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无血清培养基中脂肪酸杂交瘤对生长和产量的影响

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无血清培养基中脂肪酸杂交瘤对生长和产量的影响 journal of biotechnology Journal of Biotechnology 39 (1995) 165-173 ELSEVIER The effect of fatty acids on hybridoma cell growth and antibody productivity in serum-free cultures M. Butler *, N. Huzel Department of Microbiology, University of Manitoba, Winn...
无血清培养基中脂肪酸杂交瘤对生长和产量的影响
journal of biotechnology Journal of Biotechnology 39 (1995) 165-173 ELSEVIER The effect of fatty acids on hybridoma cell growth and antibody productivity in serum-free cultures M. Butler *, N. Huzel Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 Received 19 November 1994; accepted 13 January 1995 Abstract A murine B-lymphocyte hybridoma (CC9ClO) was adapted for growth in a serum-free medium. Supplementation of the medium with k-unsaturated fatty acids (lo-50 PM) improved the cell yield in the order oleic/linoleic > linoleic > oleic. Initial supplementation with the fatty acids also caused a significant increase (58%) in the volumetric Mab titre. Continued growth of the cells in the fatty acid supplemented media over five culture passages resulted in a gradual deterioration of the Mab yield concomitant with the appearance of lipid inclusions in the cytosol. The higher Mab yield could be restored by a limited period of growth of the lipid-loaded cells in fatty acid-free medium. These effects were independent of growth rate. This suggests that the optimal intracellular lipid content is finely balanced between a reduced and an overloaded state. Specific glucose and glutamine utilisation rates were unaffected by the presence of fatty acids. Also, the optimal glucose and glutamine concentrations for growth were independent of the fatty acids. Keywords: Cell culture; Hybridoma; Antibody; Fatty acid; Serum free 1. Introduction There are numerous chemically defined serum-free formulations that have been devel- oped for the in vitro growth and productivity of mammalian cells and particularly for antibody- secreting hybridomas (Kawamoto et al., 1983; Cleveland et al., 1983; Kovar and Franek, 1986; Tharakan et al., 1986; Jager et al., 1988; Mu- rakami, 1989). Several of these formulations con- tain unsaturated fatty acids which are required * Corresponding author. Elsevier Science B.V. SSDI 0168-1656(95)00017-8 for optimal cell growth. The fatty acids are nor- mally bound to bovine serum albumin (BSA) which acts as a carrier molecule and to substitute for the role of a serum supplement (Ashbrook et al., 1975). Growth enhancement has been shown to be more likely with c&unsaturated fatty acids rather than saturated fatty acids (Rosenthal, 1987; Barnes and Sato, 19801, although in some cases combinations of saturated and unsaturated fatty acids have been used (Spieker-Polet and Polet, 1981). The optimal fatty acid concentration for growth enhancement of hybridomas is between 15 and 40 PM and higher concentrations may 166 M. Butler, N. Huzel/Journal of Biotechnology 39 (1995) 165-I 73 cause growth inhibition (Kovar and Franek, 1986). Incorporation of fatty acids into phospholipids is required in the synthesis of plasma membrane structure. However, cells appear to be unable to regulate fatty acid uptake and thus may become overloaded with an intracellular excess often recognised by the appearance of cytoplasmic lipid droplets (Schneeberger et al., 1971). Because of this, it is important to characterise the effect of fatty acid addition to cultures on the long-term growth of cells. In this report we examine the effect of two unsaturated fatty acids (oleic and linoleic acids) on the growth and monoclonal antibody production of a murine hybridoma over several culture passages. 2. Materials and methods Cell line The murine B-lymphocyte hybridoma (CC9ClO), which secretes a monoclonal antibody (a) (IgG,) against bovine insulin, was obtained from the American Type Culture Collection (ATCC No. HB123). The cells were shown to be my- coplasma-free by routine testing in an indepen- dent laboratory (Rh Pharmaceuticals, Winnipeg). Cuttut-e The cells were grown in serum-free medium consisting of DMEM/ Ham’s F12 in the ratio of 1:l v/v (Gibco). This basal medium was supple- mented with 10 wg ml-’ bovine insulin, 10 pg ml -’ bovine transferrin, 0.1% (w/v) Pluronic F- 68, 100 /.LM phosphoethanolamine, 10 FM ethanolamine and 10 nM sodium selenite. The cultures were also supplemented, where indi- cated, with fatty acid-free bovine serum albumin (0.1 mg ml-‘) complexed with a specific concen- tration of oleic or linoleic acid. A BSA-fatty acid concentrate ( X 1001 was formed by mixing 1 ml of BSA (10 mg ml-‘) with varying volumes (l-10 ~1) of fatty acid (200 mg ml-‘) in ethanol. The concentrate was mixed for 30 min at room tem- 8; (W I . OLL- / I 1._- 1 0 20 40 60 80 100 Oleic acid @M) 0 A_~~_ 1 .