CONSTRUCTION OF PEPTOLIPOSOMES FOR THE INCORPORATION OF N U T R I E N T L I P I D S U P P L E M E N T S IN I N S E C T C E L L C U L T U R E M E D I A

Ronald H . Goodwin

Rangeland Insect Laboratory, USDA, Agricultural Research Service, Montana State University, Bozeman, Montana 59717 Nutrient liposomes that incorporate peptones instead of serum proteins can be used for the lipid supplementation of insect cells in lieu of or in addition to vertebrate serum to enhance the growth or differentiation of specific insect cell types in culture. The technique described here for the preparation of nutrient peptoliposomes involves the combination of both natural and synthetic phosphatidyl cholines (containing essential polyunsaturated fatty acids) with appropriate sterols. These are protected against premature oxidation by antioxidants {glutathione, alanine, protein hydrolysates) and antioxidant synergists {citrate, ascorbate, plus other amino acids) in the basal tissue culture medium as well as in the liposomal preparation itself {alphatocopherol acetate and protein hydrolysates). The lipid components are filter sterilized in chloroform and then dried before aqueous sonication, allowing the subsequent formation of a random array of unsized uni- and multilamellar liposomes, which are suitable as direct suppliers of lipid nutrients as well as nutrient carriers for lipid-soluble supplements. SUMMARY:

Key words: nutrient liposomes; polyunsaturated fatty acid supplementation; epithelioid cells; Acrididae; cell and tissue differentation; Primaria. I.

struction technique, utilizing peptone-containing phosphatidyl choline vesicles and allowing a probable broad size range for the liposomes, may be responsible in part for effective delivery of the selected polyunsaturated fatty acids as nutrients in a nontoxic form to the cells that require them (4-7). Preservation of the phosphatidyl choline-containing polyunsaturated fatty acids in these nutrient liposomes was also enhanced by the incorporation of antioxidants {glutathione, alanine, protein hydrolysates) and antioxidant synergists {citrate, ascorbate, plus other amino acids) in the basal tissue culture medium as well as in the peptone-Iiposome concentrate preparation (alpha-tocopherol acetate and protein hydrolysates) {15).

INTRODUCTION Lipid supplementation of certain vertebrate cell culture media has promoted growth {1) as well as differentiation {3) of specific cell types. Lecithin-based liposomes have been used by some vertebrate {10,11) and invertebrate (4) cell eulturists for the nutrient supplementation of both serum-free and serumcontaining {5j cell cultures. Liposomes promote the morphologic differentiation of murine neuroblastoma cells {2) and various unusual insect cell types that form tissuelike arrays in culture (5,6). When mechanically dissociated embryonic tissues were used as primary culturing material, some previously uncultivable and at least partially differentiated insect cell types {including tracheolar duct, muscle, ducted glandlike, and some unusual epithelioid cell types) were isolated and subcultured in serum-containing liposome-supplemented media {5,6). Subculturing these special cell types also required a modified growth surface (Primaria: Falcon Plasticsq {5). Although vertebrate cell culture systems have utilized serum proteins to ensure the nutrient activity of supplementing liposomes in serum-free media (10,11), similar lipid supplemented insect cell culture systems have allowed continuous cell passagiug with bacteriologic-grade peptones in lieu of proteins to obtain nutrient lipid activity {4-7). It is important that nutrient liposomes be based on phosphatidyl choline, as other phospholipids and small unilamellar vesicles {SUVs) have shown various levels of toxicity to cells in culture {12). This suggests that the present liposome con-

Journal of Tissue Culture Methods

Vol. 12, No. 1, 1989

II.

MATERIALS A. Equipment Vertical laminar-flow hood, Biogard {exhaust port must be vented to building exterior to discharge chloroform vapors), Baker Company2 Waterbath, adjustable, Hot Pot, 36 oz. {or scientific source rotary evaporator with appropriately constructed connections and adjustable waterbath), West Bend 3 Dessicator jar, Pyrex glass, 3100 {for storage of lipid ingredients under nitrogen gas to prevent oxidation), Coming 1 Nitrogen gas, compressed air tank, Liquid Air Corp." Nitrogen gas, pressure regulator {with attached needle valve for slow gas metering), Victor Equipments

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©1989 Tissue Culture Association, Inc.

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Laboratory pipetting cannula, 14 gauge )< 4 in. in length, BD 1789, Becton-Dickinson6 Hose end tubing connector, female LUER-LOK tip to hose end, BD 9040~ Cuphorn sonicator 431B, with Sonicator Cell Disrupter generator W385, 475 watts, Heat SystemsUltrasonics 7 B. Supplies Syringe filters, nylon, 0.2-pm pore size, or other chloroform compatible types such as no. 195-2020 (for filtering nitrogen gas source and chloroformcontaining lipids), Nalgene 1 Pyrex glass beads, 3 ram, 7268 (washed with tissue culture detergent), Coming ~ Erlenmeyer flasks, screw capped, FEP-Teflon, 50 ml, 4106-0050 (rinsed before use with ultrograde chloroform), Nalgene l C. Chemicals Chloroform-d1, 99.8%, DC1080, (purified reagent grade: keep tightly capped and refrigerated to prevent phosgene formation), NorelP Cholesterol, C8253, chromatography standard grade, Sigma9 f3-Sitosterol (60%t $5753, recrystallized (Recrystallization of 3-Sitosterol product to delete toxic materials described previously by Svoboda, et al. (20): three serial recrystallizatious using absolute ethanol and filtration for crystallized product recovery. )9 Ia-Lecithin, soybean lecithin, 95%, 441601, Avanti 1° La-Lecithin, egg lecithin, >95% 1316011° l~-Leeithin, in chloroform, 20:4 diarachidonoyl, synthetic >99%, 8503971° I~-Lecithin, in chloroform, 18:3 dilinolenoyl, synthetic >99%, 850395 '0 Dl-a-Tocopherol acetate, T-33769 Tryptose, L47, Oxoid 1~ Peptic peptone, 20040, U.S. BiochemicaP 2

III. PROCEDURE A. Lipid chloroform concentrate solution, for PEGSAL2 (5); use a vertical laminar-flow hood. 1. Dissolve the following ingredients in chloroform-d1, bringing the total volume up to 20 ml chloroform sitosterol . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 mg cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . 25 mg soybean lecithin . . . . . . . . . . . . . . . . . . . . . 100 mg egg lecithin . . . . . . . . . . . . . . . . . . . . . . . . . 100 mg diarachidonyl lecithin . . . . . . . . . . . . . . . . . . 25 mg dilinolenoyl lecithin . . . . . . . . . . . . . . . . . . . . 50rag alpha tocopherol acetate . . . . . . . . . . . . . . 1.75 mg

2. Sterile filter the dissolved lipids through a nylon syringe filter (0.2 ~m), by nitrogen pressure filtration, into a sterile glass media bottle (100 ml). Note: Other chloroform and lipid soluble experimental factors may be advantageously incorporated at this point before the sterilizing filtration of this solvent phase (see Discussion). 18

