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Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.
KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
2006 (English)In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 3, 653-659 p.Article in journal (Refereed) Published
Abstract [en]

The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.

Place, publisher, year, edition, pages
2006. Vol. 22, no 3, 653-659 p.
Keyword [en]
recombinant protein-production, baculovirus expression system, fed-batch culture, medium replacement, sf-9 cells, growth, line, suspension, infection, oxygen
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-5221DOI: 10.1021/bp050252+ISI: 000237973500007Scopus ID: 2-s2.0-33745027387OAI: oai:DiVA.org:kth-5221DiVA: diva2:8042
Note

Tidigare titel: Conditioned medium factors regulate productivity and cell physiology in Trichoplusia ni insect cell cultures. Uppdaterad från submitted till published(20101125) QC 20101125

Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free cultures
Open this publication in new window or tab >>Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free cultures
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium.

Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 36 p.
Keyword
Biotechnology, Insect cells, BEVS, Spodoptera frugiperda, Sf9, Trichoplusia ni, BTI-Tn-5B1-4, specific productivity, conditioned medium, conditioned medium factors, cell cycle, synchronization, G2/M, Bioteknik
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-243 (URN)91-7178-058-0 (ISBN)
Presentation
2005-06-03, FA32, Albanova, Roslagstullsbacken 21, 10:00
Opponent
Supervisors
Note
QC 20101125Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2010-11-25Bibliographically approved
2. Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free cultures
Open this publication in new window or tab >>Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free cultures
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.

Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.

Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.

In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.

Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 47 p.
Keyword
Biotechnology, Trichoplusia ni, High Five, insect cells, BEVS, serum-free medium, yeast extract, conditioned medium, autocrine regulation of proliferation, growth factors, cell cycle, metalloproteinase, specific productivity, Bioteknik
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-191 (URN)91-7178-056-4 (ISBN)
Presentation
2005-05-27, FB52, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note

QC 20101129

Available from: 2005-05-11 Created: 2005-05-11 Last updated: 2016-12-16Bibliographically approved

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