retinoids, part b

Methods in Enzymology Volume 190 Retinoids Part B Cell Dlrerentiation and Clinical Applications EDITED BY Lester Packer DEPARTMENT OF MOLECULAR AND CELL BIOLOGY UNIVERSITY OF CALIFORNIA, BERKELEY BERKELEY. CALIFOKNIA Editorial Advisory Board Frank Chytil Leonard M&tone Dewitt Goodman Concetta Nicotra Maria A. Livrea James A. Olson Stanley S. Shapiro 0 AP ACADEMIC PRESS, INC. Harcourl Brace Jovanovich, Publishers San Diego New York Boston London Sydney Tokyo Toronto Preface Spectacular progress and unprecedented interest in the field of retinoids prompted us to consider this topic for two volumes in the Methods in Enzymology series: Volume 189, Retinoids, Part A: Molecular and Meta- bolic Aspects and Volume 190, Retinoids, Part B: Cell Differentiation and Clinical Applications. From a historical perspective we know that studies in the 1930s showed that vitamin A (retinol) and retinal had a role in the visual process. It was also recognized that some link between vitamin A and cancer incidence existed. Several decades ago it was discovered that retinoic acid had a dramatic effect on the chemically induced DMBA mouse skin carcinogen- esis model in which enormous reductions in the tumor burden were ob- served. This led to the realization that retinoids had important effects on cell differentiation. This resulted almost immediately in the synthesis and evaluation of new retinoids. Indeed, the effects of retinoids on cell differ- entiation appear to be more universal and of greater importance than their light-dependent role in vision and microbial energy transduction. Progress has been rapid, and the importance of accurate methodology for this field is imperative to its further development. The importance of methodology applies to the use of retinoids in basic research in molecular, cellular, and developmental biology, and in clinical medicine. In medicine, applications have been mainly to cancer and in dermatology to the treat- ment of skin diseases and skin aging. As new retinoids are being tested in biological models and in clinical medicine, interest in the nutrition and pharmacology of retinoids has arisen. Moreover, the beneficial effects of retinoids in pharmacological treatment have led to a recognition of the "double-edged sword" of toxicity (teratogenicity). In Section I of this volume, Cell Differentiation, the effects of retinoids in various cell differentiation systems are covered. Many new systems in which retinoids exhibit their effects have been employed. Both normal diploid cells and cell lines in vitro have been used, and the methods and systems employed are presented. In addition, tissue and organ culture are important areas for retinoid methodology. The effects of retinoids as mor- phogens and teratology agents are also included. In Sections II, Nutrition, Tissue and Immune Status, and Antioxidant Action, and III, Pharmacoki- netics, Pharmacology, and Toxicology, nutritional and pharmacological methods are presented. Retinoids in the treatment of skin disease and in cancer chemotherapy are probably the most important areas in which methodological developments have occurred. New methodology has also xiii xiv PREFACE revealed the antioxidant activity of retinoids, and since any antioxidant may also be a pro-oxidant such considerations may be important for clinical pharmacology and therapeutics. Volume 189 covers structure and analysis, receptors, transport, and binding proteins, and enzymology and metabolism. I am very grateful to the Advisory Board Frank Chytil, DeWitt Goodman, Mafia A. Livrea, Leonard Milstone, Concetta Nicotra, James A. Olson, and Stanley S. Shapiro m for their unique input, advice, counsel, and encouragement in the planning and organization of this volume. In most instances, I met with every member of the board on one or more occasions to discuss the topics and to identify the most important contribu- tors. Indeed, we found almost universal acceptance, and virtually no one turned down our invitation to contribute to this volume. In fact it was somewhat autocatalytic in that many contributors, realizing the timeliness and significance of having all of the methods dealing with retinoids in- cluded, made suggestions for additional contributions which were evalu- ated by the board. In a few instances we may have been somewhat over- zealous, and more than one article on a method has been included. We do apologize for this slight redundancy for the sake of completeness. LESTER PACKER Contributors to Volume 190 Article numbers are in lmrentheses following the names of contributon;. Afffllations listed are current. SERGIO ADAMO (9), Department of Experi- mental Medicine, University of L'Aquila, 67100 L "Aquila, Italy BRAD AMOS (23), Department of Tumor Bi- ology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030 EVA ANDERSSON (18), Department of Der- matology, University Hospital, S-581 85 LinkOping, Sweden ADRIANNE BENDICH (27), Department of Clinical Nutrition, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 TROND BERG (6), Institute for Nutrition Re- search, University of Oslo, N-0316 Oslo 3, Norway RUNE BLOMHOFF (6), Institute for Nutrition Research, University of Oslo, N-0316 Oslo 3, Norway STEVEN A. BOOK (43), Health and Welfare Agency, State of California, Sacramento, California 95814 D. LUCILLE BREDBERG (17), Department of Ophthalmology, University of Washing- ton, Seattle, Washington 98195 THEODORE R. BREITMAN (13), Laboratory of Biological Chemistry, Division of Cancer Treatment, National Cancer Insti- tute, National Institutes of Health, Be- thesda, Maryland 20892 C. D. B. BRIDGES (15), Department of Bio- logical Sciences, Purdue University, West Lafayette, Indiana 47907 CONSTANCE E. BRINCKERHOFF (19), Depart- ment of Medicine and Biochemistry, Dart- mouth Medical School, Hanover, New Hampshire 03756 ADRIAAN BROUWER (5), TNO Institute for Experimental Gerontology, 2280 HV Rijs- wijk, The Netherlands GRAEME F. BRYCE (38), Roche Dermatolo- gics, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 ROSERT J. CAPETOLA (37), Research Labo- ratories, The R. W. Johnson Pharmaceu- tical Research Institute, Raritan, New Jersey 08869 ANOREW L. DARROW (12), Department of Molecular Pharmacology, State University of New York at Stony Brook, Stony Brook, New York 11794 WILLEM J. DE GRIP (1), Center of Eye Re- search Nijmegen, University of Nijmegen, 6500 HB Nijmegen, The Netherlands Luioi M. DE LUCA (9, 10), National Cancer Institute, Bethesda, Maryland 20892 THOMAS I. DORAN (35), Preclinical Re- search, Roche Dermatologics, Hoffmann- La Roche Inc., Nutley, New Jersey 07110 R. EHLERT (3 l), Department of Dermatology and Venereology, University Medical Center Steglitz, The Free University of Berlin, Berlin, Federal Republic of Ger- many GREGOR EICHELE (21), Department of Cel- lular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115 DENISE A. FAHERTY (27), Department of Immunopharmacology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 SHAO-LING FONG (15), Departments of Oph- thalmology and Biochemistry and Molecu- lar Biology, Indiana University, Indianap- olis, Indiana 46202 ELAINE V. FUCHS (2), Department of Molec- ular Genetics and Cell Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637 GERARD J. GENDIMENICO (37), Research Laboratories, The R. W. Johnson Phar- maceutical Research Institute, Raritan, New Jersey 08869 ix X CONTRIBUTORS TO VOLUME 190 MARGARET A. GEORGE (4), Cell Biology Section, Laboratory of Pulmonary Patho- biology, National Institute of Environmen- tal Health Sciences, Research Triangle Park, North Carolina 27709 G~ORGE J. GIUDlCE (2), Department of Der- matology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 H. GOLLNICK (31), Department of Dermatol- ogy and Venereology, University Medical Center Steglitz, The Free University of Berlin, Berlin, Federal Republic of Ger- many JOANNE BALMER GREEN (32), Nutrition De- partment, Pennsylvania State University, University Park, Pennsylvania 16802 MICHAEL H. GREEN (32), Nutrition Depart- ment, Pennsylvania State University, Uni- versity Park, Pennsylvania 16802 JOSEPH F. GRIPPO (16), Department of Toxicology and Pathology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 MICHAEL D. GRISWOLD (7), Department of Biochemistry, Washington State Univer- sity, Pullman, Washington 99164 GARY L. GROVE (39), KGL's Skin Study Center, Broomall, Pennsylvania 19008 MARY Jo GROVE (39), KGL's Skin Study Center, Broomall, Pennsylvania 19008 LORRAINE J. GUDAS (14), Department of Biological Chemistry and Molecular Phar- macology, Harvard Medical School and Dana-Farber Cancer Institute, Boston, Massachusetts 02115 HENK F. J. HENDRIKS (5), TNO Institute for Experimental Gerontology, 2280 HV Rijs- wijk, The Netherlands MIDORI HIRAMATSU (29), Department of Neurochemistry, Institute for Neurobiol- ogy, Okayama University Medical School, Okayama 700, Japan W. BRIAN HOWARD (44, 45, 46), La Jolla Cancer Research Foundation, La Jolla, California 92037 FREESIA L. HUANG (10), National Institute of Child Health and Human Development, Endocrinology and Reproduction Research Branch, National Institutes of Health, Be- thesda, Maryland 20892 JAMES HURLEY (34), Business Development, Roche Dermatologics, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 ANTON M. JETTEN (4), Cell Biology Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 ALICE F. KARL (7), Department of Biochem- istry, Washington State University, Pull- man, Washington 99164 SHIGEMI KATO (9), St. Marianna University School of Medicine, Miyamae-Ku, Kawa- sam 213, Japan TERENCE I~ALEY (36), Department of Clin- ical Biochemistry, Cambridge University, Addenbrooke's Hospital, Cambridge CB2 2QR, England ANDREAS KISTLER (44, 45, 46), Clinical Re- search, F. Hoffmann-La Roche Ltd., CH- 4002 Basel, Switzerland LORRAINE H. KLIGMAN (40), Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, Pennsyl- vania 19104 DICK L. KNOOK (5), TNO Institute for Ex- perimental Gerontology, 2280 HV Rijs- wijk, The Netherlands DEVENDRA M. KOCHHAR (33), Department of Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 DAFNA LOTAN (11), Department of Tumor Biology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030 REUBEN LOTAN (11, 23), Department of Tumor Biology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030 MALCOLM MADEN (20), Anatomy and Human Biology Group, Kings College, London WC2R 2LS, England LAWRENCE J. MARNETT (30), Department of Biochemistry, Vanderbilt University Medi- cal Center, Nashville, Tennessee 37232 CONTRIBUTORS TO VOLUME 190 xi JOhN L. McGuw.~ (37), Research Laborato- ries, The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869 RAJENDRA G. MEHTA (42), Laboratory of Pathophysiology, HT Research Institute, Chicago, Illinois 60616 JAMES A. MEZICK (37), Research Laborato- ries, The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869 LEONARD M. MILSTONE (8), Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510 RICHARD C. MOON (42), Laboratory of Pathophysiology, HT Research Institute, Chicago, Illinois 60616 HEINZ NAU (47), Institute of Toxicology and Embryopharmacology, The Free Univer- sity of Berlin, D-IO00 Berlin 33, Federal Republic of Germany KATHLEEN M. NAUSS (28), Health Effects Institute, Cambridge, Massachusetts 02139 C. E. ORFANOS (3 l), Department of Derma- tology and Venereology, University Medi- cal Center Steglitz, The Free University of Berlin, Berlin, Federal Republic of Ger- many [,ESTER PACKER (29), Department of Molec- ular and Cell Biology, University of Cali- fornia, Berkeley, Berkeley, California 94720 MARIA PONEC (3), Department of Dermatol- ogy, University Hospital Leiden, 2300 RC Leiden, The Netherlands JAMES I. REARICK (4), Department of Bio- chemistry, Kirksville College of Osteo- pathic Medicine, Kirksville, Missouri 63501 RICHARD J. RICKLES (12), Department of Biochemistry and Molecular Biology, Har- vard University, Cambridge, Massachu- setts 02138 G. RINCK (31), Department of Dermatology and Venereology, University Medical Center Steglitz, The Free University of Berlin, Berlin, Federal Republic of Ger- many OLA ROLLMAN (18), Department of Derma- tology, University Hospital, S-751 85 Upp- sala, Sweden DENNIS R. ROOP (10), Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 MARVIN E. ROSENTHALE (37), Research Laboratories, The R. W. Johnson Phar- maceutical Research Institute, Raritan, New Jersey 