THE UPTAKE OF VITAMINS BY MOUSE FIBROBLAST CELLS (STRAIN LS) DURING GROWTH IN A CHEMICALLY DEFINED MEDIUM

Transcription

1 J. Cell Sri. 8, (1971) 701 Printed in Great Britain THE UPTAKE OF VITAMINS BY MOUSE FIBROBLAST CELLS (STRAIN LS) DURING GROWTH IN A CHEMICALLY DEFINED MEDIUM G.J.BLAKER AND S.J.PIRT Department of Microbiology, Queen Elizabeth College (University of London), Campden Hill, London W. 8, England SUMMARY The uptake of biotin, choline, folic acid, hypoxanthine, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin, thiamine and vitamin B,, by mouse LS cells in suspension culture was determined by microbiological assay methods. Based on the extent of uptake during cell growth, vitamin growth yields (cells produced/unit mass of vitamin utilized) were estimated for all of the vitamins, except folic acid, thiamine and B 12. The growth yields were lower during the early phases of culture. No uptakes of folic acid or B H could be demonstrated. During the period of incubation about half of the thiamine was irretrievably lost through spontaneous decomposition. INTRODUCTION Whilst the qualitative vitamin requirements of a number of mammalian cell lines have been established, a knowledge of the quantitative requirements, expressed as growth yields (the ratio of the amount of cell mass produced to nutrient utilized), and the influence of vitamin concentration on. cell growth rate, is almost entirely lacking. The growth yields are of fundamental importance to the understanding of metabolism and function in normal and abnormal growth, and to supplement studies on the whole animal. As a consequence of this lack of quantitative data, vitamins are added to most cell-culture media at arbitrary concentrations. Eagle (1955) showed qualitative requirements for choline, folic acid, nicotinamide, pantothenic acid, pyridoxal, riboflavin and thiamine for mouse L cell proliferation in a medium containing 1 % (v/v)dialysed horse serum. Haggerty & Sato (1969) demonstrated a qualitative requirement for biotin for the growth of L cells in a chemically denned medium. Although Higuchi (1970) found some evidence for a B 12 requirement by L cells, an absolute requirement has not been conclusively demonstrated. L cells are capable of growing in the absence of added inositol (Eagle, Oyama, Levy & Freeman, 1956; Sanford, Dupree & Covalesky, 1963), and can synthesize all their inositol from glucose (Eagle, Agranoff & Snell, i960). L cells do not require added hypoxanthine. The problem of vitamin stability in both inoculated and uninoculated cell culture media during incubation has received little attention, though certain vitamins were studied by Mohberg & Johnson (1963) in an investigation of possible growth limitation by these vitamins in MB 752/1 medium (Waymouth, 1959). 45 CEL8

2 702 G. J. Blaker and S. J. Pirt In the present study, the uptake of vitamins by LS cells growing in a chemically defined medium was followed using microbiological assay methods. Based on these determinations, vitamin growth yields were estimated, and the stability of the vitamins during cell culture investigated. MATERIALS AND METHODS Cell line The mouse LS cell line has been described elsewhere (Griffiths & Pirt, 1967). Routine tests, previously described (Birch & Pirt, 1969) failed to show any evidence of mycoplasma infection. Medium The defined medium was based on that of Birch & Pirt (1970). Preliminary determinations of the vitamin uptake during LS cell growth in this medium (medium A) showed that certain vitamins were completely exhausted, whilst others were not utilized to a significant extent. The modified vitamin mixture of medium B, shown in Table i, was employed in our subsequent studies. Penicillin was the only antibiotic added to experimental cultures. Table 1. Vitamin composition of culture media A and B Vitamin mg/1. in medium A mg/1. in medium B d-biotin Choline chloride Folic acid Hypoxanthine i-inositol Nicotinamide Calcium-d -pantothenate Pyridoxine hydrochloride Riboflavin Thiamine hydrochloride Vitamin Bi. o-i 2O-O* i 2-o O-2 2-O 003 2O-O* o-isf 30-oJ o-c; Added as a 100 x aqueous solution. Stored at 4 C. f Added as a 1000 x solution, prepared by the addition, of 001 N NaOH to an aqueous suspension to give a folic acid solution at ph 7-8. Stored at 20 C. \ Added as a 100 x solution, prepared by dissolving hypoxanthine in distilled water with heating and vigorous stirring. Stored at 4 C. Added as a 100 x solution, prepared by dissolving riboflavin in oi N acetic acid, heated on a steam bath. Stored at 20 C. The remaining vitamins for the preparation of media A and B were kept as dried powders, and prepared for addition by dissolving the amount required for 1 1. of medium in 20 ml of 006 N NaOH. Culture procedure Static suspension cultures were grown as described by Birch & Pirt (1969). Agitated cultures were grown in a 5-I. spinner vessel containing 2 1. of medium, continuously gassed with 5 % ( v / v ) CO 2 in air at a rate of 10 ml/min. An uninoculated control culture, in an identical vessel, was gassed in the same way to determine evaporation and any vitamin decomposition during incubation. Evaporation from the culture vessel was estimated as 2 ml per day.

3 Measurement of cell growth Vitamin uptake by LS cells 703 Cell counts were made in a haemocytometer, using trypan blue dye exclusion to distinguish viable cells. Cell size determinations were made using a model A Coulter Counter fitted with a ioo-/tm aperture tube. Phosphate-buffered saline containing 05 % (w/v) methylcellulose was employed as the electrolyte. Cell dry weights were determined by the method of Birch & Pirt (1971). The ratio of cell dry weight to cell number was determined for a population of known mean cell volume. Corrections were made to this ratio for changes in cell volume, in calculating cell dry weight values for all cell counts. Preparation of samples for assay Cells were removed from culture samples by centrifugation (at 150 g for 5 min, followed by 250 g for 10 min) and, after gassing the samples with 5 % (v/v) CO, in air, they were stored in foil-covered bottles at 20 C. Penicillin was destroyed in the samples before bacterial assay using the penicillinase method of Griffiths & Pirt (1967). Penicillinase treatment of the assay samples did not significantly increase their vitamin content. Interfering methionine was removed from choline assay samples using Permutit columns as described by Horowitz & Beadle (1943). A 90 % (w/w) recovery of choline from the columns was obtained. Samples for the assay of pyridoxine, riboflavin and vitamin B 12 were handled under reduced lighting conditions to minimize light-induced degradation. Vitamin assay Vitamins were determined by microbiological assay. Prepared dehydrated media (Difco) were employed for all assays except riboflavin and hypoxanthine. The medium of Kornberg, Langdon & Cheldelin (1948) was used to assay riboflavin, and the basal medium of Mitchell & Houlahan (1946) was used to assay hypoxanthine. The assay organisms are described in Table 2. In the assays, bacterial and yeast populations were determined turbidimetrically in a spectrophotometer at 620 nm; Neurospora was determined by mycelial dry weight. Table 2. Microbiological vitamin assay organisms Organism Collection number Vitamins assayed Lactobacillus arabinosus 17 J5 ATCC 8014* Biotin, pantothenic acid, nicotinamide Lactobacillus fermenti A TCC 9338 Thiamine Lactobacillus leiclimannii ATCC 7830 B 12 Streptococcus faecalis ATCC 8043 Folic acid Streptococcus faecalis var. liquefaciens ATCC Riboflavin Saccliaromyces carlsbergensis ATCC 9080 Inositol, pyridoxine Neurospora crassa ATCC 9277 Choline Neurospora crassa FGSC isf Hypoxanthine American Type Culture Collection, f Fungal Genetic Stock Centre. RESULTS Method of vitamin addition The method of adding vitamins in the preparation of medium A resulted in loss of folic acid, hypoxanthine and riboflavin because of vitamin instability or low solubility in cold alkaline solutions. As shown in Table 3, the modified method of addition, used 45-2

4 704 G. J. Blaker and S. J. Pirt Table 3. The effect of the method of vitamin addition in the preparation of medium A Vitamin Expected level, mg/1. Alkaline addition,* mg/1. Modified addition,'}' mg/1. Folic acid Hypoxanthine Riboflavin 10 i S-6 o-i I'O IO'I O'lO Addition in 0-06 N NaOH. f As routinely employed in the preparation of medium B fdo JT 00 ID a E D u 50 s cs z: Incubation time (h) Incubation time (h) Fig. 1 Fig. 1. Growth of LS cells in agitated suspension culture. O 0 #, viable cell count. Fig. 2. Change in mean cell volume during culture. Fig. 2 O, Cell dry weight; in medium B preparation, overcame these problems. The vitamin stock solutions were stable under the storage conditions described for periods in excess of 3 months. Cell growth The growth of the LS cell culture from which samples were removed for vitamin assay is shown in Fig. 1. Growth was logarithmic from 1 to over 20 x io 5 cells/ml, with a doubling time of 38 h. During the culture, the mean cell volume changed as shown in Fig. 2. This sequence of changes in cell size is similar to that reported by Merchant, Kuchler & Munyon (i960), though their volume changes were greater. Estimated cell dry

