Growth and differentiation of Friend erythroleukemia cells in serum-free culture

LeukemtoRest~rch Vol. 7, No. 5, pp. 591-$97, 1983.

0143-2126/83 $3.00+ 0.O0 ¢) 19113Perpmon PressLtd.

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GROWTH AND DIFFERENTIATION OF FRIEND ERYTHROLEUKEMIA CELLS IN SERUM-FREE CULTURE CRISTINA JEMMA and GIOVANNIBATTISTAROSSI Laboratorio di Virologia, lstituto Superiore di SanitY, Rome and Gruppo di Microbiologia e Patologia Generale, Universit~ degli studi di Roma, Rome, Italy

(Received 17 January 1983. Revised 18 April 1983. Accepted 19April 1983) Ahtraet--A synthetic medium allowing indefinite optimal growth of Friend erythroleukemia cells (FLC) is described. It consists of lscove's modified Dulbecco's medium supplemented with bovine serum albumin, transferrin, and a lipid mixture. Transferrin and lipids are essential for Friend cells growth. Under these conditions, FLC erythroid differentiation, promoted by a number of inducers, is less efficient than in cultures with serum-rich medium, suggesting that unknown serum factors may play an additional role in this phenomenon. Conversely, the enhancement of erythroid differentiation induced by low doses of Interferon is superimposable in both types of cultures.

Key words: Friend leukemia cells, serum-free medium, differentiation.

INTRODUCTION In vitro GROWTHof eukaryotic cells requires addition of serum to a nutrient medium con-

taining salts, sugars, amino acids and vitamins. Serum is a mixture of various proteins, which include albumin and transferrin, as well as growth factors and hormones [7]. Among the latter components some are still poorly characterized, others are presumably unidentified as yet. Furthermore, their concentration may widely vary among different batches of serum. It is, thus, apparent that presence of serum in culture medium obscures both methodology and interpretation. Different approaches have been attempted to eliminate serum requirement in culture, including purification of its active components [11] and development of serum-free culture systems. In this last regard, Barnes and Sato have set up different synthetic media for a number of cell lines (HeLa, SV40-3T3, TM4, etc.) [3, 4] whereby serum is replaced by a mixture of hormones modulating cell proliferation. With respect to hemopoietic cells, Breitman et al. [6] have established a synthetic medium, consisting of RPMI 1640 supplemented with insulin, trar~ferrin and selenium, which promotes growth of a human promyelocytic line. More important, Iscove et aL [12, 13] have developed a serum-free system allowing growth of normal lyrnphoeytes and hemopoietie progenitors in semisolid culture. Serum is replaced by deionized and delipidated albumin (BSA), selenium present in IMDM, iron-saturated transferrin and a lipid mixture. BSA may act in vitro as a detoxifying agent and a buffer system; more important, it functions as a carrier for lipids, metals and hormones, thus rendering them

Abbreviations: A.A.S., atomic absorption spectrophotometry; B +, benzidine-positive; BSA, bovine serum albumin; BSA/L ÷, undelipidated; BSA/L--, delipidated; DM$O, dimethylsulfoxide; FC$, fetal calf serum; FLC, Friend leukemia cells; FL I/, Friend leukemia virus; HMBA, hexamethylenebisacetamide;IFN, Interferon; IMDM, lscove's modified Dulbecco's medium; L, liptds; RT, reverse transcriptase; T, transferrin. Corr~pondencc to: G. B. Rossi, Laboratorio di Virologia, Istituto Superlore di Sanitt, 299 viale Regina Elena, 00161 Rome, Italy. 591

592

CRISTINAJEMMAand GIOVANNIBATTISTARossl

available to cells [2]. Transferrin (T), the major iron-carrying serum protein, (normal concentration 3 - 4 m g / m l ) is essential for in vitro cell growth, in that it supplies red cell precursors with Fe for h e m o g l o b i n and c y t o c h r o m e synthesis [12]. T h e role o f lipids (L) in culture is not yet clarified, a l t h o u g h it has long been recognized as essential. Their g r o w t h s u p p o r t i n g action in serum-free m e d i u m m a y be related to their role in m e m b r a n e renewal

[13]. we have recently developed a serum-free system based on that established by Iscove, which allows growth a n d differentiation o f Friend e r y t h r o l e u k e m i a cells (FLC). These cell lines, established by Friend [9] f r o m leukemic livers or spleens o f mice inoculated with the Friend leukemia virus (FLV) complex, g r o w in suspension, are chronically infected by FLV, and c o n t i n u o u s l y shed viral particles. F L C are erythroid precursors blocked in their differentiation p a t h w a y at approximately the p r o e r y t h r o b l a s t stage. T h e addition o f various agents (i.e. dimethylsulfoxide ( D M S O ) , hexamethylenebisacetamide ( H M B A ) , short chain fatty-acids, etc.) causes a massive shift o f F L C towards terminal erythtoid differentiation [16]. T r e a t m e n t with hemin also increases the percentage o f differentiated cells, but does n o t trigger terminal erythroid differentiation [17]. Hemoglobin-producing cells can be assayed b y benzidine staining [14], and are designated as benzidine-positive

(B+). MATERIALS AND METHODS Cells Friend erythroleukemia cells (FLC), clone 745A, grown in serum-supplemented or in serum-free medium, were seeded every 3-4 days at 5 x I04 or IOs cells/ml, and cultured in a 5 % C02 humidified atmosphere at 37°C.

Culture media 745A were grown in (1) Dulbecco's modified Eagle's minimum essential medium (D-MEM) (Eurobio, Paris) supplemented with 501ofetal calf serum (FCS) and antibiotics; or (2) in a synthetic medium including Iscove's modified Dulbccco's medium 0MDM) (Gibco) that contains additional amino acids, vitamins and more important, selenium. FCS batches, carefully pre-test~d as emphasized in the Results section, were stored at --20°C. Aliquots, to be used within one week, were thawed and centrifuged at :5000g to remove aggregates and possible growth inhibitors that may originate in sera frozen at 20"C for more than three months (MttHe,r-Berat, personal communication). In serum-free media FCS was totally replaced by (a) bovine serum albumin (BSA) (Sigma, fraction V, 95.-99010 pure) added to the culture medium either after de.ionization by moans of ambcrlite (BSA/L +) or after deiontsation + delipidation with activated charcoal (BSA/L-). In both cases the final concentration in the medium was 6 mg/ml [12]; (b) pure human transferrin (T) (Bchring) guppicmaaeed at a final concentration of 420/~g/ml after saturation with FeCI3, and (c) a lipid complex (lccitin, cholesterol and iinolcic acid) added to the culture medium after sonication, at a final concentration of: linolci¢ acid 4.2 /~g/ml, cholesterol 1!.7/ag/ml and Iccitin 12/ag/ml [12]. In t erfero n Details of a/[J IFN production have been published elsewhere [1]. Inducers of erythroM dU'ferentiation Dimethylsulfoxide (DMSO) (Merck, stock solution undiluted, at room temperature); hexamcthylcnebisacetamide (HMBA) (gift of Dr. Carlo Delfini, Istituto Superior¢ di Sanit/k, Rome, Italy, stock solution: 200 mM in tridistilled water, stored at + 4°C); heroin (He) (bovine, Sigma Chemical Co., H-2250; 13 mg were dissolved in 0.2 ml 0.5 N NaOH; then neutralized with 0.25 ml 1 M Tris, pH 7.8, and finally diluted to a volume of 5 mi to yield a 4 mM stock solution; this solution was stored at --20°C). Each inducer was added to culture medium prior to cells seeding. Percentages of hemoglobin-producing (B+)-cells were determined by the wet benzidine staining method of Orkin et al. [14]. Reverse transcriptase (R. T. ) assay Details of R.T. assay have been published elsewhere [1].

RESULTS Cell g r o w t h F L C are grown in I M D M supplemented with (a) B S A / L ÷ only, (b) B S A / L - , T and L, and (c) B S A / L - and no T a n d L. Culture systems as in (a) a n d (b) allow optimal g r o w t h

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1~6. I. Growth of FLC serially passaged in vitro either in serum-supplemented or in serum-free medium. Cells were seeded at 105 or 5 × 104 c/ml every 3 days after evaluation of cell growth.

Percentage of cell mortality, evaluated by trypan blue dye exclusion method, was always less than 5%. Panel A: cell growth in serum-supplemented medium. Panel B, C, D: cell growth in different serum-frce media (see Materials and Methods).

of FLC, as compared with serum-rich medium (Fig. 1, panels A-C); the number of cells/ml reaches 2 x 10~ three days after seeding 105 or 5 x 104 cells/ml. In contrast, FLC growth in BSA/L--supplemented IMDM, in absence of any T or L (c) is soon, and drastically, impaired (Fig. 1, panel D). It is evident that BSA/L + effectively replaces T and L in culture, probably because of excess amounts of lipids and iron bound to albumin. On the other hand, delipidation of BSA drastically reduces iron content (i.e. from 11.5 parts x 106 to 1.3 parts x 10~, as determined by A. A. S.). Consequently, the addition of T and L is strictly necessary to ensure optimal cell growth in BSA/L-supplemented medium. Cell saturation densities and doubling time (13 h) of 745A-cells in serum-free cultures are essentially the same as those attainable in 5 o7oFCS-supplemented medium (Fig. 2).

Erythroid differentiation A number of compounds, known to induce erythroid differentiation of FLC [16], have been tested on 745A-cells growing in serum-rich or serum-free media. The former was supplemented only with 5o70 FCS as every batch of this component is carefully pre-tested in our laboratory to ensure the best cost-benefit ratio. This means that the capacity of every FCS batch under scrutiny to promote FLC growth and differentiation is assessed by continuously passaging the cells in medium supplemented with each batch for 7 weeks at limiting (1, 2.5, 5, 7.5, 10 and 15070) FCS concentrations. Only FCS batches promoting optimal (as compared with cells grown in medium additioned with a known "control" FCS batch) growth and differentiation of FLC at the end of a 7-week period of testing are

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selected and used at the lowest optimal serum concentration. Data in Fig. 2 and Table I show that optimal growth and differentiation are achieved under these experimental conditions. DMSO concentration is kept at 1.5% as it may occasionally cause cell toxicity when used at higher dosages. Hemin is less effective in absence of FCS than in serum-containing medium. Moreover, other inducers such as DMSO and HMBA are toxic in FCS-free cultures at the doses normally used in serum-rich medium (Table I) [I0, l 5]. Dose-response experiments show that DMSO can be added to serum-free cultures up to a final concentration of 1.2% without affecting cell growth. At this dose it still induces a significant level of differentiation. On the other hand, HMBA is uneffective when added at concentrations not altering cell growth in serum-free cultures (Table l). Moreover, at any inducer dose, B+-cell values in serum-free cultures are always lower than in FCS-supplemented ones.

Effect of Interferon on FLC differentiation in serum-free medium Low doses of Interferon (IFN) have been reported to stimulate DMSO-induced erythroid differentiation of FLC growing in serum-rich medium [8, 18]. Preliminary experiments have been carried out to investigate IFN effects on DMSOinduced differentiation of 745A-cells growing in serum-free medium. IFN addition results in enhanced erythroid differentiation (Table 2). Friend virus production and release As a measure of FLV release, the presence of reverse transcriptase (RT) activity was assayed in supernatants of FLC serum-free cultures. It appears that 745A-cells in serumfree culture initially release FLV at levels comparable to those of FLC growing in serumsupplemented medium, but after a long period of culture in serum-free conditions (about 20 passages) FLV-related R.T. activity was not detectable any more.

Friend erythroleukemia cells in serum-free culture

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TABLE I . ERYTHROID DIFFERENTIATION OF F L C IN SERUMFREE MEDIUM

Treatment with inducers

%B + cells on day 4 of culture in serum-rich (5% FCS)

or

serum-free medium (BSA/L- + TL)

None Heroin 10-4M

1 (38)* 44 (35)

2.5 (26) 35 (28)

DMSO 1.5% DMSO 1.2% DMSO 1% DMSO 0.8%

77 (27) 70(25) 60(26) 39 (27)

50 41 36 20

HMBA 4 mM HMBA 4 mM HMBA 3 mM HMBA 2 mM HMBA I mM

87 (27) 93 (26) 81 (30) 64 (24) 21 (28)

- - --1" 17 (6.3) 11 (9.8) 14.5 (20) 5 (26)

(20) (20) (21) (23)

*Between brackets, cell numbers x lO-S/ml. tPronounced cell death.