08869 A. CATHARINE ROSS (28), Department of Physiology and Biochemistry, Division of Nutrition, Medical College of Pennsylva- nia, Philadelphia, Pennsylvania 19129 JOHN C. SAARI (17), Department of Ophthal- mology, University of Washington, Seattle, Washington 98195 PETER G. SACKS (11), Department of Tumor Biology, M. D, Anderson Cancer Center, University of Texas, Houston, Texas 77030 VICTOR M. SAMOKYSZYN (30), Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, California 94143 STANLEY S. SHAPIRO (34, 35, 38), Preclini- cal Research, Roche Dermatologics, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 MICHAEL I. SHERMAN (16), Department of Cell Biology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 KENNETH L. SIMPSON (25), Food Science and Nutrition Research Center, West Kingston, Rhode Island 02892 JOHN EDGAR SMITH (24), Nutrition Depart- ment, Pennsylvania State University, Uni- versity Park, Pennsylvania 16802 DAVID L. STOCUM (20), Department of Biol- ogy, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana 46205 xii CONTRIBUTORS TO VOLUME 190 SIDNEY STRICKLAND (12), Department of Molecular Pharmacology, State University of New York at Stony Brook, Stony Brook, New York 11794 KAMPE TEELMANN (41), Pharmaceutical Division, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland CHRISTINA THALLER (21), Department of Cellular and Molecular Physiology, Har- vard Medical School Boston, Massachu- setts 02115 ADRIAN M. TIMMERS (1), Department of Ophthalmology, University of Florida, Gainesville, Florida 32610 HANS TORM.~ (18, 22), Department of Der- matology, University Hospital, S-581 85 LinkOping, Sweden SALLY S. TWINING (28), Department of Bio- chemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 BARBARA A. UNDERWOOD (26), National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 ANDERS VAHLQUIST (18, 22), Department of Dermatology, University Hospital S-581 85 LinkOping, Sweden SARAH WEDDEN (21), Department of Anat- omy, University Medical School, Edin- burgh EH8 9AG, Scotland ARIJ WEERHEIM (3), Department of Derma- tology, University Hospital Leiden, 2300 RC Leiden, The Netherlands CALVIN C. WILLHITE (43), Toxic Substances Control Program, Department of Health Services, State of California, Berkeley, California 94710 [ 1 ] SHORT-TERM INCUBATION OF ISOLATED RPE CELLS 3 [ 1 ] Analysis of the Visual Cycle by Short-Term Incubation of Isolated Retinal Pigment Epithelial Cells By ADRIAN M. TIMMERS and WILLEM J. DE GRIP Introduction The recycling of retinoids in retina and retinal pigment epithelium (RPE), effectuating the regeneration of bleached visual pigment to the photoactive state, is called the visual cycle. The trigger reaction for the process of vision as well as the visual cycle is photoinduced isomerization (11-cis to all- trans) of the chromophore of vertebrate visual pigments) Major parts of the pathway of the visual cycle remained obscure for decades. Only re- cently has insight been obtained into some of the long-standing enigmas: which type of retinoid is isomerized where and how and transported in what form to the outer segments to eventually regenerate the visual pig- merit, rhodopsin. 2 This regeneration pathway is initiated in the RPE where all.trans-refinol is enzymatically isomerized to 11-cis-retinol followed later by conversion to 11-cis-retinaldehyde. The isomerization reaction is driven by the free energy of hydrolysis of the retinyl ester? Retinoid metabolism in the RPE, with respect to the visual cycle, encompasses several steps: uptake of retinol, intracellular transport, acyla- tion, isomerization, oxidation, and secretion of retinoid. In order to study such a complex set of metabolic pathways in RPE cells, the development of a reliable in vitro system would be highly desirable. Investigations on the multifaceted retinoid metabolism in RPE cells in vitro require isolated RPE cells in which the complexity of cellular organization is preserved to a high extent and which are viable and metabolically active? In order to meet these requirements of physiological fitness, we optimized the isola- tion of bovine RPE cells and the in vitro incubation conditions by applying a variety of criteria. These criteria included morphology (ultrastructure of the cells), viability (exclusion of viability stains and retention of small cellular proteins), and metabolic activity (energy charge). Here we describe an approach to study retinoid metabolism in isolated bovine RPE cells during short-term in vitro incubation. t G. Wald, Nature (London) 219, 800 (1968). 