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Protease activity in protein-free NSO myeloma cell cultures
KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
2005 (English)In: In vitro Cellular & Developmental Biology-Animal, ISSN 1071-2690, E-ISSN 1543-706X, Vol. 41, no 10, 330-336 p.Article in journal (Refereed) Published
Abstract [en]

Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that thiscell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identifiedas aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular massesof 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using anenzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid andcysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product oraffecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, butaddition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibodyproduct was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acidproteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM,one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotininsignificantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminalamino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitorand cystatin C.

Place, publisher, year, edition, pages
2005. Vol. 41, no 10, 330-336 p.
Keyword [en]
conditioned medium, zymography, aprotinin, cathepsin L, SLPI, cystain C
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-6313DOI: 10.1007/s11626-005-0004-4ISI: 000236070000004PubMedID: 16448222Scopus ID: 2-s2.0-33645019941OAI: oai:DiVA.org:kth-6313DiVA: diva2:10993
Note

QC 20100920

Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2014-12-01Bibliographically approved
In thesis
1. Development of a protein-free fed-batch process for NS0 cells: studies on regulation of proliferation
Open this publication in new window or tab >>Development of a protein-free fed-batch process for NS0 cells: studies on regulation of proliferation
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 44 p.
Keyword
NS0 myeloma cells, protein-free medium, fed-batch, conditioned medium, autocrine growth factors, abortive proliferation, protease activity, aprotinin, gp130
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4162 (URN)91-7178-466-7 (ISBN)
Public defence
2006-11-17, FB54, AlbaNova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Supervisors
Note
QC 20100920Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2010-09-20Bibliographically approved

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