TABLE2. ERYTHROIDDIFFERENTIATIONINSERUM-FREEMEDIUMOF FLC TREATEDWITHIFN Treatments

¢/0B + cells on day 4 of culture in serum-rich (5% FCS)

None DMSO 1% DMSO 1% + 30 U/ml IFN DMSO 1% + 60 U/ml IFN DMSO 1% + 120 U/inl t.FN

1 (41)* 65 (41) 73 (33) 76 (35) 78 (24)

serum-free (BSA/L- + TL)

serum*free medium (BSA/L + - - T L )

3 (39) 27 (19) 25 (18) 42 (13) 33 (19)

3 (45) 28 (19) 47 (13) 40 (21) 43 (12)

*Between brackets, cell numbers × lO-'S/ml.

DISCUSSION A s e r u m - f r e e c u l t u r e system, s i m i l a r to t h a t d e v e l o p e d f o r m u r i n e h e m o p o i e t i c p r o g e n i t o r s [13] h a s b e e n successfully e s t a b l i s h e d f o r F L C . I n d e e d , results r e p o r t e d here s h o w t h a t s e r u m c a n b e c o m p l e t e l y r e p l a c e d in c u l t u r e s fed w i t h I M D M s u p p l e m e n t e d with B S A , t r a n s f e r r i n a n d lipids, thus allowing g r o w t h a n d e r y t h r o i d d i f f e r e n t i a t i o n o f F L C . Cells t o b e a s s a y e d in s e r u m - f r e e m e d i u m were a l l o w e d to a d a p t to the new c u l t u r e c o n d i t i o n s while g e t t i n g rid o f t r a c e a m o u n t s o f s e r u m f a c t o r s . 745A-cells have b e e n g r o w n in this ~ r u m - f r e e m e d i u m f o r as m a n y as 50 p a s s a g e s , i n d i c a t i n g that these c u l t u r e c o n d i t i o m allow i n d e f i n i t e o p t i m a l cell g r o w t h . Cell s a t u r a t i o n d e n s i t y a n d d o u b l i n g time a r e essentially the s a m e in s e r u m - f r e e a n d in s e r u m - s u p p l e m e n t e d m e d i u m . H o w e v e r , t h e p r e s e n c e o f T a n d L is essential f o r g r o w t h o f 745A-cells in m e d i u m c o n t a i n i n g d e i o n i z e d and delipidated BSA.

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CRISTINA JEMMAand GIOVANNIBAI"rlSTA ROSSl

Erythroid differentiation, measured as %0 of B÷-cells, is always lower in serum-free than in serum-rich media. Moreover, DMSO and HMBA concentrations routinely used in FCS-supplemented media are toxic in serum-free cultures. The effects of both inducers have been, thus, tested at lower doses. These findings suggest that factors present in the serum play a role in modulating erythroid differentiation and can, at the same time, eliminate or lower any toxic activity due to the inducers employed. Similar observations have been reported by Pessano et al. [15] and Gazitt [10]. Pessano et al. used a synthetic medium in which serum was replaced by insulin, transferrin and selenium dioxide. However, in this system the saturation densities typical for FLC growth in FCS-contalning medium were not reached. Data reported by Gazitt [10] support the hypothesis of a modulating action on erythroid differentiation by unknown serum factors. Yet the data are not strictly comparable to those of Pessano et al. and ours, as in those experiments FLC, always grown in medium with 15% FCS, were only washed and resuspended in MEM with 1% BSA just before being assayed for induction of differentiation. FCL were, thus, not adapted to the new culture conditions. Since IFN effects on DMSO-induced differentiation of FLC in serum-free medium are similar to those obtained in serum-supplemented cultures, it follows that IFN action, at least in this case, is not mediated by serum factors. Finally, Friend virus release seems to be lower and eventually suppressed in cells grown in serum-free conditions for a long period of time. In conclusion, the results reported here show that growth of FLC in serum-free media is a useful approach to the study of the biology of FLC system. Furthermore, it must be kept in mind that in vivo a few cells actually grow in the presence of serum. Culture in serumfree media could therefore represent a more physiological way to grow cells in vitro. Acknowledgements--We are indebted to Drs. C. Peschle, A. Covelli and F. Lettied for generous supply of materials and stimulating discussion. Dr. A. Alimonti performed the determination of iron content in bovine serum albumin. This work was supported in part by grant from Consiglio Nazionale ddle Ricerche, Progetto Finalizzato "Controllo della Creacita Neoplastica" (82.00406.96).

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