Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity
KTH, School of Biotechnology (BIO), Bioprocess Technology.
2006 (English)In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 2, 394-400 p.Article in journal (Refereed) Published
Abstract [en]

The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages > 100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.

Place, publisher, year, edition, pages
2006. Vol. 22, no 2, 394-400 p.
Keyword [en]
Growth kinetics, Physiology, Productivity, Proteins, Synchronization, Baculovirus, Conditioned medium factors, Lag phase, Maximum cell density, Yeastolate limitation, Cell culture, animal, armyworm, article, cell aging, cell line, cell proliferation, chemistry, culture medium, cytology, drug effect, metabolism, molecular weight, Animals, Culture Media, Conditioned, Spodoptera, Lepidoptera, Spodoptera frugiperda
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-24419DOI: 10.1021/bp050297aISI: 000236783400008Scopus ID: 2-s2.0-33646018011OAI: oai:DiVA.org:kth-24419DiVA: diva2:349666
Note
QC 20100908Available from: 2010-09-08 Created: 2010-09-08 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Physiology and productivity of serum-free Spodoptera frugiperda Sf9 insect cell cultures
Open this publication in new window or tab >>Physiology and productivity of serum-free Spodoptera frugiperda Sf9 insect cell cultures
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The objective of this study was to investigate the mechanisms and factors controlling growth and proliferation in serum-free Spodoptera frugiperda Sf9 cultures as well as the implications of these factors for protein production in the baculovirus expression system.

The physiology of recently thawed, low passage (Lp) Sf9 cultures, were compared to high passage (Hp) cultures at p>100. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µN,max, from 0.03/h to 0.04/h. Conditioned medium (CM), 10 kDa CM filtrate and 10 kDa CM concentrate promoted proliferation of Lp Sf9 cells, but had no effect after the switch. Sf9 cell cycle dynamics were characterized by an initial G2/M arrest, which synchronized the cells and this feature was more pronounced for Hp than for Lp cells. CM addition decreased the initial arrest for Lp cultures, but did not affect Hp cells. Late in the culture, a final G2/M accumulation occurred. An octaploid population emerged during G2/M arrests. Further, a 49 kDa proform and a 39 kDa active form of Sf9 cathepsin L was identified from gelatine zymography of Sf9 CM, on basis of inhibitor profile and substrate range. Removal of procathepsin L during the course of an Sf9 culture had a negative effect on Sf9 proliferation. Procathepsin L was also identified in Trichoplusia ni High five CM. High five CM promoted growth of Sf9 cells, but when procathepsin L was removed no effect was observed. It is suggested that these observations are due to an autocrine system controlling proliferation. One Sf9 mitogen might be a <10 kDa peptide, while the effect of 10 kDa CM concentrate may originate from procathepsin L. A hypothesis is therefore that procathepsin L acts as a mitogen in Sf9 cultures, perhaps in concert with the <10 kDa peptide.

The volumetric product yield (P) in baculovirus infected Sf9 cells increased linearly up to 68-75 h of culture. Beyond this point almost no product was detected. Medium renewal at infection prolonged the productivity phase until 117 h, but generated only a 10% increase in P. The specific product formation rate (YP/N) was highest at µN,max. YP/N of Lp cells decreased by 30-50% when 20% CM or 10 kDa CM filtrate was added, whereas addition of CM to cells having passed the switch on growth kinetics did not affect productivity. Further, Hp cells exhibited a two-fold higher YP/N than Lp cells, when infected during the initial 48 h of culture. This coincided with a high degree of synchronization. Yeastolate limitation was used to achieve artificial synchronization of an Lp culture, and YP/N could thereby be maintained high during a prolonged time, resulting in a 69% increased P. This suggests that a decreasing degree of synchronization during the course of a culture partly explains the cell-density dependent drop in productivity in Sf9 cells.

Finally, ~10 kDa gel filtration fractions from Sf9 and High five CM were found to be bactericidal. Exposure of a Bacillus megaterium culture for eight min to an Sf9 CM fraction killed 99% of the population, and 60 min exposure killed 35% of an Escherichia coli population. In both cases cell lysis was observed. B. megaterium incubated in an High five CM fraction lost 97% viability in 40 min. The effect of the High five CM fraction most probably originated from a lysozyme precursor protein, whereas the Sf9 executor remains unknown.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 53 p.
Keyword
Animalcellteknologi
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4033 (URN)91-7178-383-0 (ISBN)
Public defence
2006-06-15, Sal FB52, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100908Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2010-09-08Bibliographically approved
2. Physiological effects of conditioned medium and passage number on Spodoptera frugiperda Sf9 serum free cultures
Open this publication in new window or tab >>Physiological effects of conditioned medium and passage number on Spodoptera frugiperda Sf9 serum free cultures
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system.

CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction.

Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 43 p.
Keyword
Biotechnology, Spodoptera frugiperda Sf9, insect cell cultures, conditioned medium, cell cycle phase dynamics, proteolytic activity, antibacterial activity, recombinant protein production, Bioteknik
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-247 (URN)91-7178-074-2 (ISBN)
Presentation
2005-06-01, FA32, AlbaNova, Roslagstullsbacken 21B, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101222Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2012-03-21Bibliographically approved
3. 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

Open Access in DiVA

No full text

Other links

Publisher's full textScopushttp://www.scopus.com/inward/record.url?eid=2-s2.0-33646018011&partnerID=40&md5=a743b0a4f3ecfd2d02efb4a6ad615734

Search in DiVA

By author/editor
Calles, KarinLindskog, EvaHäggström, Lena
By organisation
Bioprocess Technology
In the same journal
Biotechnology progress (Print)
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 115 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf