al. Metabolic heterogeneity of the proximal and distal kidney tubules. Life Sci. 33:855-861; 1983. 6. Cojocel, C.; Laeschke, K. H.; Inselrnann, G,, et al. Inhibition of cephaloridine-induced lipid peroxidation. Toxicology 35:295-305; 1985. 7. Gilbert, S. F.; Migeon, B. R. D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 5:11-17; 1975. 8. Glasgow, E. F.; Hancock, W. W.; Atkins, R. C. The technique of glomerular culture. In: Allen, D, E.; Dowling, J. P., eds. Techniques for nephropathology. Boca Raton: CRC Press 1981:87-103. 9. Hassall, C. D.; Gandolfi, A. J.; Brendel, K. Correlation of the in vivo and in vitro renal toxicity of S-O,2-dichlorovinylbL-cysteine. Drug Chem. Toxicol. 6:507-520; 1983. 10. Inui, K.; Saito, H.; Hori, R. The use of kidney epithelial cell line {LLCPK~) to study aminoglycoside nephrotoxicity. Dev. Toxicot. Environ. Sci. 14:217-226: 1986.

11. Leffert, H. L.; Paul, D. Serum dependent growth of primary cultured differentiated fetal rat hepatoyetes in arginine-deficient medium. J. Cell Physiol. 81:113-124; 1973. 12. Ruegg, C. E.; Gandolfi, A. J.; Nagle, R. B., et al. Preparation of positional renal slices for study o5 cell-specific toxicity. J. Pharmacol. Methods 17:111-123; 1987. 13. Sakhrani, L. M.; Pine, L. G. Renal tubular cells in euhure. Mineral Electrolyte Metab. 9:276-281; 1983. 14. Schwertz, D. W.; Kreisberg, J. I.; Venkataehalam, M. A. Gentamieininduced alterations in pig kidney epithelial (LLC-PK~) eeUs in culture. J. Pharmaeol. Exp. Ther. 236:254-262; 1986. 15. Smith, M. A.; Acosta, D.; Bruekner, J. V. Development of a primary culture system of rat kidney cortical cells to evaluate nephrotoxocity of xenobiotics. Food Chem. Toxieol. 24:551-556; 1986. t6. Smith, M. A.; Aeosta, D.; Bruekner, J. V. Cephaloridine toxicity in primary cultures of rat renal cortical epithelial cells. In Vitro 1:23-29; 1987. 17. Smith, M. A.; Hewitt, W. R.; Hook, J. B. In vitro methods in renal toxicology. In: Atterwill, C. K.; Steele, C. E., eds. In vitro methods in toxicology. New York: Cambridge University Press; 1987:13-35. 18. Smith, M. W.; Ambudkar, I. S.; Phelps, P. C., et al. HgCl2-1nduced changes in eytosolic Ca "2of cultured rabbit renal tubular cells. Biochem. Biophys. Acta 93h130-142; 1987. 19. Smith, W. L.; Garcia-Peres, A. Immunodissection: use of monoelonal antibodies tx~ isolate specific types of renal cells. Am. J. Physiol. 248:FI-F7; 1985. 20. Trifillis, A. L.; Regee, A. L ; Trump, B. F. Isolation culture and characterization of human renal tubular cells. J. Urol. 133:324-329; 1985. 21. Waymouth, C. Studies on chemically defined media and the nutritional requirements of cultures of epithelial cells. In Katsuta, H., ed. Nutritional requirements of cultured cells. Baltimore: University Park Press; 1978:39-61. 22. Weinberg, J. M,; Davis, J. A.; Abarzua, M., et al. Cytoprotective effects o1 glyeine and glutathione against hypoxic injury to renal tubules. J. Clin. Invest. 80:1446-1454; 1987. 23. Williams, P. D.; Trimble, M. E.; Crespo, L., et at. Inhibition of renal Na +, K'-adenosine triphosphatase by gentamicin. J. Pharmacol. Exp. Ther. 23h248-253; 1984. 24. Wilson, P. D. Use of cultured renal tubular cells in the study ot cell injury. Mineral Electrolyte Metah. 12:71-84; 1986.

Daniel Aeosta is a Burroughs Wellcome scholar in Toxicology. Fisher Scientific Co., Fair Lawn, NJ 2 MCB, Cincinnati, OH 3 Sigma Chemical Company, St. Louis, MO ' Worthington Biomedical, Freehold, NJ GIBCO, Grand Island, NY 6 J.T. Baker Chemical Co., Phillipsburg, NJ Hazleton Research Products, Inc., Lenexa, KS 8 Pfizer Inc., New York, NY 9 E. R. Squibb & Sons, Inc. Princeton, NJ '° Baxter Scientific Products, Houston, TX l~ Falcon, Beeton Dickinson Labware, Lincoln Park, NJ 1: Wheaton Scien~ic, Millvilh, NJ 13 Hotpack Corporation, Philadelphia, PA

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~' E. Leitz, Inc., Roekleigh, NJ ~s Labeonco Corporation, Kansas City, MO s6 Pure Aire Corporation, Van Nuys, CA tr International Equipment Company, Needham Heights, MA tB Precision Scientific Corporation, Houston, TX ~9 Becton Dickinson Labware, Oxnard, CA 20 Cole Panner, Chicago, IL 2, Corning Glass Works, Coming NY ~2 Ford Dixon Company, Dallas, TX 23 Animal Resources Center, University of Texas, Austin, TX 24 Millipore Corporation, Bedford, MA z~ Gelman, Ann Arbor, MI

Journal of Tissue Culture Methods Vol. 11, No. 4, 1988