Bleeding is largely unavoidable following syringe needle puncture of biological tissues and, while inconvenient, this typically causes little or no harm in healthy individuals. However, there are certain circumstances where syringe injections can have more significant side effects, such as uncontrolled bleeding in those with haemophilia, coagulopathy, or the transmission of infectious diseases through contaminated blood. Herein, we present a haemostatic hypodermic needle able to prevent bleeding following tissue puncture. The surface of the needle is coated with partially crosslinked catechol-functionalized chitosan that undergoes a solid-to-gel phase transition in situ to seal punctured tissues. Testing the capabilities of these haemostatic needles, we report complete prevention of blood loss following intravenous and intramuscular injections in animal models, and 100% survival in haemophiliac mice following syringe puncture of the jugular vein. Such self-sealing haemostatic needles and adhesive coatings may therefore help to prevent complications associated with bleeding in more clinical settings.

Low culture temperature is known to enhance the specific productivity of Chinese hamster ovary (CHO) cells expressing erythropoietin (EPO) (LGE10-9-27). Genomic and proteomic approaches were taken to better understand the intracellular responses of these CHO cells resulting from use of low culture temperature (33 degrees C). For transcriptome analysis, commercially available rat and mouse cDNA microarrays were used. The data obtained from the rat and mouse cDNA chips were only somewhat informative in understanding the gene expression profile of CHO cells because of their different sequence homologies with CHO transcriptomes. Overall, transcriptome analysis revealed that low culture temperature could lead to changes in gene expression in various cellular processes such as metabolism, transport, and signaling pathways. Proteome analysis was carried out using 2-D PAGE. Based on spot intensity, 60 high intensity protein spots, from a total of more than 800, were chosen for MS analysis. Forty of the 60 protein spots, which represent 26 different kinds of proteins, were identified by MALDI-TOF-MS and validated by MS/MS. Compared to the reference temperature (37 degrees C), the expression levels of seven proteins (PDI, vimentin, NDK B, ERp57, RIKEN cDNA, phosphoglycerate kinase, and heat shock cognate 71 kDa protein) were increased over twofold at 33 degrees C and those of two proteins (HSP90-beta and EF2) were decreased over twofold at 33 degrees C. Taken together, the results demonstrate the potential of combined analysis of transcriptome and proteome analyses as a tool for the systematic comprehension of cellular mechanisms in CHO cells.

To better understand intracellular responses to hyperosmotic pressure of recombinant Chinese hamster ovary (rCHO) cells expressing an antibody, we have taken a proteomics approach. Using two-dimensional electrophoresis and mass spectrometry, a proteome profile of rCHO cells comprising 23 identified proteins was established. On the basis of this proteome profile, we found three proteins of which expression levels were significantly changed at 450 mOsm/kg. Compared to the results at 300 mOsm/kg, two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, were found to be up-regulated, probably leading to an increased metabolic energy for antibody synthesis. The elevation of specific glucose consumption rate at 450 mOsm/kg agreed with the up-regulation of these glycolytic enzymes. On the other hand, tubulin expression was down-regulated, reflecting a depressed cell growth rate at 450 mOsm/kg. Taken together, this study shows the potential of the proteomics approach in understanding intracellular and physiological changes in cells and seeking a better insight into possible environmental or genetic manipulation approaches for increasing foreign protein production in rCHO cells.

When subjected to hyperosmotic pressure resulting from NaCl addition, KR12H-2 transfectoma, like most hybridomas, displayed a decrease in specific growth rate (µ) and an increase in specific antibody productivity (q Ab ). Elevation of medium osmolality from 285 to 425 mOsm/kg decreased µ by 20%, while it increased q Ab by 376%. Although cell mass also increased at higher osmolality, it was not the main factor in increasing q Ab . Hyperosmotic pressure was found to enhance transcription levels of immunoglobulin (Ig) mRNAs preferentially, compared with non-IgG mRNA. The transcription levels of both heavy chain (HC) and light chain (LC) mRNAs were enhanced as much as q Ab . This result suggests that enhanced q Ab at higher osmolality was mainly due to enhanced transcription levels of Ig mRNA. However, these increased transcription levels of Ig mRNAs were not due to the enhanced stability of Ig mRNA. In fact, the stability of Ig mRNAs decreased at higher osmolality. Elevation of osmolality from 285 mOsm/kg to 425 mOsm/ kg decreased the half-lives of HC and LC mRNAs by 37% and 36%, respectively. A simple mathematical model revealed that transcription rates of Ig mRNAs increased by more than 476% at 425 mOsm/kg. These elevated transcription levels could, in turn, increase the translation rates of Ig polypeptides. However, the translation rates of Ig polypeptides were not enhanced as much as the transcription levels of Ig mRNAs and q Ab . The elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg increased HC and LC mRNA specific translation rates by 172% and 240%, respectively. Taken together, the data suggest that (1) enhanced q Ab of KR12H-2 transfectoma at higher osmolality is due to elevated transcription rates of Ig mRNAs and expedited post-translational processing of Ig, and (2) antibody secretion by KR12H-2 transfectoma is most likely controlled at the level of Ig translation, particularly HC translation.

The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (mu) and increased specific antibody productivity (q(Ab)) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in q(Ab) and less reduction in mu at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and q(Ab), indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G(1)-phase arrest, suggesting that the increase of cell population in G(1)-phase may contribute in part to enhanced q(Ab) at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in q(Ab) at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage.