Document Detail


Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions.
MedLine Citation:
PMID:  2758049     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids. The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature. Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l. Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8. Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory. The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber). This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion. It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures. At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied. Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion. Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession. Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established. This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process. The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins. Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.
Authors:
J J Schmitt; U Zimmermann
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  983     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  1989 Jul 
Date Detail:
Created Date:  1989-09-08     Completed Date:  1989-09-08     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  42-50     Citation Subset:  IM    
Affiliation:
Lehrstuhl für Biotechnologie, Universität Würzburg, F.R.G.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Fusion
Cell Line
Culture Techniques / methods
Electrophysiology / methods
Hybridomas / cytology*
Hypotonic Solutions
Lymphocytes / cytology
Mice
Mice, Inbred BALB C
Osmolar Concentration
Plasmacytoma / pathology
Chemical
Reg. No./Substance:
0/Hypotonic Solutions

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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