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Impact of autocrine factors on physiology and productivity in Trichoplusia ni 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 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 [en]
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
Keyword [sv]
Bioteknik
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-191ISBN: 91-7178-056-4 (print)OAI: oai:DiVA.org:kth-191DiVA: diva2:7856
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
List of papers
1. Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures
Open this publication in new window or tab >>Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures
2005 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, no 1, 76-86 p.Article in journal (Refereed) Published
Abstract [en]

Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B 1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45 kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25 kDa, as well as precursor forms above 48 kDa. Metalloproteinase bands below the main band at 48 kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor DL-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10 kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.

Keyword
Trichoplusia ni, serum-free medium, conditioned medium, gel filtration fractions, metalloproteinase activity, autocrine regulation of proliferation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5093 (URN)10.1016/j.jbiotec.2005.05.020 (DOI)000231491000009 ()2-s2.0-23444452392 (Scopus ID)
Note

QC 20100922. Uppdaterad från Manuskript till Artikel (20100922).

Available from: 2005-05-11 Created: 2005-05-11 Last updated: 2017-12-05Bibliographically 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
3. Yeast extract from express five serum-free medium contains factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells
Open this publication in new window or tab >>Yeast extract from express five serum-free medium contains factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells
2005 (English)In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 27, no 20, 1623-1627 p.Article in journal (Refereed) Published
Abstract [en]

The yeast extract (of unknown origin) present in the commercially available serum-free medium 'Express Five' contains factors ('yeast extract factors') up to 35 kDa which are essential for growth of Trichoplusia ni insect cells. A yeast extract brand lacking these components could not support growth of T. ni cells. However, cell proliferation was restored by adding chromatographic fractions containing the yeast extract factors. The yeast extract factors were not solely responsible for the growth enhancing effect of yeast extract but some other components, which seem to be generally present in yeast extracts, are also required for T. ni proliferation.

Keyword
Essential nutrients, Express Five, Insect cells, Serum-free medium, Trichoplusia ni, Yeast extract
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5095 (URN)10.1007/s10529-005-2603-2 (DOI)000232799300014 ()
Note

QC 20101005. Uppdaterad från Manuskript till Artikel (20101005). Tidigare Titel:"Yeast extract from Express Five SFM contains essential factors for growth of Trichoplusia ni insect cells".

Available from: 2005-05-11 Created: 2005-05-11 Last updated: 2017-12-05Bibliographically approved

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