A Comparison of Automated Perfusion- and Manual Diffusion-Based Human Regulatory T Cell Expansion and Functionality Using a Soluble Activator Complex

Absence or reduced frequency of human regulatory T cells (Tregs) can limit the control of inflammatory responses, autoimmunity, and the success of transplant engraftment. Clinical studies indicate that use of Tregs as immunotherapeutics would require billions of cells per dose. The Quantum® Cell Expansion System (Quantum system) is a hollow-fiber bioreactor that has previously been used to grow billions of functional T cells in a short timeframe, 8–9 d. Here we evaluated expansion of selected Tregs in the Quantum system using a soluble activator to compare the effects of automated perfusion with manual diffusion-based culture in flasks. Treg CD4+CD25+ cells from three healthy donors, isolated via column-free immunomagnetic negative/positive selection, were grown under static conditions and subsequently seeded into Quantum system bioreactors and into T225 control flasks in an identical culture volume of PRIME-XV XSFM medium with interleukin-2, for a 9-d expansion using a soluble anti-CD3/CD28/CD2 monoclonal antibody activator complex. Treg harvests from three parallel expansions produced a mean of 3.95 × 108 (range 1.92 × 108 to 5.58 × 108) Tregs in flasks (mean viability 71.3%) versus 7.00 × 109 (range 3.57 × 109 to 13.00 × 109) Tregs in the Quantum system (mean viability 91.8%), demonstrating a mean 17.7-fold increase in Treg yield for the Quantum system over that obtained in flasks. The two culture processes gave rise to cells with a memory Treg CD4+CD25+FoxP3+CD45RO+ phenotype of 93.7% for flasks versus 97.7% for the Quantum system. Tregs from the Quantum system demonstrated an 8-fold greater interleukin-10 stimulation index than cells from flask culture following restimulation. Quantum system–expanded Tregs proliferated, maintained their antigenic phenotype, and suppressed effector immune cells after cryopreservation. We conclude that an automated perfusion bioreactor can support the scale-up expansion of functional Tregs more efficiently than diffusion-based flask culture.

Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Background: Trypsin has a plenty application in food and pharmaceutical manufacture. While, the commercial trypsin is usually extracted from pork pancreas, which has the risk of infectious and immunogenicity. Therefore, the microbial Streptomyces griseus trypsin (SGT) is a prior alternation because it processes efficient hydrolysis activity without the aforementioned risk. The remarkable hydrolysis efficiency of SGT caused its autolysis, and five autolysis sites R21, R32, K122, R153, and R201 were identified from its' autolysate. Results: The tbcf (K101A, R201V) mutant was screened by directed selection approach for improved activity in flask culture (60.85 ± 3.42 U·mL -1 , increased 1.5-fold). From the molecular dynamics simulation, the K101A/R201V mutation shortened the distant between catalytical residues D102 and H57 from 6.5 Å vs 7.0 Å, which afforded the improved specific activity 1527.96 ± 62.81 U·mg -1 . Further, the production of trypsin was increased 302.8% (689.47 ± 6.78 U·mL −1 ) in 3-L bio-reactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: The SGT protein could be an adequate trypsin for insulin production. When working with hydrolysates analysis and direction selection, the tbcf (K101A, R201V) mutant increased 1.5-fold activity. Further, the production of trypsin was improved 3-fold by overexpressing chaperone protein in Pichia pastoris . The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical.

Humus composition of mineral–microbial residue from microbial utilization of lignin involving different mineral types

This study explored the mineral contribution of lignin to humus (HS) formation through the change of HS composition in microbial–mineral residue (MMR). The liquid shake flask culture method was adopted to collect the MMR formed through the microbial utilization of lignin in the presence of goethite, bayerite, δ-MnO2, kaolinite, and montmorillonite. The carbon (C) contents of humic-like acid (HLA), fulvic-like acid (FLA), and humin-like (HLu) in MMR, represented as CHLA, CFLA, and CHLu, respectively, coupled with the ΔlogK of the HLA alkali-soluble extract and CHLA/CFLA ratio were analyzed at 10, 30, 60, and 110 d. In terms of improving HLA aggregated on minerals, the following rule was observed: goethite > bayerite > montmorillonite > kaolinite ≈δ-MnO2. Goethite was most likely to adsorb organic molecules with a high degree of polymerization. Compared with kaolinite and montmorillonite, goethite, bayerite, and δ-MnO2 were more helpful for decreasing the molecular weight and the degree of HLA condensation. Goethite, δ-MnO2, and montmorillonite presented the greatest advantages in enhancing the relative proportions of CHLA, CFLA, and CHLu, respectively, in MMR. In MMR formed in the presence of kaolinite, goethite, and bayerite, CHLA was decreased by 14.8%, 12.0%, and 5.8%, respectively, at the end of culture, whereas the CHLA associated with δ-MnO2 was increased by 12.0%. δ-MnO2 contributed the most to the conversion of CFLA to CHLA. Due to expandability and a much greater adsorption capacity, montmorillonite was most beneficial to the accumulation of CHLu.

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

Fermentation of Oil Palm Trunk Acid Hydrolysate to Ethanol

Studies on the feasibility of using oil palm trunk acid hyd10lysates as a substrate for ethanol p10duction using the yeast Saccha10myces cerevisiae were carried out in shake flask culture. The effect of pH and fermentation time on the rate of ethanol p10duction were investigated. Results showed that with 3 hours fermentation period, the highest ethanol yield was obtained at pH 4.75. This yield was about 14% of the dry weight of the sample used; thus giving a fermentation efficiency of 94.7%.