Cytotechnology 8:5-11, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Use of fluorescence anisotropy determinations9 for indicating. the physiological status of hybridoma cell cultures Vdronique Eyl I, Sylvaine Muller ~, Mireille Donner ~, Michel Maugras 1,3 and Iean-Fi'an~ois Stolt;Zi=3 ~INSERM U 284; ~-Service Fractionnement C.R.T.S., Plateau de Brabois, 54511 Vandoeuvre Cedex; and SUniversitd Nancy, 1 B.P.239, 54506 Vandoeuvre Cedex, France Received 12 August 1991; accepted in revised form 8 January 1992
Key words: membrane fluidity, hybridoma culture, fluorescence anisotropy, fluorescence quenching Abstract The evolution of lipid compartment fluidity during culture of hybridoma cells Was. studied:by, flUoreScence polarization measurements. The probe partition between the plasma.membrane.and," intraeytoplas'-.'. mic compartments was determined by a quenching fluorescence method. A progressive-deerease~of~,'~e plasma membrane fluidity was observed during the growth phase with an increase during sta~iot~ary ~andt degeneration phases of the culture. These data Suggest that fluidity parameters could be used'to :follow; the' behaviour of hybridoma cell cultures.
Introduction The production of monoclonal antibodies requires good control of hybridoma cell cultures, since the cell growth state must constantly be known. The cell cultures are usually characterized by three different stages: growth, stationary and degeneration phases. The latter is related to a loss of cell viability which itself is connected with modifications in the membrane permeability. In the case of transformed cells (Fourcade et al., 1980) it has been reported that other membrane cell properties. such as lipid fluidity, varied in the course of a culture. To our knowledge, no such information has been described for hybridoma cells. In this paper, we report the study of lipid fluidity of hybridoma cells during cultures: performed with, and without renewal of. medium. Lipid cohesion was estimated from fluorescence: polarization measurements using two lipophilic
probes 1,6-diphenyl: 1,3,5;hexalTiene ~DPH), ata~ 1,[4~(trimethylarnino) phenyl]: 6-phenyl' hdxg tJiL, 3,5-triene:(TMA-DPH.). The: first probe'charaeter'-' izes the. hydrophobic areasr while-.the Ser ~, which possesses a cationic group~:.is, l~cated,:in: more polar compartments (D0riner.,atid..St~l~ t985).. The anisotropy.,of ~ae,fluoresC~ne6~'eixi~tt~d; by the probes is. inversely related', to:the fl~idity,iSi~ their lipid surroundings. The fact that the probes enter the cells and label the cytoplasmi'6,elem'en.~, allows a study of the probe partition .between the~ plasma membrane and, mtracytoplasrmc~r ompart~; ments. In our work,:: this. parameter:':haS,-~be~ determined by the, assoe.iation,.Of fluorescence '" ......... ~* ' polarizafioff, with-~quen'cN~g, in6th'6~.,aSin~:2~'4~6:2 trinitrobenzene.su Ifon at6~(,~4"BS~):~'I~i~eo/t~lb0tifa~:l~ which, is Ve~ reacfi,r ard:s',~.?~s,~b~ds~l~o~. bus ic.~amino~ar grOUl~S,0 f ~ e ~ l k s u d ~ a ~ t ~ ' ~ l absorplaon speetrum.;o~dm.~tropl:aeny,l|g~oups~#,~ laps the fluorescence ,spectrum, ~of ',flae.;!prolP~st .
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located in its proximity, it is possible to discriminate the light emitted by probes located in the lipid cytoplasmic compartment from that emitted from the membrane lipid one (Grunberger, 1982). Using this quencher, the evolution of the fluidity of the different compartments has been determined and related to the different phases of the culture.
Materials and methods
Cell labelling Daily sampled cells were centrifugated for 5 min at 400 g. The cell pellet was suspended at 2 106 cells • ml -j in a phosphate buffer solution (PBS) pH 7.2. One 1 ktl of probe solution was added to 1 ml of cell suspension so that its final concentration was 2 10-6 mol • 1-I The suspensions were incubated at 25~ in the dark, 10 rain for TMADPH and 20 min for DPH. The fluorescence polarization determinations were performed immediately.
