Three-Dimensional Culture of Hybridoma Cells Secreting Anti-Human Chorionic Gonadotropin by a New Rolling Culture System

Abstract

Cell growth rate and production of monoclonal antibody (MAb) of hybridoma cells producing anti-human chorionic gonadotropin (hCG) MAb have been used as investigation criteria in double-mouthed rolling bottle (DMRB). Compared with T-flask cell culture, both of the cell number and MAb production increased by approximately 42.5% when the medium was supplemented with 5% fetal calf serum (FCS) and DMRB rotated at 2 turns per minute. Yield of MAb was experimentally related to the number of viable cells. Interestingly, MAb yield was four times as high as that cultured in T-flask in the first 24 hours, and about 75% yield of total MAb was secreted by 48 hours during the culture. It appears that the promoted cell growth and MAb yield are resulted from the three-dimensional growth of hybridoma cells under a suitably revolving condition.

Figure 1

Diagram of a DMRB for culture of hybridoma cells. The cured surface of the bottle touches on the rolling spindles horizontally and rotates at a suitable revolution speed. It is placed in an incubator during cell culture.

Figure 2

Cell growth status of murine hybridoma cells perching on microcarriers. The DMEM medium was supplemented with 50 μM gentamycin, at pH 7.4 (adjusted with 7.4% NaHCO₃ when necessary). Viable cells were seeded at a density of 1.5–2 × 10⁵/mL for both T-flasks (50 mL, 25 cm² containing 10 mL of medium) and DMRBs (containing 10 mL of medium). Microcarrier beads (0.3 g/100 mL medium) were added to the medium and cells were allowed to grow in DMEM medium (5% CO₂, at 37^°C) with 1%, 5%, and 10% FCS, respectively. Cells were cultured in static DMRBs supplemented with 10% FCS for 72 hours. Cells growing in T-flask were as control (bar = 0.1 mm). For rotating DMRB culture (supplemented with 10% FCS), cells were under a revolution speed of 2 turns/min for 72 hours. For each experiment, three bottles, at least, were as a group.

Figure 2

Cell growth status of murine hybridoma cells perching on microcarriers. The DMEM medium was supplemented with 50 μM gentamycin, at pH 7.4 (adjusted with 7.4% NaHCO₃ when necessary). Viable cells were seeded at a density of 1.5–2 × 10⁵/mL for both T-flasks (50 mL, 25 cm² containing 10 mL of medium) and DMRBs (containing 10 mL of medium). Microcarrier beads (0.3 g/100 mL medium) were added to the medium and cells were allowed to grow in DMEM medium (5% CO₂, at 37^°C) with 1%, 5%, and 10% FCS, respectively. Cells were cultured in static DMRBs supplemented with 10% FCS for 72 hours. Cells growing in T-flask were as control (bar = 0.1 mm). For rotating DMRB culture (supplemented with 10% FCS), cells were under a revolution speed of 2 turns/min for 72 hours. For each experiment, three bottles, at least, were as a group.

Figure 2

Cell growth status of murine hybridoma cells perching on microcarriers. The DMEM medium was supplemented with 50 μM gentamycin, at pH 7.4 (adjusted with 7.4% NaHCO₃ when necessary). Viable cells were seeded at a density of 1.5–2 × 10⁵/mL for both T-flasks (50 mL, 25 cm² containing 10 mL of medium) and DMRBs (containing 10 mL of medium). Microcarrier beads (0.3 g/100 mL medium) were added to the medium and cells were allowed to grow in DMEM medium (5% CO₂, at 37^°C) with 1%, 5%, and 10% FCS, respectively. Cells were cultured in static DMRBs supplemented with 10% FCS for 72 hours. Cells growing in T-flask were as control (bar = 0.1 mm). For rotating DMRB culture (supplemented with 10% FCS), cells were under a revolution speed of 2 turns/min for 72 hours. For each experiment, three bottles, at least, were as a group.

Figure 3

Comparison of cell growth in DMRBs and T-flasks. Total (left) and viable (right) numbers of hybridoma cells cultured in both DMRBs and T-flasks in medium containing 10% (a), 5% (b), and 1% (c) FCS, respectively. The black curve represents cell number in DMRBs and the red curve represents cell number in T-flasks. Each point is the mean of three independent parallel cultures. Vertical bars are SDs.

Figure 3

Comparison of cell growth in DMRBs and T-flasks. Total (left) and viable (right) numbers of hybridoma cells cultured in both DMRBs and T-flasks in medium containing 10% (a), 5% (b), and 1% (c) FCS, respectively. The black curve represents cell number in DMRBs and the red curve represents cell number in T-flasks. Each point is the mean of three independent parallel cultures. Vertical bars are SDs.

Figure 3

Comparison of cell growth in DMRBs and T-flasks. Total (left) and viable (right) numbers of hybridoma cells cultured in both DMRBs and T-flasks in medium containing 10% (a), 5% (b), and 1% (c) FCS, respectively. The black curve represents cell number in DMRBs and the red curve represents cell number in T-flasks. Each point is the mean of three independent parallel cultures. Vertical bars are SDs.

Figure 4

Comparison of MAb production rates between rolling DMRBs and T-flasks. The MAb concentrations are measured in the medium supplemented with 5% FCS in both DMRBs and T-flasks. (a) Accumulated MAb production. (b) Daily MAb production. The values are mean ± SD of three independent experiments. Vertical bars are SDs.

Figure 4

Comparison of MAb production rates between rolling DMRBs and T-flasks. The MAb concentrations are measured in the medium supplemented with 5% FCS in both DMRBs and T-flasks. (a) Accumulated MAb production. (b) Daily MAb production. The values are mean ± SD of three independent experiments. Vertical bars are SDs.