ABSTRACT
With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.
Subject(s)
Humans , HEK293 Cells , Genetic Vectors/genetics , Batch Cell Culture Techniques , Bioreactors , PerfusionABSTRACT
In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.
Subject(s)
Animals , Batch Cell Culture Techniques , Bioreactors , Reference Standards , CHO Cells , Cricetulus , Mammals , PerfusionABSTRACT
Objective@#To investigate the feasibility of in vitro perfusion culture of human adipose tissue and its induction into muscle tissue.@*Methods@#Human abdominal adipose tissue were cultured in vitro by perfusion culture. After 1, 3, 5 or 7 weeks, FAD/PI staining was used to detect the tissue vitality. Histological staining was used to observe the changes of its histomorphology. Protein expressions of myogenic molecules Myf-5 and myoD1 as well as muscle specific protein Desmin were measured by immunohistochemistry and Western blot assay.@*Results@#The adipose tissues cultured in myogenic induction media were still in the appearance of adipose tissue at 7 weeks. While in the basal medium without inducing, vascular pedicles shed after 7 weeks and could not continue to be cultured. FAD/PI staining showed that the tissue cultured in the induction media remained viable at 7 weeks, while the viability of the tissue in the basal culture medium decreased significantly at 5 weeks. Histologically, Myf-5, myoD1 and Desmin were all positively expressed in muscle tissues, while in adipose tissues, some mesenchymal and vascular endothelial cells expressed Myf-5 but not myoD1, and only separate vascular smooth muscle cells expressed Desmin. Interestingly, in adipose tissues cultured in myogenic induction medium, partial muscle-like tissue formed, evidenced as positive expression of Myf-5, myoD1 and Desmin. There was no muscle-like tissue formation in adipose tissue cultured in basal medium and the expression patterns were similar to that of the control group. Western blot results showed that the expression levels of Myf-5 and Desmin in muscle tissue were significantly higher than that of the other groups (P<0.05). The expression level of Myf-5 was slightly higher in inducing group than that in non-inducing group at the same time points, but the difference was not statistically significant. Similarly, the expression of Desmin was higher in inducing group than that in non-inducing group, yet there was statistically significant difference only in the first week. The protein expression of myoD1 was not detected in any group using Western blot.@*Conclusions@#The adipose tissue cultured in the perfusion bioreactor can survive for up to 7 weeks, and it can be partially induced into muscle-like tissue in the myogenic induction medium.
ABSTRACT
We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31ºC) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32 percent at 34ºC and 55 percent at 31ºC, compared to cultures at 37ºC. Similar behaviour was observed in cultures at 34ºC using galactose as a carbon source. Nonetheless, at 31ºC, the specific productivity of rh-tPA strongly decreased (about 58 percent) compared to the culture at 37ºC. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34ºC. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112 percent over that obtained in semi-perfusion culture carried out at 37ºC with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.
Subject(s)
CHO Cells , Cold Temperature , Galactose , Glutamic Acid , Tissue Plasminogen Activator , Cell Culture Techniques , TemperatureABSTRACT
In the field of regenerative medicine,much consideration has been given to stem/progenitor cells for the future treatment of acute and chronic renal failure.For this strategy to be effective,however,cell biological information about tubule development within the diseased organ is needed.Unresolved cell-biological issues relating to this kind of treatment include①the integration of stem/progenitor cells,②their differentiation into site-specific cell types,and③the spatial formation of new tubules.To better understand the mechanisms related to this technology,renal tubules were generated at the interphase of an artificial interstitium by using advanced culture techniques.Stern/progenitor cells derived from neonatal rabbit kidney were covered with layers of polyester fleece,placed in a perfusion culture container,and superfused for 13 days with fresh and chemically defined Iscove's Modified Dulbeccos Medium(IMDM) containing aldosterone (1×10-7mol/L).The spatial growth of tubules was registered by scanning electron microscopy(SEM) and on whole mounts or cryosections labeled with soybean agglutinin,silver stain and monoclonal antibodies reacting with collagen type Ⅲor laminin γ1.SEM revealed that the generated tubules were completely covered by a basal lamina.The lamina fibroreticularis exhibited numerous fibers connecting the basal aspect of generated tubules with the surrounding polyester fibers.Cryosections labeled with monoclonal antibodies anti-collagen type Ⅲ and silver stain demonstrated the formation of numerous fibers spanning between the basal lamina of generated tubules and neighboring polyester fibers.In matured kidney tubules the samg arrangement of collagen type Ⅲ fibers is observed as that for generated tubules.This work shows that collagen type Ⅲ is a relevant molecular linker between the basal aspect of generated renal tubules and the polyester fibers of the artificial interstitium.
ABSTRACT
Objective To verify the application of anterior segment perfusion culture and trabecular meshwork (TM) organ culture for glaucoma study. Methods TM tissue was cultured by perfused anterior segment and TM organ culture, light microscopy was used to observe the TM cells and intercellular spaces.Results IOP of the porcine anterior segments perfused under constant flow at 0.1 mL/h could bekept in normal range(10～12 mmHg). The IOP was elevated with the increasing of perfusion rate, while the morphology and structure of the tissue were well preserved. TM cultured by TM organ culture could also reserve the tissues well, but the intercelluar spaces collapsed. Conclusion Anterior segment perfusion model could be a short-term high-pressure model and may simulate the normal physical state. Adequate perfusion was necessary for normal TM.