5 Vitamin uptake by LS cells 705 weights, corrected for volume changes during culture were within the range of /tg per io 6 cells. This value compares closely with L-cell dry weights obtained by other authors (Westfall, Evans, Shannon & Earle, 1954; Birch & Pitt, 1971). Extent of vitamin uptake during cell growth The stability of the medium vitamins and the extent of their utilization during culture are shown in Table 4. No decrease in medium content, either as a result of cell culture or incubation could be shown for folic acid or vitamin B 12. The slight instability of pyridoxine and riboflavin possibly represents their light-induced degradation during incubation. Based on the amount of vitamin uptake by the cells during Table 4. Extent of vitamin uptake and vitamin growth yields during cell culture in medium B Vitamin Biotin Choline Folic acid Hypoxanthine Inositol Nicotinamide Pantothenate Pyridoxine Riboflavin Thiamine B la The growth yield itilized (g). % (w/w) decrease 1 ULUUcltCU control i 'i A. 11LCU UalCU culture o h 3-92 x io x io OI X IO 4 Growth yield* \ h 1-53 x io 6 45-o X IO x io 4 is the ratio of the amount of cell dry weight produced (g) to substrate culture, growth yields were estimated for 8 vitamins. As shown in Fig. 3, the growth yields were lower during the early phases of culture. This decrease in the extent of uptake per cell during culture has also been reported for both amino acid (Griffiths & Pirt, 1967) and glucose utilization (Munyon & Merchant, 1959) by L cells. The growth yields are given by the slopes of the graphs in Fig. 3. During culture, there appeared to be at least 2 distinct growth yields for each vitamin. In most cases, the growth yield during the later phase of culture was twice that obtained during the initial phase (Table 4). The decrease in the medium thiamine content during incubation is shown in Fig. 4. Mohberg & Johnson (1963) reported that during incubation of Waymouth's MB 752/1 medium, thiamine was rapidly oxidized to thiamine disulphide, and that L cells could reduce the disulphide back to native thiamine. The experiments here show that about 50% of the thiamine was lost, and could not be reduced back to thiamine. In the biological assay system used in the present study it was found that both thiamine and

6 706 G. J. Blaker and S. J. Pirt thiamine disulphide were growth stimulatory ( mol of thiamine disulphide was equivalent to 1-95 mol of thiamine hydrochloride). Thus, the extensive loss of thiamine during incubation must represent formation of biologically inactive thiamine derivatives, other than the disulphide. The instability of thiamine precluded an estimation of a thiamine growth yield O Biotin Pantothenate () C) CK 750 Inositol / P Riboflavin p U J / ) () Chollne Hypoxanthine JA* > X y O Decrease in vitamin content (ng/ml) of medium Fig. 3. Vitamin uptake during cell growth. DISCUSSION The results show the amounts of vitamins taken up by LS cells from a denned culture medium when the vitamins are supplied in excess. The growth yields should be regarded as the minimum values, since if the vitamins are supplied in excess, they could be accumulated as intracellular vitamin reserves; such 'storage' could suffice for continued cell multiplication in a medium lacking a particular vitamin, as described by Eagle (1955). One would expect to obtain the maximum growth yield only when growth is limited by the vitamin supply. A comparison of choline growth yields, when