~_1._ 1 ---2L 0 20 40 60 80 100 Lmoleic acid (uM) Fig. 1. The effect of oleic or linoleic acid on cell yield. Cells (CCYClO) were inoculated at 0.5 X lo5 ml-’ into 2 ml of culture media supplemented with BSA-bound oleic acid (a) or linoleic acid (b) at O-100 PM. Daily viable cell counts were determined during the culture period. Cell yields for single cultures are shown after 4 d growth. Control cultures established with and without BSA showed no significant differences in cell yield. M. Butler, N. Huzel /Journal of Biotechnology 39 (1995) 165-173 167 perature prior to addition to the cultures. Cul- tures were established in T-flasks or in 24-well plates and grown under a 10% CO, non-humidi- fied atmosphere. All chemicals were purchased from Sigma Chemical Co., unless otherwise stated. Sample analysis Monoclonal antibody concentrations were de- termined by HPLC using an affinity column (Pro- AnaMabs from Hyclone). Glucose concentrations were determined with a specific analyzer (Model YSI 27, Yellow Springs Instrument Co.). Glu- tamine was measured using an enzymatic assay (Lund, 1985). Cell concentrations were deter- mined by a Coulter counter. Specific productivities (AC//N dt) were calculated as the cumulative concentration change divided by the viability in- dex (integral of cell concentration and culture time). 3. Results 3.1. Concentration-dependent effect of fatty acid addition The effect of fatty acid addition on the growth of the CC9ClO cells was initially determined from 2 ml cultures incubated in 24-well plates. The cell yield was determined after 4 d for cultures con- taining O-100 PM oleic or linoleic acid (Fig. 1). Control cultures were established with and with- out supplemented BSA. The presence of BSA had no effect on cell yield. Cell growth was enhanced in the presence of oleic acid at an optimal concentration of 50 PM or in the presence of linoleic acid at an optimal concentration of 25 PM. The growth enhance- ment was equivalent for either fatty acid. How- ever, concentrations of either fatty acid > 75 PM caused growth inhibition. As a result of this ex- periment it was decided to continue serum-free cell growth in the presence of 25 PM oleic, linoleic acid or an equimolar mixture of oleic and linoleic acids. Fig. 2. Growth of cells in serum-free media supplemented with fatty acids. Cells (CC9ClO) were inoculated at 10’ ml-’ into 50 ml of culture media in 75 cm2 T-flasks. Daily viable cell counts were determined from cultures supplemented with 25 PM oleic acid (W ), 25 PM linoleic acid ( A 1, 12.5 PM oleic and 12.5 PM linoleic acid (0) or control cultures (0). Each point represents a single independent cell count. 3.2. The effect of cell passage Subsequent experiments were conducted in T- flasks at an inoculum of lo5 cells per ml and cells were sub-cultured after 4 d. Cell growth in the presence of the unsaturated fatty acids is shown in Fig. 2. Cell yields were higher in the presence of fatty acids with oleic/linoleic > linoleic > oleic > control cultures. Compared to the control culture, the fatty acid supplemented cultures also showed a higher growth rate, Jo (Table 1). The relative differences in cell yield between cultures was maintained over five passages indi- cating a consistent enhancement of final cell den- Table 1 Growth rate fi cd-‘) of hybridomas in media supplemented with fatty acids (+ SEM for n = 4) Media: control + oleic + linoleic + oleic/linoleic 0.63 f 0.03 0.71 k 0.04 0.80 k 0.07 0.85 k 0.07 +oleic acid 0 1 TimE (d) 3 4 168 M. Butler, N. Huzel/Journal of Biotechnology 39 (1995) 165-173 sity in cultures supplemented with fatty acids (Fig. 3). Regular microscopic examination of the cells over this growth period indicated the grad- ual formation of cytoplasmic inclusions for cells from fatty acid supplemented cultures. Staining with Nile red showed that these inclusions had a lipid content. The antibody concentration of culture super- natants was determined at the end of each cul- ture period (Fig. 4). At passage 1, cells grown in the presence of fatty acids produced a signifi- cantly higher (58%) antibody concentration. However, this difference was not significant after passage 3. This was also reflected in a change in the specific antibody productivity (qMMah) over cul- ture passage. The qMab was not significantly dif- ferent for the four cultures in passage 1, but by passage 5 the productivity of cultures supple- mented with fatty acids was significantly lower (42%) than the control cultures (Fig. 5). Antibody production of the controls was constant through- out the five passages with a maximum concentra- tion of 56.7 k 1.4 pg ml-‘. nber - ConfrY lZ%‘, +a,e,c CC,d _1:, +, “/,P i r<.,,i .IIl(_SI lh11>l1~~ II Fig. 3. Cell yield in CCYClO cultures over five consecutive passages. At each passage, cells were inoculated at 10’ ml-’ into 10 ml cultures in 75 cm* T-flasks. The final cell concen- tration was determined after 4 d incubation. Error bars indi- cate the SEM where n = 2 or 3. 