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Physiology and productivity of serum-free Spodoptera frugiperda Sf9 insect cell cultures
KTH, School of Biotechnology (BIO).
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 [en]
Animalcellteknologi
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4033ISBN: 91-7178-383-0 (print)OAI: oai:DiVA.org:kth-4033DiVA: diva2:10491
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
List of papers
1. Cell cycle progression in serum-free cultures of Sf9 insect cells: Modulation by conditioned medium factors and implications for proliferation and productivity
Open this publication in new window or tab >>Cell cycle progression in serum-free cultures of Sf9 insect cells: Modulation by conditioned medium factors and implications for proliferation and productivity
Show others...
2000 (English)In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 16, no 5, 837-846 p.Article in journal (Refereed) Published
Abstract [en]

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (mu(N,max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining mu(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase mu(N,max), decrease the time to reach mu(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean;population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

Keyword
recombinant protein-production, nuclear polyhedrosis-virus, autographa-californica nucleopolyhedrovirus, baculovirus expression system, spodoptera-frugiperda sf9, fed-batch culture, drosophila embryogenesis, dna-replication, vector system, amino-acids
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-20090 (URN)10.1021/bp000108i (DOI)000089797600025 ()
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved
2. 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
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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
3. A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells
Open this publication in new window or tab >>A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells
2006 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 71, no 4, 444-449 p.Article in journal (Refereed) Published
Abstract [en]

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

Keyword
nuclear polyhedrosis-virus, baculovirus expression system, cysteine proteinase, bombyx-mori, procathepsin-l, in-vivo, recombinant proteins, proteolytic activity, structural proteins, protease inhibitors
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-15847 (URN)10.1007/s00253-005-0181-9 (DOI)000239020100009 ()2-s2.0-33745968407 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
4. 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
5. Extracellular proteolytic activity innon-infected Sf9 and Trichoplusia ni Hi5 insect cell cultures: Implications for proliferation.
Open this publication in new window or tab >>Extracellular proteolytic activity innon-infected Sf9 and Trichoplusia ni Hi5 insect cell cultures: Implications for proliferation.
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-24421 (URN)
Note
QC 20100907Available from: 2010-09-08 Created: 2010-09-08 Last updated: 2010-09-08Bibliographically approved

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