7 Vitamin uptake by LS cells Incubation time (h) Fig. 4. The effect of incubation on the thiamine content of medium B. O O, Control;, culture. it is growth-limiting and when it is supplied in excess, shows such a difference. Under conditions of growth limitation by choline, Birch &Pirt (1969) found a choline growth yield for LS cells of approximately 250/ig dry weight/^g choline chloride. In the present study, with excess choline, the growth yield was /^ dry weight//ig choline chloride. This difference in growth yield could represent (a) less efficient utilization under conditions of choline excess, and/or (b)' storage' of choline, possibly in the form of phosphorylcholine. Although several vitamins approach exhaustion from the medium after LS cell growth, vitamin 'storage' by the cell precludes a determination of their growthlimiting levels. There was an excess of all vitamins in medium B. Even on the basis of minimum growth yields, the medium concentrations of folic acid and B 12 could be reduced to at least 20% (w/w) of the levels present in medium B. Since L cells do not require added hypoxanthine, the extensive uptake during growth might result in a sparing effect on a precursor of purine metabolism. Similarly, perhaps, inositol taken up by LS cells during growth may exert a sparing effect on glucose metabolism, as demonstrated by Eagle et al. (i960). The stability of the vitamins in the medium during incubation was in agreement with the observations of Mohberg & Johnson (1968); some 60% (w/w) of the medium thiamine was degraded during incubation to unidentified, biologically inactive products. This work was supported by a grant from the Cancer Research Campaign (British Empire Cancer Campaign).

8 708 G. J. Blaker and S. J. Pirt REFERENCES BIRCH, J. R. & PIRT, S. J. (1969). The choline and serum protein requirements of mouse fibroblast cells (strain LS) in culture. J. Cell Sci. 5, BIRCH, J. R. & PIRT, S. J. (1970). Improvements in a chemically denned medium for the growth of mouse cells (strain LS) in suspension. J. Cell Sci. 7, BIRCH, J. R. &PIRT, S. J. (1971). The quantitative glucose and mineral nutrient requirements of mouse LS (suspension) cells in chemically defined medium. J. Cell Sci. 8, EAGLE, H. (1955). The minimum vitamin requirements of the L and HeLa cells in tissue culture, the production of specific vitamin deficiencies, and their cure. J. exp. Med. 102, EAGLE, H., AGRANOEF, B. W. & SNELL, E. E. (i960). The biosynthesis of meso-inositol by cultured mammalian cells and the parabiotic growth of inositol-dependent and inositolindependent strains. J. biol. Chem. 235, EAGLE, H., OYAMA, V. I., LEVY, M. & FREEMAN, A. (1956). Myo-inositol as an essential growth factor for normal and malignant human cells in tissue culture. Science, N. Y. 123, GRIFFITHS, J. B. & PIRT, S. J. (1967). The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate. Proc. R. Soc. B 168, HAGGERTY, D. F. & SATO, G. H. (1969). The requirement for biotin of mouse fibroblast L cells cultured on serumless medium. Biochem. biophys. Res. Comrnun. 34, HIGUCHI, K. (1970). The requirements of cultured mammalian cells for B ls and biotin. Fedn Proc. Fedn Am. Socs exp. Biol. 28, HOROWITZ, N. H. & BEADLE, G. W. (1943). A microbiological method for the determination of choline by use of a mutant of Neurospora. J. biol. Chem. 150, KORNBERG, H. A., LANGDON, R. S. & CHELDELIN, V. H. (1948). Microbiological assay for riboflavin. Analyt. Chem. 20, MERCHANT, D. J., KUCHLER, R. J. & MUNYON, W. H. (i960). Population dynamics in suspension cultures of an animal cell strain. J. biochem. microbiol. Technol. Engng 2, MITCHELL, H. K. & HOULAHAN, M. B. (1946). Adenine-requiring mutants of Neurospora crassa. Fedn Proc. Fedn Am. Socs exp. Biol. 5, MOHBERG, J. & JOHNSON, M. J. (1963). Stability of vitamins in a chemically defined medium for 929-L fibroblasts. J. natn. Cancer Inst. 31, MUNYON, W. H. & MERCHANT, D. J. (1959). The relation between glucose utilization, lactic acid production and utilization and the growth cycle of L strain fibroblasts. Expl Cell Res SANFORD, K. K., DUPREE, L. T. & COVALESKY, A. B. (1963). Biotin, B 1S and other vitamin requirements of a strain of mammalian cells grown in a chemically defined medium. Expl Cell Res. 31, 345~37S- WAYMOUTH, C. (1959). Rapid proliferation of sublines of NCTC clone g2g (strain L) mouse cells in a simple chemically defined medium (MB 752/1). J. natn. Cancer Inst. 22, WESTFALL, B. B., EVANS, V. J., SHANNON, J. E. & EARLE, W. R. (1954). The glycogen content of cell suspensions prepared from massive tissue culture: comparison of cells derived from mouse connective tissue and mouse liver. J. natn. Cancer Inst. 14, (Received 19 September 1970)