2 p. S. Bemstein, C. W. Law, and R. R. Rando, Proc. Natl. Acad. Sci. U.S.A. 84, 1849 (1987). 3 R. R. Rando, J. Canada, P. S. Deigner, and C. W. Law, Invest. Ophthalmol. Visual Sci. 30, 331 (1989). 4 A. M. M. Timmers, W. J. De Grip, and F. J. M. Daemen, in "Proceedings on Retinal Proteins" (N. G. Abdulaev and Y. A. Ovchinnikov, eds.), p. 381. VNU Science Press, Utrecht, 1987. Copyright © 1990 by A~l~mi¢ ~ Inc. METHODS IN ENZYMOLOGY, VOL. 190 AllfishtsofreProducfionin~ay form t~xl. 4 NORMAL CELLS [ 1 ] Isolation of Viable Bovine Retinal Pigment Epithelial Cells Principle Isolation of intact RPE cells requires release of cells from their intimate contact with both retina and BrOchs membrane. Strong reduction of the attachment of RPE to retina and BrOchs membrane is achieved by perfu- sion of the intact bovine eye through the central ophthalmic artery with a divalent cation-free isotonic salt buffer (perfusion buffer; Ca 2+, Mg2+-free Hanks-EDTA s) kept at 0 °.6 A high recovery of RPE cells which are not contaminated by either red blood cells or rod outer segments is obtained after 12-18 min ofperfusion. The yield of 1-2 × 106 RPE cells per bovine eye represents 20-40% of the total RPE cell population. Furthermore, over 80% of the cells exclude didansylcystine (viability stain), 6 and 85% of the cellular retinol-binding protein (CRBP) is retained in the cells as assayed with the Lipidex 1000 binding assay.7 The ultrastructure of the isolated cells is very well preserved (Fig. 1). The yield, purity, and integrity of this RPE cell population meet the high standards for in vitro studies. Procedure Bovine eyes are enudeated 20 min after the death of the animal; the optic nerve is kept at least 2 cm long. The eyes are immediately transported in a fight-tight container to the laboratory, where excess fat and muscle are trimmed off under dim red light. The eyes are wrapped in aluminum foil, leaving the optic nerve accessible, and kept in a dark container at 8-10 °. The central ophthalmic artery is cannulated with a blunt 21-gauge needle connected to a reservoir of perfusion buffer (137.8 mM NaCI; 5.4 mM KC1; 0.3 mM Na,zHPO4; 0.4 mM KH2PO4; 2 mM EDTA; 5.5 mM glucose; 10 mM HEPES, pH 7.4) positioned 100- 120 cm above the eye (Fig. 2). The central ophthalmic artery, which supplies the entire eye and runs along the optic nerve, can be readily identified by its translucent white color and the blood clot at its end. Further differentiation from fat tissue is achieved by pulling it gently with forceps; this ruptures fat tissue but not the artery. The artery is grasped with two small forceps (5SA, Technical Tools, Rotterdam), pulled over the blunt needle, and tied with a suture. The wrapped eye is peffused with ice-cold peffusion buffer for 13- 17 min at a flow rate of 0.5-1 ml/min. Routinely, eyes are processed within 2 hr 5 j. Heller and P. Jones, Exp. Eye Res. 30, 481 (1980). 6 A. M. M. Timmers, E. A. Dratz, W. J. De Grip, and F. J. M. Daemen, Invest. Ophthalmol. Visual Sci. 25, 1013 (1984). 7 A. M. M. Timmers, W. A. H. M. van G-roningen-Luyben, F. J. M. Daemen, and W. J. De Grip, J. LipidRes. 27, 979 (1986). [...]... SHORT-TERM INCUBATION OF ISOLATED RPE CELLS 9 TABLE I METABOLIC FITNESS OF ISOLATED RETINAL PIGMENT EPITHELIAL CELLS DURING SHORT-TERM INCUBATION Incubation time (hr) 0 2 4 8 Metabolic parameters Incubation medium ATP~ Total A ° Energy charge Perfusion buffer K r e b s - Ringer RPMI1640DM(4) b Perfusion buffer Krebs - Ringer R P M I 1640 D M Perfusion buffer Krebs-Ringer R P M I 1640 D M (3~' Perfusion buffer... cells/ml RPE cells incubated in either RPMI 1640 DM (Flow Labs, Irvine, Scotland) or perfusion buffer (see above) maintain equally well a steady level of CRBP (120 + 15 pmol/10~ cells) during at least 8 hr of incubation (data not shown) The energy chargc is measured in R P E cellsincubated in R P M I 1640 D M , Krcbs-Ringcr, or perfusion buffer (Table I) During incubation in pcrfusion buffer thc cncrgy... Rothblat, L Y Arborgast, L Ouellet, and B V Howard, In Vitro 12, 554 (1976) 22 NORMAL CELLS [2] volume of phosphate-buffered saline (PBS) such that the original serum protein concentration is restored, as judged by Lowry protein analysis H Preparation of Fibroblast Feeder Layer Optimal growth of human epidermal cells is dependent on exogenous dermal factors, which can be supplied by 3T3 mouse fibroblasts,... KERATINOCYTES 25 background staining An antikeratin antibody, diluted as necessary in BSA-PBS, is incubated with the sections for 1 hr at room temperature After three 10-min washes in PBS, sections are incubated overnight at room temperature with gold-conjugated secondary antibodies (15-nm gold particles; Janssen Life Science Products, Piscataway, NJ) diluted in BSAPBS Sections are then washed in PBS (6 times,... and describe some of the analytical tools which have been used to study the 4 C E Bloch, J Hyg 19, 283 (1921) s S Mori, Bull Johns Hopkins Hosp 33, 357 (1922) 6 S B Wolbaeh and P R Howe, J Exp Med 43, 753 (1925) 7 C N Frazier and C K Hu, Arch Intern Med 48, 507 (1931) s S B Wolbaeh and P R Howe, J Exp Med 57, 511 (1933) 9 D Burk and R J Winzler, Vitam Horm 2, 305 (1944) ~oH B Fell and E Mellanby, J Physiol... ready for hybridization Probe Preparation 35S-Labeled UTP-containing eRNA antisense probes are prepared by in vitro transcription using a construct containing a bacteriophage promoter (the promoters most commonly used for this purpose are derived from SP6, T3, and TT) and the appropriate polymerase 22 Prior to use, probes are hydrolyzed to an average size of 150 base pairs (bp) by sodium carbonate treatment... the air-liquid interface Bar: 30/zm [(A, B, and C) From R Kopan, G Traska, and E Fuchs, J Cell Biol 105, 427 (1987) (D) From A Stoler, R Kopan, M Duvic, and E Fuchs, J.CellBiol 107, 427 (1988).] biosynthetic changes that take place during induction or inhibition of keratinization Submerged Keratinocyte Culture By using a feeder layer o f growth-arrested 3T3 m o u s e fibroblasts, Rheinwald and G r... within 15 sec Short-Term Incubation System for Retinal Pigment Epithelial Cells Studies on cellularmetabolism in vitro do not only require viable cells but also require an incubation system in which the cells can be kept metabolically fit for an appropriate time period) 3 T w o parameters arc applied to evaluate incubation media: (I) retention of CRBP, a small cytoplasmic rctinol-bindingprotein ( M r 14,000),... differentiation program is dearly abnormal and is completely suppressed by retinoids 23 The following protocol for culturing keratinocytes at the air-liquid interface is essentially as described by Asselineau et al 2° Type I collagen (Seikagaku America, Inc., St Petersburg, FL) is combined with keratinocyte growth medium as described by the manufacturer A confluent culture of 3T3 fibroblasts is trypsinized and... concentrations become lyric for membranes? 9 These features therefore demand the use of a carrier Unfortunately, the putative "natural" carrier in the interretinal space, interphotoreceptor matrix retinoid-binding protein (IRBP), is not easily available and is rather unstable Instead, we opted for an aseleetive system, phosphatidylcholine vesicles, which have been demonstrated to be reliable and stable retinol . 190 Retinoids Part B Cell Dlrerentiation and Clinical Applications EDITED BY Lester Packer DEPARTMENT OF MOLECULAR AND CELL BIOLOGY UNIVERSITY OF CALIFORNIA, BERKELEY BERKELEY. CALIFOKNIA. Health, Be- thesda, Maryland 20892 C. D. B. BRIDGES (15), Department of Bio- logical Sciences, Purdue University, West Lafayette, Indiana 47907 CONSTANCE E. BRINCKERHOFF (19), Depart-. Perfusion buffer 390 540 0.79 Krebs- Ringer 370 370 0.58 RPMI1640DM(4) b 320_ 37 600+ 50 0.69_0.02 2 Perfusion buffer Krebs - Ringer RPMI 1640 DM 390 720 0.73 4 Perfusion buffer 30 90 0.47 Krebs-Ringer