1 3 Passage number = C>lltrU i _,/r c “i,d , :_,, +,,nu,i c “rid , +Oi~,c,I,,,Oi~$i “-0 Fig. 4. Monoclonal antibody concentration in CC9ClO cul- tures over five consecutive passages. At each passage, cells were inoculated at lo5 ml-’ into 10 ml cultures in 75 cm2 T-flasks. The final antibody concentration was determined after 4 d incubation. The error bars indicate the SEM where n = 2 or 3. (1) Indicates determinations from single cultures. 3.3. Removal of fatty acid supplement It was decided to investigate whether the ef- fects of fatty acid supplementation on cell growth and Mab productivity were reversible. Cells sub- cultured for at least 20 passages in fatty acid supplemented media were inoculated into control media (without fatty acid). After two culture pas- sages, there were still significant differences in cell yields between cells previously grown in fatty acid cultures and controls (Fig. 6). However, sub- sequent passages reduced the cell yield of fatty acid supplemented cultures, so that by passage 5 there was no significant difference in cell yields between cultures. The relative antibody production of cultures was also affected by removal of the fatty acid supplement (Fig. 7). No significant differences were found in the final antibody concentrations of cultures at passage 1, but by passage 2 and 3 the antibody yield from cells previously grown in fatty acid supplemented cultures was significantly higher (23%) compared to controls. However, M. Butler, N. Huzel /Journal of Biotechnology 39 (1995) 165-173 169 this difference was eroded by passages 4 and 5. These changes in final antibody concentrations were also reflected in qMvlab values which were significantly higher (36%) in fatty acid supple- mented cultures at passage 3 (Fig. 8). 3.4. Utilisation rates of glucose and glutamine Fig. 9 is representative of the pattern of glu- cose and glutamine utilisation for a series of cultures grown for five passages in the absence of a fatty acid supplement or in the presence of oleic acid, linoleic acid or a linoleic/oleic mix. The example is of a culture supplemented with linoleic acid at passage 5. Both glucose and glu- tamine were almost completely utilised over the 4 d growth period. The initial rate of glutamine utilisation (q ,,,) was calculated as 1.32 f 0.10 pmol per 10B cells per d for the cells grown in linoleic acid supplemented media. The corre- sponding value for the initial rate of glucose utilisation (q,,J was 2.17 + 0.07 pmol per lo5 1 +Ol 2 Passage number 5 Fig. 5. Specific antibody productivity (qMab) in CC9ClO cul- tures over five consecutive passages. At each passage, cells were inoculated at 10’ ml-’ into 10 ml cultures in 75 cm2 T-flasks. The qMab was determined from the cell and anti- body concentration changes over the 4 d of culture. The error bars indicate the SEM where n = 2 or 3. (1) Indicates deter- minations from single cultures. 2 4 Passage number n - con,ro, m +oleic acid m +ll”Olelc OCld [ +oie,c,l,n”le,c rmr Fig. 6. The effect of fatty acid removal on final cell yields. Cells which had been grown for at least 20 passages in fatty acid-supplemented media were inoculated (10’ ml-‘) into control growth media, i.e., with no fatty acid supplement. The effect of growth in the absence of fatty acid was monitored over five consecutive culture passages. The error bars indicate the SEM where n = 2. (1) Indicates determinations from single cultures. cells per d. No significant difference was found in the initial qgln or qaC values between the control and fatty acid supplemented cultures. Further- more, the qgln or qglC values were unaffected by passage number. 3.5. The effect of initial glucose and glutamine concentrations on cell growth A matrix of cell cultures was established by varying the glucose concentration (O-35 mM) in one dimension and the glutamine concentration (OS-24 mM) in a second dimension. The cell inocula were taken from a single culture grown in serum-free medium but in the absence of a fatty acid. The experimental cultures were grown with or without added fatty acid (Fig. 10). The relative final cell yields were consistent under all condi- tions with respect to fatty acid supplementation with linoleic = linoleic/oleic > oleic = control(+linoleic) > control. This showed that M. Butler, N. Huzel /Journul of Biotechnology 39 (1995) 165-173 ce,,r denufi, 1111111 Passage number m COnfrOl r2-.; tdri< I>/ Mi ;,;j tiirll k/S XII rl 1 +,-,*, ,,,IdI., /‘I Fig. 7. The effect of fatty acid removal on monoclonal anti- body concentrations. Cells which had been grown for at least 20 passages in fatty acid-supplemented media were inoculated (10” mll’) into control growth media, i.e., with no fatty acid supplement. The effect of the absence of fatty acid was monitored over five consecutive culture passages. The error bars shown indicate the SEM where n = 2. (1) Indicates determinations from single cultures. there was a significant improvement (18%) in cell yield in cultures supplemented with linoleic acid rather than oleic acid for cells adapted over sev- eral passages to each respective media formula- tion. The addition of linoleic acid to the control cultures also improved the cell yield significantly (60%), but only up to the level shown for cells continuously passaged with oleic acid. The presence of a fatty acid supplement in cultures did not alter the concentration-depen- dent effect of glucose or glutamine on cell growth. The highest cell yields were found at a glucose concentration of 17.5-25 mM and glutamine of 24 mM. Tolerance of such a high glutamine con- centration suggests that these cells are relatively insensitive to ammonia, a conclusion consistent with previous experiments (not presented here). 4. Discussion Fatty acids are often added in micromolar concentrations to serum-free culture formulations to maintain optimal growth of mammalian cells. The cellular requirement for fatty acids is not apparent in serum-supplemented cultures where it is presumed that sufficient concentrations of fatty acids are attached to serum proteins. In serum-free media the fatty acids are normally included as a complex with BSA (Ashbrook et al., 1975). In this report, we show that the addition of the c&unsaturated fatty acids, oleic and linoleic, to serum-free cultures of a murine hybridoma can increase the growth rate and final cell yield signif- icantly. However, the fatty acid concentration is critical. The optimal concentrations for growth stimulation by oleic and linoleic acids were 25 and 50 PM, respectively. At higher concentra- tions the stimulatory effect decreased sharply and at concentrations > 75 PM the fatty acids were growth inhibitory. There are extensive reports on the effects of fatty acids on mitogen-stimulated lymphocytes showing stimulation at low concentration (N 5 4 2 3 4 Passage number II = con’r”I ‘;:.‘I +o,eic “cNc i-.:2 +iilolelC “Cld 1 +o,eic,i Il”lCIC !/INS Fig. 8. The effect of fatty acid removal on specific antibody productivity (qMab). Cells which had been grown for at least 20 passages in fatty acid-supplemented media were inoculated (10’ ml-‘) into control growth media, i.e., with no fatty acid supplement, The effect of the absence of fatty acid was monitored over five consecutive culture passages. The error bars shown indicate the SEM where n = 2. (1) Indicates determinations from single cultures. M. Butler, N. Huzel /Journal of Biotechnology 39 (1995) 165-173 171 FM) (Kelly and Parker, 1979; Cuthbert and Lip- sky, 1989) but inhibition at higher concentration (> 30 PM; Calder et al., 1991). Similar concen- tration effects have been reported previously for hybridomas in serum-free medium (Kovar and Franek, 1986), although the actual concentrations causing stimulation and inhibition are different. Jager et al. (1988) showed that the require- ment of hybridomas for fatty acids is not immedi- ately apparent following adaptation from serum- supplemented medium. They reported that the stimulatory effect was only shown following 10 culture passages in serum-free medium. This may be related to a period in which an endogenous store of fatty acid is utilised. This is consistent with the data presented here for the CC9ClO cells which had been grown in a serum-free, fatty acid-free formulation for at least 10 passages prior to the addition of a fatty acid. At the concentrations used, linoleic acid in- creased the growth rate of the CC9ClO cells more than oleic acid but a synergistic effect was ob- served with a combination of oleic and linoleic acid providing the greatest growth stimulation (Fig. 2). The growth stimulation of the fatty acids was maintained over at least 20 culture passages. When the fatty acid was removed from the cul- ture medium, it took two culture passages before the growth rate of the stimulated cells decreased to the value of the control cultures (Fig. 6). Thus, it would seem that a constant supply of fatty acid in the medium is desirable to maintain a con- stantly high growth rate. The relationship between antibody production and availability of fatty acid was more complex. The initial addition of fatty acid caused an in- crease in the antibody yield. However, the calcu- lated specific antibody productivities (qMMab) were not significantly different and the increased anti- body yield could be explained by an increase in cell concentration. After five passages of growth in the presence of fatty acid, the qMulab declined significantly compared to control cultures (Fig. 5). This effect was reversed when the fatty acid was removed from the medium, so that the antibody productivity and yield increased after two pas- sages but then declined to control levels after five passages (Fig. 8). The antibody productivity data is compatible with the idea that there is an optimal intracellular fatty acid concentration that maximises qMab. In our experiments, the optimal intracellular fatty acid concentration was achieved by
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