Cell cultures Fluorescence inhibition with TNBS The hybridoma strain A49 used produces IgM antihuman A blood group (Mau~as et al., 1987). Cells were maintained in T 75 cm / plastic cell culture flasks at 37~ with 5% CO2, 95% air under fully humidified conditions. The cell culture medium was RPMI 1640 basal medium supplemented with 2% v/v ULTROSER HY (I.B.F., Villeneuve La Garenne, France) and 0.5% v/v fetal calf serum (FCS) (Flow Laboratories, Puteaux, France). The culture used in experiments without medium renewal were seeded at the concentration of 105 viable cells • ml -I. A flask was used each day for fluorescence determinations. The experiments with medium renewal were conducted with cultures seeded at a similar concentration of viable cells. To get cultures always in exponential phase, two parts of the cell suspension were daily discarded and replaced by the same volume of new culture medium. In these conditions, the viability determined by the trypan blue exclusion test was maintained around 100%. Fluorescent probes 1,6-diphenyl 1,3,5-hexatriene (DPH) (Aldrich, Strasbourg, France) and its cationic derivative 1-[4-(tfimethylamino) phenyl] 6-phenyl hexa 1,3,5-triene (TMA-DPH) (Molecular Probes, Eugene City, Oregon, USA) were respectively dissolved in tetrahydrofuran (THF) and N-N'dimethylformamide (DMF). These solutions can be maintained at 4~ in the dark for two weeks.
After the probe incorporation in the cellular lipidic compartments, the cell suspensions were separated into two parts which were maintained at 4~ In the first sample, 100 I.tl of TNBS solution (Aldrich Strasbourg, France) were added to obtain a 2.5 10-3 mg x ml -l final concentration. One hundred ktl of PBS solution were added to the second sample which was considered the control. Cells were washed and resuspended in cold PBS after one hour incubation. All polarization measurements were carried out at 4~ Fluorescence meosul'e177ent3 Fluorescence polarization was assessed by means of a continuous excitation instrument (Fluofluidimeter Sefam, Vandoeuvre-Les-Nancy, France). Signals which are measured are the emission intensities determined with the emission polarizer orientated parallel II[ and perpendicular I_1_to the direction of polarization of the excitation light, respectively. In all cases, non-labelled cell suspensions were measured before each series of experiments in order to calculate the correcting factors. After determining the signals Ib I[, Ib_l_ due to 'blanks' (non-labelled samples), the fluorescence intensities Ifll, ~f& of the sample were assessed. Fluorescence steady-state anisotropy and fluorescence intensity were assessed, based on the following relationship:
.
(100-P) + P
[2]
with
Use of fluorescence anisotropy determinations9 for indicating. the physiological status of hybridoma cell cultures Vdronique Eyl I, Sylvaine Muller ~, Mireille Donner ~, Michel Maugras 1,3 and Iean-Fi'an~ois Stolt;Zi=3 ~INSERM U 284; ~-Service Fractionnement C.R.T.S., Plateau de Brabois, 54511 Vandoeuvre Cedex; and SUniversitd Nancy, 1 B.P.239, 54506 Vandoeuvre Cedex, France Received 12 August 1991; accepted in revised form 8 January 1992
Key words: membrane fluidity, hybridoma culture, fluorescence anisotropy, fluorescence quenching Abstract The evolution of lipid compartment fluidity during culture of hybridoma cells Was. studied:by, flUoreScence polarization measurements. The probe partition between the plasma.membrane.and," intraeytoplas'-.'. mic compartments was determined by a quenching fluorescence method. A progressive-deerease~of~,'~e plasma membrane fluidity was observed during the growth phase with an increase during sta~iot~ary ~andt degeneration phases of the culture. These data Suggest that fluidity parameters could be used'to :follow; the' behaviour of hybridoma cell cultures.
Introduction The production of monoclonal antibodies requires good control of hybridoma cell cultures, since the cell growth state must constantly be known. The cell cultures are usually characterized by three different stages: growth, stationary and degeneration phases. The latter is related to a loss of cell viability which itself is connected with modifications in the membrane permeability. In the case of transformed cells (Fourcade et al., 1980) it has been reported that other membrane cell properties. such as lipid fluidity, varied in the course of a culture. To our knowledge, no such information has been described for hybridoma cells. In this paper, we report the study of lipid fluidity of hybridoma cells during cultures: performed with, and without renewal of. medium. Lipid cohesion was estimated from fluorescence: polarization measurements using two lipophilic
probes 1,6-diphenyl: 1,3,5;hexalTiene ~DPH), ata~ 1,[4~(trimethylarnino) phenyl]: 6-phenyl' hdxg tJiL, 3,5-triene:(TMA-DPH.). The: first probe'charaeter'-' izes the. hydrophobic areasr while-.the Ser ~, which possesses a cationic group~:.is, l~cated,:in: more polar compartments (D0riner.,atid..St~l~ t985).. The anisotropy.,of ~ae,fluoresC~ne6~'eixi~tt~d; by the probes is. inversely related', to:the fl~idity,iSi~ their lipid surroundings. The fact that the probes enter the cells and label the cytoplasmi'6,elem'en.~, allows a study of the probe partition .between the~ plasma membrane and, mtracytoplasrmc~r ompart~; ments. In our work,:: this. parameter:':haS,-~be~ determined by the, assoe.iation,.Of fluorescence '" ......... ~* ' polarizafioff, with-~quen'cN~g, in6th'6~.,aSin~:2~'4~6:2 trinitrobenzene.su Ifon at6~(,~4"BS~):~'I~i~eo/t~lb0tifa~:l~ which, is Ve~ reacfi,r ard:s',~.?~s,~b~ds~l~o~. bus ic.~amino~ar grOUl~S,0 f ~ e ~ l k s u d ~ a ~ t ~ ' ~ l absorplaon speetrum.;o~dm.~tropl:aeny,l|g~oups~#,~ laps the fluorescence ,spectrum, ~of ',flae.;!prolP~st .
.
. . .
.
.
.
.
.
.
located in its proximity, it is possible to discriminate the light emitted by probes located in the lipid cytoplasmic compartment from that emitted from the membrane lipid one (Grunberger, 1982). Using this quencher, the evolution of the fluidity of the different compartments has been determined and related to the different phases of the culture.
Materials and methods
Cell labelling Daily sampled cells were centrifugated for 5 min at 400 g. The cell pellet was suspended at 2 106 cells • ml -j in a phosphate buffer solution (PBS) pH 7.2. One 1 ktl of probe solution was added to 1 ml of cell suspension so that its final concentration was 2 10-6 mol • 1-I The suspensions were incubated at 25~ in the dark, 10 rain for TMADPH and 20 min for DPH. The fluorescence polarization determinations were performed immediately.
Cell cultures Fluorescence inhibition with TNBS The hybridoma strain A49 used produces IgM antihuman A blood group (Mau~as et al., 1987). Cells were maintained in T 75 cm / plastic cell culture flasks at 37~ with 5% CO2, 95% air under fully humidified conditions. The cell culture medium was RPMI 1640 basal medium supplemented with 2% v/v ULTROSER HY (I.B.F., Villeneuve La Garenne, France) and 0.5% v/v fetal calf serum (FCS) (Flow Laboratories, Puteaux, France). The culture used in experiments without medium renewal were seeded at the concentration of 105 viable cells • ml -I. A flask was used each day for fluorescence determinations. The experiments with medium renewal were conducted with cultures seeded at a similar concentration of viable cells. To get cultures always in exponential phase, two parts of the cell suspension were daily discarded and replaced by the same volume of new culture medium. In these conditions, the viability determined by the trypan blue exclusion test was maintained around 100%. Fluorescent probes 1,6-diphenyl 1,3,5-hexatriene (DPH) (Aldrich, Strasbourg, France) and its cationic derivative 1-[4-(tfimethylamino) phenyl] 6-phenyl hexa 1,3,5-triene (TMA-DPH) (Molecular Probes, Eugene City, Oregon, USA) were respectively dissolved in tetrahydrofuran (THF) and N-N'dimethylformamide (DMF). These solutions can be maintained at 4~ in the dark for two weeks.
After the probe incorporation in the cellular lipidic compartments, the cell suspensions were separated into two parts which were maintained at 4~ In the first sample, 100 I.tl of TNBS solution (Aldrich Strasbourg, France) were added to obtain a 2.5 10-3 mg x ml -l final concentration. One hundred ktl of PBS solution were added to the second sample which was considered the control. Cells were washed and resuspended in cold PBS after one hour incubation. All polarization measurements were carried out at 4~ Fluorescence meosul'e177ent3 Fluorescence polarization was assessed by means of a continuous excitation instrument (Fluofluidimeter Sefam, Vandoeuvre-Les-Nancy, France). Signals which are measured are the emission intensities determined with the emission polarizer orientated parallel II[ and perpendicular I_1_to the direction of polarization of the excitation light, respectively. In all cases, non-labelled cell suspensions were measured before each series of experiments in order to calculate the correcting factors. After determining the signals Ib I[, Ib_l_ due to 'blanks' (non-labelled samples), the fluorescence intensities Ifll, ~f& of the sample were assessed. Fluorescence steady-state anisotropy and fluorescence intensity were assessed, based on the following relationship:
.
(100-P) + P
[2]
with