3. Pipette 4 ml of the filtered material into each of four (autoclave-sterilized and dried) FEP-Teflon screwcapped flasks (50 ml) that contain a layer of washed 3-mm Pyrex glass beads (approximately 275 beads, or 7 ce of beads per flask). There must be no water droplets in the flasks when the chloroform-lipid mixture is added. The remaining, partly evaporated, chloroform-lipid mixture may be later pooled with freshly dissolved lipids, or discarded. B. Preparation of dried lipid concentrate (chloroform evaporation) I. Within the laminar floow hood, gently swirl and rotate by hand each Teflon flask containing the chloroform-lipid mixture in a slow stream (approximately 0.5 Ib gas pressure} of sterile-filtered nitrogen gas to rapidly and uniformly evaporate the chloroform solvent from the lipid mixture. [Use the laboratory pipetting cannula and hose end tubing connector to connect the nylon syringe (or other 0.2-/~m pore sized) gas filter to the nitrogen gas source. Mount the gas line filter rigidly into a clamp attached to a ring stand at a suitable angle so that the chloroform-containing flasks can be conveniently brought up to the cannula for sparging.] During the nitrogen sparging, the flasks are at the same time (or intermittently) immersed in a (60° C) water bath (precalibrated 36 oz. Hot Pot or similar) to aid the chloroform evaporation process. Insert the sterile cannula well into the flask during the sparging process. A preferable but more costly technique is to adapt a small scientific source rotary evaporator (with water bath) so that the Teflon flasks can be screwed into an autoclave-sterillzed glass tube, mounted in the evaporator jaws, and continuously rotated while immersed in the water bath. In this case the nitrogen gas pipetting cannula is inserted through the sterile glass tube connector into the flask for the evaporation process. After the chloroform is evaporated, the lipid mixture is left as a coating on the inner lower walls and bottom of the flask and on the glass beads. The chloroform removal is complete when the glass beads stick together and will not move when the flask is tilted. 2. These lipid concentrates may be stored under nitrogen gas by tightly capping and elastic taping each, then placing them into a glass desiccator jar that has a (stoppered) top access hole through which a nitrogen gas tube can be inserted for sparging and replacing the air with nitrogen gas. The desiccator jar is then kept in a chest freeezer for temporary lipid storage (Teflon flasks are not necessary if the liplds are not to be stored frozen). C. Preparation of the peptone-liposome concentrate I. Pipette 20 ml of peptone stock (tryptose 1 g, plus peptic peptone 1 g, dissolved in 500 ml of reverseosmosis water and autoclaved for sterilization) into each flask of lipid concentrate to give the final concentrations as listed in Table 1. Journal of TissueCultureMethods Vol.12, No. I, 1989

GOODWIN TABLE 1 PEPTONE-LIPOSOME CONCENTRATE COMPOSITION: PEGSAL2 (5)~ Ingredients

Source

rag/20ml

Lipids Cholesterol /3-Sitosterol Soybean lecithin Egg lecithin Diarachidonoyi lecithin Dilinolenoyl lecithin tt-Tocopherol acetate

Sigma Ch-S: C8253 Sigma $5753, recrystallized Avanti 441601 Avanti 131601 Avanti 850397 Avanti 850395 Sigma T-3376

5.0 5.0 20.0 20.0 5.0 10.0 0.35

Peptones: (dissolved together in 20 ml water and autoclaved for sterilization separately from the above ingredients) Tryptose Peptic Peptone

Oxoid L-47 U.S. Biochem. Co. 20040

40.0 40.0

"Aqueous peptone-based contents of each 20 ml teflon flask when complete. 2. Sonicate each flask for 20 min by insertion of the capped Teflon flask (held rigidly by a flask-andbottle clamp on a ring stand) into the water-filled sonicator cup as close to the horn face as is possible without direct contact with the cold water circulation system ON during sonication. The sonicator is operated at a control setting of 7, 60% duty cycle, pulsed mode, for the 20-min sonication period. (Note: These settings apply to the 300 W sonicator generator model W375, which is no longer available). 3. Distribute the nutrient peptoliposomal mixture into nutrient culture medium according to Table 2 to obtain the listed concentrations of the selected lipid preparations (5L Excess peptone-liposome concentrate may be sealed with stretch tape and stored in the freezer desiccator under nitrogen with other lipid preparations, but care should be taken not to exceed the 1-3-too. shelf life maximum for phosphatidyl cholines containing polyunsaturated fatty acids with chain lengths and unsaturations greater than 18:2 (see Avanti catalog for additional information}. IV.

demonstrated that nutrient peptoliposomesupplemented, serum-free media could support the complete replication of both a baculovirus (polyhedrosis) (4) and an entomopoxvirus (7) in a gypsy moth (Lymantria dispar) cell line. The same cell line grown in similar serum-free media without lipids or with lipid-Tween emulsions supported no replication or only partial replication, respectively, of the same baculovirus. These data suggest that certain of the more complex D N A viruses have specific lipid requirements for their replication. Numerous correlations between phospholipid metabolism and a) cell division, b) mitogenic stimulation, c) hormone responses, d) hormone production, e) cell transformation, and f) various tissuespecific functions have been noted in prior research (9,16,17). Although supplementation of the medium with specific fatty acids, usually bound to serum albumin or delipidized serum, is an accepted method for producing lipid modifications in the membranes of cultured mammalian, flbroblast, and epithelial cells, such additions generally inhibit cell growth and adversely affect cell survival. These negative effects have been most pronounced in response to the longer chain polyunsaturated fatty acids (16,17). On the other hand, some earlier (9) and other very recent investigations have linked normal cell growth, division, and cell function with the occurrence of certain polyunsaturated fatty acids in the phospholipids of both vertebrate 18)

DISCUSSION As previously reported, mechanically dissociated posthlastoklnetic grasshopper embryos of various species have given rise to several partially differentiated cell types when grown in certain peptoliposomesupplemented media; some of these cells are subcultivable in vitro (5,6L Of the several lipid combinations first reported from this work, the combination detailed in the present procedure (PEGSAL2) resulted in the longest survival in vitro of the widest array of cell types, even in the presence of a 15% fetal bovine serum supplementation (5). Subsequent work has suggested that fl-sitosterol and high serum levels are contraindicated for several grasshopper species including Melanoplus sanguinipes (6). Earlier virus studies

Journal of Tissue CultureMethods Vol.12, No. I, 1989

TABLE 2 ADDITIONS OF PEPTONE-LIPOSOME CONCENTRATES FOR VARIOUS LIPID USAGE AMOUNTS TO THE MEDIUM Medium Volumes,ml. 5 10

ConcentrateVolumesforListedLevels,mL(5) 3X 5X 7X 0.188 0.375

0.313 0.625

0.438 0.875 19

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and plant (14) cell membranes. I believe that, although the potential for liposomes in the study of cellular lipid metabolism has been recognized (16), their potential in the nutritional role has not been as generally evident, because the majority of cell culture lipid studies were based on albumin-bound free fatty acids, which have not characteristically promoted cell survival (17L Liposomes have been more generally used as a means of delivery for pharmacologic agents in vivo, or in more basic biochemical studies, again in an agent-delivery role for DNA, viruses, macromolecules, hormones and the like by encapsulation (13). The present liposome technique could also be useful in the delivery of hydropbobie or poorly soluble factors such as the juvenile hormones or beta-carotene through their incorporation into the lipid vesicle bilayer (via the chloroform phase before filtration and sonieationk Such factors, being both chloroform and lipid soluble, may be more readily protected and delivered to the cell after incorporation into the pbospholipid bilayer of the liposome rather than within the enclosed aqueous space {i.e. by encapsulation). The insertion of polyunsaturated (often essential) fatty acids as peptoliposome phospholipids into the membranes o( living cells in culture may in fact mimic the transport of lipids by naturally occurring interorganelle lipid transport vesicles (2h489-490). Although fatty acids in the cellular triglycerides are more likely- to be metabolized for energy, some fatty acids in the membrane phospholipids are more often conserved and retained for more critical structural and physiologic cellular functions in vivo (19L Inasmuch as the majority of the essential fatty acids occur in these membrane phospholipid locations in vivo and are conserved there during starvation {18L they have been provided to cultured cells in the same form, as nutrient peptoliposomes, to ensure their maximum utilization in essential roles in vitro. V.

REFERENCES

1. Bettger, W. J.; Boyce, S. T.; Walthall, B. J., et al. Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexametlmsone. Proc. Natl. Acad. Sci. USA 78:5588-5592; 1981. 2. Chen, J. S.; Del Fa, A.; Di Luzio, A., et al. Liposome-indueed morphological differentiation of murine neuroblastoma. Nature 263:604-606; 1976.

i VWR Scientific,Seattle,WA ZBaker Company, Inc., Sanford, ME 3West Bend Co., West Bend, WI "Liquid Air Corp., Walnut Creek, CA sVictor Equipment Co., Mars, PA 6Harris Hospital Supply, Broadview, IL

