Yoshiaki Matsuda

Non-woven fabrics as new cell matrices for IMR-90 human embryonic lung diploid fibroblast cells

IMR-90 cells could proliferate on non-woven fabrics (NWF) up to a cell density of about 3 × 105/cm2, which was about 7 times higher compared to that in monolayer culture with a T-flask. The production of tissue plasminogen activator (t-PA) by perfusion culture using a device which includes a cultivating pan fitted inside a sheet of NWF was performed. The t-PA activity at a dilution rate of 0.22, 0.33 and 0.45 d−1 was 245, 220 and 160 IU/ml. Compared to plastic surfaces, NWF stably retained the cells during t-PA production using serum-free medium.

Continuous production of tissue plasminogen activator (t-PA) by human embryonic lung diploid fibroblast, IMR-90 cells, using a ceramic bed reactor

Ceramic pieces composed of 99.5% Al2O3, 3 to 6 mm long, were found to be a good matrix for growth of the human embryonic lung diploid fibroblast, IMR-90 cells. The tissue plasminogen activator (t-PA) was secreted in DME medium containing proteose peptone as a t-PA inducer. In addition, production of t-PA was enhanced by increasing extracellular CaCl2, from 3.6 to 5.4 mM. In order to eliminate negative feed-back control caused by t-PA produced and thus raise productivity, perfusion cultivation was performed using a ceramic-packed bed column, with a recirculating vessel. The recirculating vessel was used to mix fresh medium with spent medium, and to control dissolved oxygen concentrations in the extracellular environment by stirring. In continuous production using the packed bed column with 2 kg of ceramics (Ø=H=150 mm), increasing dilution rate to 0.5 day-1 could reduce product inhibition at 3–4×105cells/ml. Cellular productivity of 560 IU/106cells/day was obtained over 40 days and corresponded to the volumetric productivity of 183 IU/ml/day.

Non-Woven Fabrics for Immobilizing Mammalian Cells

We have developed two types of Non-woven fabrics (NWF) with urethane binder for tissue culture use. Most of cell lines tested, L-929, BHK-21, IMR-90 and hybridoma cells, could grow well on the NWF coated with Gelatin + Fibroin + Chitosan (M type).

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

SummaryTissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm3 per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed.