No abstract

Mesenchymal stromal cells (MSCs) have considerable potential for a wide range of clinical applications and regenerative medicine and cell therapy. As a consequence, there is considerable interest in developing robust culture methods for producing large number of MSCs for use in repair of injured tissues or treatment of diseases. In general, tissue culture plates or flasks that have been precoated with substrates derived from animal tissues are used in the production of MSCs. However, these substrates can potentially cause serious problems due to contamination of the MSCs with animal-derived components. In this study, we evaluated the use of a type I collagen-based recombinant peptide (RCP) for MSC culture in an attempt to avoid the problems associated with animal cell-derived substances. This RCP is xeno free, has an increased RGD (Arg–Gly–Asp) sequence, and has high molecular weight uniformity. The effect of RCP on promotion of cellular adhesion and proliferation of MSCs was investigated in cultures in which RCP was included in the culture medium. The effects of RCP on promotion of cellular adhesion and proliferation of MSCs were investigated by comparing cultures in which the additive was present in the culture medium and those where the culture plates were coated with RCP. In addition, changes in gene expression profiles during cell culture were monitored by real time-polymerase chain reaction. Our analyses showed that RCP enhanced cellular adhesion and proliferation in cultures in which the additive was included in the culture medium. Our findings indicate that adding RCP to the culture medium could save time and cost in MSC culture. Our gene expression analysis indicated that RCP enhanced expression of genes encoding proteins associated with the extracellular matrix and cell adhesion.

The tripeptide Z-GlyPheLeuNH2 was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH2, by immobilized thermolysin (IMT) and immobilized alpha-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GlyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116 hr. The optimal conditions for the synthesis of Z-GlyPheLeuNH2 from Z-GlyPheOMe and LeuNH2 by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 microliters/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH2 was synthesized from Z-GlyPheOMe and LeuNH2 with a yield of 100%, by continuous reaction for 160 hr. The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80%. The residual activity of IMT was 94% and that of IMC was 100%.

No abstract

The tripeptide Z-GlyPheLeuNH2 was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH2, by immobilized thermolysin (IMT) and immobilized alpha-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GlyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116 hr. The optimal conditions for the synthesis of Z-GlyPheLeuNH2 from Z-GlyPheOMe and LeuNH2 by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 microliters/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH2 was synthesized from Z-GlyPheOMe and LeuNH2 with a yield of 100%, by continuous reaction for 160 hr. The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80%. The residual activity of IMT was 94% and that of IMC was 100%.