3. Cheesebeauf, M.; Padieu, P. Rat liver epithelial cell cultures in a serumfree medium: primary cultures and derived cell lines expressing differentiated functions. In Vitro 20:780-795; 1984. 4. Goodwin, R. H. Growth of insect cells in serum-free medium. In: Techniques in the lifesciences, cell biology. VoL CI, Techniques in setting up and maintenance of tissue and cell cultures. County Claire, Ireland: Elsevier Scientific Publishers Ireland Ltd.; 1985:separate C109, 28 pp. 5. Goodwin, R. H. The effect of lipids on the subculture of differentiated cells from primary cultures of grasshopper embryonic tissues. In Vitro 24:388-400; 1988. 6. Goodwin, R. H. The culture of differentiated ceils from insect embryonic tissues: the effect of peptoliposomal versus serum supplementation. In Vitro 24(3:Part 2k13A; 1988. 7. Goodwin, R. H.; Adams, J. R. Serum-free replication of the entomopoxvirus from Amsacta moorei in a gypsy moth cell line. J. Invertebr. Pathol. (In Pressk 1989. 8. Goppelt-Strnbe, M.; Resch, K. Polyunsaturated fatty acids are enriched in the plasma membranes of mitogen-sfimulatedT-lymphocytes. Biochem. Biophys. Acta 904:22-28; 1987. 9. Howard, B. V.; Howard, W. J. Lipid metabolism in cultured cells. Adv. Lipid Res. 12:51-96; 1974. 10. Iscove, N. N.; Melchers, F. Complete replacement of serum by albumin, transferrin and soybean lipid in cultures of lipopolysaccharide-reactive B-lymphocytes. J. Exp. Med. 147:923-933; 1978. 11. Iscove, N. N. Guilbert, L. J.; Weyman, C. Complete replacement of serum in primary cultures of erythropoietin-dependent red cell precursors (CFU-E) by albumin, transferrin, iron, unsaturated fatty acid, lecithin, and cholesterol. Exp. Cell Res. 126:121-126; 1980. 12. Mayhew, E. Toxicity of non-drug-containingliposomes for cultured human cells. Exp. Cell Res. 171:195-202; 1987. 13. Ostro, M. J., ed. Liposomes. New York: Marcel Dekker, Inc.; 1983. 14. Passaquet, C.; Teodorescu-Ionescu, N.; Zuilly-Fadil, Y., et al. Changes in fatty acid and lipid content in callus and protoplasts of Parthenocissus tricuspidata and Petunia hybrida during culture. Physiol. Plant. 67:211-216; 1986. t 5. Pokorny, J. Major factors affecting the autoxidation of lipids. In: Chan, H. W.-S., ed. Autexldation of unsaturated lipids. London: Academic Press; 1987:141-206. 16. Spector, A. A.; Mathur, S. N.; Kaduce, T. L., et al. Lipid nutrition and metabolism of cultured mammalian cells.Prog. Lipid Res. 19:155-186; 1981. 17. Spector, A. A.; Yorek, M. A. Membrane lipid composition and cellular function. J. Lipid Res. 26:1015-1035; 1985. 18. Stanley-Samuelson, D. W.; Dadd, R. H. Long.chain polyunsaturated fatty acids: patterns of occurrence in insects. Insect Biochem. 13:54%558; 1983. 19. Stanley-Samuelson, D. W.; Rapport, E. W.; Dadd, R. H. Effects of dietary polyunsaturated fatty acids on tissue monounsaturate and saturate proportions in two insect species. Comp. Biochem. Physiol. 81B:749-754; 1985. 20. Svoboda, J. A.; Thompson, M. J.; Robbins, W. E. 3-~-Hydroxy-24norchol-5-en-23-oic acid, a new inhibitor d the AZ4-sterol reductase enzyme system in the tobacco hornworm. Steroids 12:559-570; 1968. 21. Voelker, D. R. Lipid assembly into cell membranes. In: Vance, D. E.; Vance, J. E., eds. Biochemistry of lipMs and membranes. Menlo Park, CA: The Benjamin/Cummings Publishing Co.; 1985:475-502.

'Heat Systems-UltransonicsInc., Farmingdale, NY SNorell, Inc., Landisville, NJ 'Sigma Chemical Co., St. Louis, MO '°Avanti Polar Lipids, Inc., Pelham, AL "Hazleton Biologics, Lenexa, KS ~2United States Biochemical Corp., Cleveland, OH

I thank Dr. Spiro J. Louloudes for his introduction to me of liposomes and their construction at a time when their use in these applications was only a remote possibility.

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Journalof Tissue Culture Methods Vol. 12, No. 1, 1989