Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.

Cultivation of Chlorella sorokiniana in a bubble-column bioreactor coupled with cooking cocoon wastewater treatment: effects of initial cell density and aeration rate

Previous studies documented that Chlorella sorokiniana could grow well on cooking cocoon wastewater (CCW) with a maximum biomass of 0.49 g/L. In order to further enhance the biomass production and nutrient removals, a bubble-column bioreactor was designed and performed to cultivate C. sorokiniana in CCW, and two main cultivation parameters were investigated in this work. Results showed that a maximum algal biomass, specific growth rate, and biomass productivity of 2.83 g/L, 0.854 d−1, and 476.25 g/L/d, respectively, were achieved when this alga was cultivated in the bioreactor with an initial cell density of 0.8 g/L and an aeration rate of 3.34 L air/L culture/min; meanwhile, removal efficiencies of ammonium, total nitrogen, total phosphorus, and chemical oxygen demand reached 97.96, 85.66, 97.96, and 86.43%, respectively, which were significantly higher than that obtained in our previous studies. Moreover, chemical compositions in the algal cells varied with the changes of cultivation conditions (i.e., initial cell density and aeration rate). Thus, it is concluded that (1) the bubble-column bioreactor was suitable for cultivation of C. sorokiniana coupled with the CCW treatment; and (2) initial cell density and aeration rate affected the biomass production, nutrient removals and chemical compositions of this alga.

Initial cell density encodes proliferative potential in cancer cell populations

Individual cells exhibit specific proliferative responses to changes in microenvironmental conditions. Whether such potential is constrained by the cell density throughout the growth process is however unclear. Here, we identify a theoretical framework that captures how the information encoded in the initial density of cancer cell populations impacts their growth profile. By following the growth of hundreds of populations of cancer cells, we found that the time they need to adapt to the environment decreases as the initial cell density increases. Moreover, the population growth rate shows a maximum at intermediate initial densities. With the support of a mathematical model, we show that the observed interdependence of adaptation time and growth rate is significantly at odds both with standard logistic growth models and with the Monod-like function that governs the dependence of the growth rate on nutrient levels. Our results (i) uncover and quantify a previously unnoticed heterogeneity in the growth dynamics of cancer cell populations; (ii) unveil how population growth may be affected by single-cell adaptation times; (iii) contribute to our understanding of the clinically-observed dependence of the primary and metastatic tumor take rates on the initial density of implanted cancer cells.

Optimal Initial Cell Density that Yields the Highest Number of Primary Synovial Mesenchymal Stem Cells in a Clinical Setting

Background Synovial mesenchymal stem cell (MSC) transplantation improved clinical outcomes in knees with cartilage defects and after repairs of degenerative torn menisci. For these, nucleated cells were cultured with 10% autologous serum for 14 days. Importantly, nucleated cell number and serum volume practically varied by patient, and initial cell density and dish number affected yield/donor. Our purpose was to examine the effect of plating density within range in clinical studies on fold increase and to estimate the MSC yield/donor, which are affected by the nucleated cell number and serum volume.Methods We obtained the data of the autologous serum volume, nucleated cell number, and initial plating cell density from 10 patients who participated in our previous clinical studies. Experimentally, synovial nucleated cells harvested from three patients with osteoarthritis during total knee arthroplasty were cultured with 10% mixed allogenic human serum for 14 days to examine the effect of initial cell density on fold increase. We used both our previous clinical data and our current experimental data to predict the yields of synovial MSCs in nine cases within the range of clinical studies.Results In the clinical studies, collected serum ranged between 87 and 186 ml, harvested nucleated cells ranged between 6.2 and 23 million, and the top five total MSC yields were obtained from donors when the initial plating cell density ranged from 919 to 1905 cells/cm2. Experimentally, fold increase significantly decreased between 700 and 1,900 cells/cm2. In cases where the cell number was low and the serum volume was high, the estimated yield/donor decreased along with the initial density, while in cases where the cell number was high and the serum volume was low, the estimated yield/donor increased along with the initial density. In cases both where some nucleated cells were discarded at low density and no cells were discarded at high density, the maximum yield/donor were obtained when the initial density was the lowest density that does not discard cells.Conclusions There was an optimal initial density that yields the highest number of primary MSCs in a clinical setting when cell numbers and human serum volumes are limited.

Optimal initial cell density that yields the highest number of primary synovial mesenchymal stem cells in a clinical setting

Background Synovial mesenchymal stem cell (MSC) transplantation improved clinical outcomes in knees with cartilage defects and after repairs of degenerative torn menisci. For these, nucleated cells were cultured with 10% autologous serum for 14 days. Importantly, nucleated cell number and serum volume practically varied by patient, and initial cell density and dish number affected yield/donor. Our purpose was to examine the effect of plating density within range in clinical studies on fold increase and to estimate the MSC yield/donor, which are affected by the nucleated cell number and serum volume. Methods We obtained the data of the autologous serum volume, nucleated cell number, and initial plating cell density from 10 patients who participated in our previous clinical studies. Experimentally, synovial nucleated cells harvested from three patients with osteoarthritis during total knee arthroplasty were cultured with 10% mixed allogenic human serum for 14 days to examine the effect of initial cell density on fold increase. We used both our previous clinical data and our current experimental data to predict the yields of synovial MSCs in nine cases within the range of clinical studies. Results In the clinical studies, collected serum ranged between 87 and 186 ml, harvested nucleated cells ranged between 6.2 and 23 million, and the top five total MSC yields were obtained from donors when the initial plating cell density ranged from 919 to 1905 cells/cm 2 . Experimentally, fold increase significantly decreased between 700 and 1,900 cells/cm 2 . In cases where the cell number was low and the serum volume was high, the estimated yield/donor decreased along with the initial density, while in cases where the cell number was high and the serum volume was low, the estimated yield/donor increased along with the initial density. In cases both where some nucleated cells were discarded at low density and no cells were discarded at high density, the maximum yield/donor were obtained when the initial density was the lowest density that does not discard cells. Conclusions There was an optimal initial density that yields the highest number of primary MSCs in a clinical setting when cell numbers and human serum volumes are limited.

Study on the biosynthesis and structure characterization of exopolysaccharide from Lactobacillus fermentum MC3

<p><strong>Abstract: </strong>The effects of carbohydrate sources in various concentration (2%, 3%, 4%, 5%, 6%) and fermentation conditions (such as initial cell density, temperature, pH and incubation time) on EPS synthesis of <em>Lactobacillus fermentum </em>MC3 were also studied. The results showed that adding different sugars (including glucose, lactose and sucrose) to culture medium significantly increased the EPS production. In comparison with other concentrations, EPS amounts were maximized in the medium supplemented with 4% (w/v) of sugars. The outcome was the highest for glucose, which was 178.207 mg/L, the obtained figures for lactose and for sucrose were 148.614 mg/L and 152.272 mg/L respectively. The results indicated that EPS production by <em>L. fermentum </em>MC3 reached the maximum values in the medium supplemented with 4% (w/v) glucose at 40⁰C, pH 6.0, initial cell density of 10⁶CFU/ml for 48 h cultivation with amount of 200.728 mg/L. By methylation analysis and gas–liquid chromatography–mass spectrometry (GLC–MS), the exopolysaccharide was found to be composed of D-mannose: D-glucose: D-galactose in a molar ratio of 1 : 0.74 : 0.09.</p>