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
Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free cultures
KTH, School of Biotechnology (BIO), Bioprocess Technology.
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 [en]
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
Keyword [sv]
Bioteknik
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-243ISBN: 91-7178-058-0 (print)OAI: oai:DiVA.org:kth-243DiVA: diva2:8043
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
List of papers
1. Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity
Open this publication in new window or tab >>Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity
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.

Keyword
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:nbn:se:kth:diva-24419 (URN)10.1021/bp050297a (DOI)000236783400008 ()2-s2.0-33646018011 (Scopus ID)
Note
QC 20100908Available from: 2010-09-08 Created: 2010-09-08 Last updated: 2017-12-12Bibliographically approved
2. Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.
Open this publication in new window or tab >>Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.
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.

Keyword
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:nbn:se:kth:diva-5221 (URN)10.1021/bp050252+ (DOI)000237973500007 ()2-s2.0-33745027387 (Scopus ID)
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

Open Access in DiVA

fulltext(4322 kB)1425 downloads
File information
File name FULLTEXT01.pdfFile size 4322 kBChecksum MD5
500eee615eaefb6b39790262f2e8ec2477b955293efc503633016a0fb0d68e9102dca88e
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Calles, Karin
By organisation
Bioprocess Technology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1425 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 702 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