Cell-dislodging methods under serum-free conditions |
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Authors: | H J Cruz E M Dias J L Moreira M J T Carrondo |
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Institution: | (1) IBET/ITQB – Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Apartado 12, P-2780 Oeiras, Portugal Fax: (3511) 442 11 61, PT;(2) Faculdade de Ciências e Tecnologia/Univ. Nova de Lisboa, P-2825 Monte da Caparica, Portugal, PT |
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Abstract: | In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail,
growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation,
currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation
solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control);
(5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results
indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI
followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved
results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml−1), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing
with this new, alternative dislodging method did not affect cell growth, viability and productivity.
Received: 23 September 1996 / Received revision: 27 December 1996 / Accepted: 30 December 1996 |
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