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
Physiological effects of conditioned medium and passage number on Spodoptera frugiperda Sf9 serum free cultures
KTH, School of Biotechnology (BIO).
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 [en]
Biotechnology, Spodoptera frugiperda Sf9, insect cell cultures, conditioned medium, cell cycle phase dynamics, proteolytic activity, antibacterial activity, recombinant protein production
Keyword [sv]
Bioteknik
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-247ISBN: 91-7178-074-2 (print)OAI: oai:DiVA.org:kth-247DiVA: diva2:8061
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
List of papers
1. Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells
Open this publication in new window or tab >>Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells
Show others...
2005 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 69, no 1, 92-98 p.Article in journal (Refereed) Published
Abstract [en]

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

Keyword
antibacterial peptides, expression, membrane, protein, growth
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-5225 (URN)10.1007/s00253-005-1958-6 (DOI)000233502300014 ()2-s2.0-28344454084 (Scopus ID)
Note
QC 20100830 Uppdaterat från manuskript till artikel (20100830)Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2017-12-04Bibliographically approved
2. Cathepsin L and its proform identified in conditioned medium from Sf9 insect cells
Open this publication in new window or tab >>Cathepsin L and its proform identified in conditioned medium from Sf9 insect cells
(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5227 (URN)
Note
QC 20101222Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2010-12-22Bibliographically approved
3. 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

Open Access in DiVA

fulltext(1520 kB)1611 downloads
File information
File name FULLTEXT01.pdfFile size 1520 kBChecksum MD5
2a800f740ae83408d9653176b34814451f2e9bc98701843c23bd187f070aef076ab925a3
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Svensson, Ingrid
By organisation
School of Biotechnology (BIO)
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1611 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: 607 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