Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production

Abstract

Osmolarity is an important process variable during the cultivation of mammalian cells in vitro. Cell culture medium is designed to have osmolarity in the range of 260 and 320 milliosmoles (mOsm), basically to mimic the osmolarity of serum at 290 mOsm/kg. Fragmented information on the response of hybridoma cells to elevated osmolarity is available. A decrease in cell growth and an increase in antibody production were reported at high o~molarities.~~ Influence of osmolarity on antibody productivity seems to be cell line dependent.12 Here we report the effects of osmolarity on hybridoma cell growth, metabolism, and antibody synthesis. The osmolarity of the medium was altered by the addition of both ionic and non-ionic substances. MATERIALS AND METHODS Cell lines, Media, and Culture MaintenanceA murine hybridoma cell line, 167.465.3, was used in the experiments. This cell line was provided by Dr. Latham Claflin from the Medical Center at The University of Michigan. The antibody produced by this cell line is an kG1, directed against phosphorylcholine.2 Hybridoma :ells were made by fusion of BALB/c spleen cells with the nonsecreting plasmacytoma fusion line P3X63-488.653. Antibody was generated from mice immuiized with PC-keyhole limpet henemocyanin (KLH). "11s were propagated in T-flasks (Bellco Glass, Inc., Vineland, NJ) at 37°C in a humidified incubator under 5% COz. The media used was Iscove's Modified Dul-)ecco7s Medium (IMDM, Gibco Laboratories, Grand sland, NY) containing 5% Fetal Bovine Serum (FBS, 3ibco). The media was supplemented by 100 Units/mL )otassium penicillin G, and 100 pg/mL streptomycin ulfate (Sigma Chemical, St. Louis, MO). Effect of Osmolarity on Cell Physiology:ells were inoculated into IMDM medium at different jsmolarities. Osmolarity in IMDM medium was in-:reased by the addition of either an ionic (NaC1 or PBS) )r nonionic (sucrose) species. The IMDM medium was repared from powder and the pH was adjusted to 7.4 by the addition of sodium bicarbonate. Medium osmolarity was measured using an Osmometer (Osmette, model 2007, Precision Systems, Inc., Natick, MA) as 290 mOsm. Media with osmolarities of about twice this value (580 mOsm) were prepared by dissolving IMDM in (1) 9 g/L NaC1, (2) in PBS, and (3) in 0.31M sucrose solution. The pH was also adjusted to 7.4 in these media. The osmolarities in these media preparations were measured. Then these IMDM preparations were mixed with standard IMDM media at 290 mOsm to give final osmolarities of 290, 338, 386, 435, and 580 mOsm.Cells growing exponentially in ordinary IMDM with 5% FBS were spun down at 200g for 10 min. They were then washed with fresh IMDM and were inoculated with IMDM with different osmolarities. Fetal bovine serum was added to all the media preparations at 5%. Batch cultivations were carried out in 100-mL spinner flasks at an initial cell density of 4 x lo4 cells/mL. The volume in spinner flasks was 60 mL and they were kept at 37°C in a humidified incubator under 5% C02. Agitation at 100 rpm was provided by a MultiStir magnetic stir...

Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H-2 transfectoma

“…However, because hyperosmotic pressure suppresses cell growth, the enhanced q Ab of hybridoma cells in a hyperosmolar batch culture does not result in a substantial increase in the final antibody concentration Oh et al, 1993Oh et al, , 1995Ozturk and Palsson, 1991;Øyaas et al, 1994a, 1994bReddy and Miller, 1994).…”

Application of hypoosmolar medium to fed-batch culture of hybridoma cells for improvement of culture longevity

“…Each addition of 10× DMEM concentrates resulted in a significant increase in medium osmolality. The final medium osmolality reached up to 540 mOsm/kg which is known to be lethal to most hybridomas (Bibila et al, 1994;Oh et al, 1993;Øyaas et al, 1994;Ozturk and Palsson, 1991;Reddy et al, 1992;Reddy and Miller, 1994;Ryu and Lee, 1997). Figure 2 shows the results of fed-batch culture using a hypoosmolar medium (223 ± 3 mOsm/kg) as an initial basal medium.…”

Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions

“…A number of studies have shown that hypertonic stress can increase the specific MAb productivity of hybridoma cells (Oh et al, 1993;Øyaas et al, 1994a;1994b;Ozturk and Palsson, 1991;Reddy et al, 1992;Reddy and Miller, 1994;Yang et al, 1996). This has been attributed in part to increased amino acid uptake via the Na + -dependent symport (Oh et al, 1995).…”